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1
Douglas, Chanel Catherine. "A study into the protein/protein interactions involved in HIV-1 capsid assembly." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2008r/douglas.pdf.
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Seckler, James Malcolm. "The Structural Dynamics of Human Immunodeficiency Virus Type I Reverse Transcriptase." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1298562809.
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Apcher, Géraud-Sébastien. "HIV-1 Tat protein and proteasomal Subunit Interactions." Clermont-Ferrand 2, 2003. http://www.theses.fr/2003CLF21470.
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Etant la source centrale de production de peptides antigéniques, le protéasome, un complexe multiprotéique qui est un constituant majeur de la voie protéolytique non-lysosomale, semble être une des cibles principales pour les virus. En réponse à une production d'interféron-y lors d'une infection virale, les nouveaux immunoprotéasomes produisent des peptides provenant de la dégradation de protéines virales qui vont alors interagirent avec les molécules de classes I du CMH afin de déclencher une réponse cytosolique en vue d'éliminer les cellules infectées. La réponse immunitaire cellulaire à une infection par le VIH est inhabituelle. Ce phénomène serait dû à la protéine Tat du VIH. Un des buts précis de ce projet était de démontrer les interactions entre les différentes sous-unités alpha du protéasome 20S qui n'ont pas été prédictes auparavant dans l'étude de la structure crystal du protéasome 20S de levure. A l'aide de la technique du double hybride nous avons pu montrer que la sous-unité alpha7 intéragit fortement avec la sous-unité alpha4 ainsi qu'avec les 5 autres sous-unités alpha. Ces résultats ont été également justifié par l'interaction des sous-unités alpha-radiomarquées, aux protéines de fusions GST-alpha. L'autre but de ce projet était de déterminer quelles sous-unités du protéasome 20S interagissaient avec la protéine Tat. Utilisant la technique de chromatographie d'affinité nous avons pu montrer que la protéine Tat intéragissait avec les sous-unités alpha4 et alpha7 ainsi qu'avec 8 pro-sous-unités bêta. En outre, la protéine Tat inhibe partiellement l'interaction entre ces deux sous-unités. D'autre part la protéine Tat inhibe l'activité chymotrypsine du protéasome 20S in vivo. La protéine Tat peut ainsi, d'une part, interférer avec l'assemblage du protéasome 20S et, d'autre part, inhiber la production de peptides antigéniques et donc leurs présentations aux molécules de classes I des cellules infectées
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Abdurahman, Samir. "Studies on HIV-1 core assembly /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-363-4/.
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Leung, Sze-ki, and 梁詩琪. "Mechanism of human immunodeficiency virus induced immunedysregulation: TAT & IL-18 interaction." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B34605472.
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Antonucci, Jenna Marie. "Mechanisms of HIV-1 Restriction by the Host Protein SAMHD1." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1524006072232491.
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Leung, Sze-ki. "Mechanism of human immunodeficiency virus induced immune dysregulation TAT & IL-18 interaction /." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B34605472.
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Postupalenko, Viktoriia. "Ratiometric fluorophores for peptides and oligonucleotides labeling : application to the HIV-1 nucleocapsid protein." Strasbourg, 2011. https://publication-theses.unistra.fr/public/theses_doctorat/2011/POSTUPALENKO_Viktoriia_2011_ED414.pdf.
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Ce travail porte sur le développement de sondes fluorescentes 3-hydroxychromones pour suivre l’interaction de protéines avec des oligonucléotides (ODNs) et des membranes. Ces sondes ont deux bandes d'émission bien séparées et sensibles à l'environnement, résultant d’un transfert de proton intramoléculaire à l’état excité, d’où une détection des interactions via leur rapport d'intensité. Un analogue d’acide aminé dérivé de la sonde 3HC a été synthétisé puis inséré à des positions ciblées de la protéine de la nucléocapside (NC) du VIH-1. Nous avons ainsi obtenus des informations site-spécifique sur les modifications induites à proximité du site marqué lors de l’interaction avec des ODNs, une alternative étant d’utiliser des nucléosides fluorescents introduits en différentes positions des ODNs. Pour étudier les interactions peptide-membrane, une sonde 3HC sensible aux changements de polarité en solvants apolaires a été couplée à l’extrémité N-terminale de peptides synthétiques (mélittine, magaïnine, poly-L-lysine) connus pour interagir avec les biomembranes. Le rapport d'intensité se corréle bien avec la profondeur d'insertion de cette région N-terminale. Enfin, nous avons appliqué cette sonde pour détecter des interactions possibles de la NC avec les membranes. Nous avons montré que NC se lie avec une forte affinité par voie électrostatique aux membranes contenant des lipides anioniques, permettant de localiser NC au niveau des têtes lipidiques. NC déstabilise la membrane de manière dose-dépendante. Les complexes NC-ADN se lient également aux membranes comme la NC libre, permettant de suggérer une participation de NC à l'import nucléaire du complexe de pré-intégrationThis work focuses on the development of a methodology for sensing interactions of proteins with oligonucleotides (ODNs) and membranes based on fluorescent probes from the 3-hydroxychromone (3HC) family. Due to an excited state intramolecular proton transfer, these dyes exhibit two highly resolved emission bands, differently sensitive to the environment, thus allowing to sense interactions through their intensity ratio changes. An amino acid analogue based on 3HC was synthesized and inserted at selected positions of the HIV-1nucleocapsid protein (NC), to further characterize the peptide-ODNs interaction and provide site-specific information on the environmental changes induced by the interaction close to the labeling site. As an alternative, fluorescent nucleosides were synthesized and applied to different positions of ODNs. To study the peptide-membrane interactions, 3HC probe sensitive to polarity changes in apolar solvents was coupled to the N-terminus of melittin, magainin and poly-L-lysine peptides, known to interact with lipid membranes. The observed intensity ratio of the probe correlates well with the insertion depth of the N-terminal region of the peptides. Finally, we applied this dye for the detection of possible interactions of NC with membranes. We have shown that NC binds with high affinity to membranes containing negatively charged lipids. This interaction is mainly driven by electrostatic forces and locates NC at the level of lipid heads. Moreover, NC destabilizes the membrane in a concentration-dependent manner. As free NC, NC-DNA complexes can also bind to membranes, suggesting an involvement of NC in the nuclear import of the pre-integration complex
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Zheng, Yingfeng. "Functional analysis on the interactions of the human immunodeficiency virus type 1 integrase with its cofactors that regulate viral replication." BioMed Central Ltd, 2008. http://hdl.handle.net/1993/16248.
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Like all viruses, the replication of HIV-1 relies heavily on host proteins due to its limited genome products. HIV-1 integrase (IN) catalyzes the integration of viral DNA into host genome and also impacts other steps of viral replication cycle, all of which are assisted by various cellular proteins. Among them, LEDGF/p75 acts as the IN-to-chromatin tethering factor. However, whether other cellular cofactors also participate in this process still remains elusive. To gain insight into the mechanism of action of HIV-1 IN during viral integration, we used a previously described IN/yeast lethality system and our results revealed that the HIV-1 IN-induced yeast lethality absolutely required its chromatin binding ability. Since there is no yeast homolog of LEDGF/p75, it raises the possibility that IN may recruit other cellular cofactors for its chromatin targeting. Consistently, further analysis in mammalian cells indicated that HIV-1 IN was able to mediate chromatin binding independent of IN-LEDGF/p75 interaction and that HIV-1 fitness relied more on chromatin binding than LEDGF/p75 binding of IN. These data greatly enrich our current knowledge on the dynamic interplay within the ternary complex IN/LEDGF/chromatin.
HIV-1 exploits multiple cellular cofactors not only to facilitate viral replication, but also to evade the host defense system in favor of the virus. IN is known to be an unstable protein, degraded by the host ubiquitin-proteasome pathway. To investigate how IN avoids the host degradation machinery in the context of viral infection, we showed that IN interacted with host protein Ku70 and protected itself from the Lys48-linked polyubiquitination proteasomal pathway. More importantly, Ku70 was shown to be incorporated into the progeny virus in an IN-dependent manner, and both cell- and virus- associated Ku70 were essential for HIV-1 replication. Finally, the data demonstrated that the interactions between HIV-1 IN and host cofactors can be regulated through its SUMO-interacting motifs (SIMs). Three putative SIMs (72VILV75; 200IVDI203 and 257IKII260) in IN were examined and shown to be essential for IN-LEDGF/p75 but not IN-Ku70 interaction.
In summary, this study advances our knowledge of the interaction network between IN and its cofactors, which would have important implications for the design of anti-HIV drugs.
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Zhang, Wei Hong. "Studies on structure and function of human immunodeficiency virus type one (HIV-1) Gag protein." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320175.
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Djekic, Uros V. "Coupling selection of the HIV-1 tRNA primer used for reverse transcription with viral translation and encapsidation." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2008r/djekic.pdf.
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Dupont, Stefan A. "The Functional Roles of the Human Immunodeficiency Virus Type-1 Matrix Protein during Viral Life Cycle: A Dissertation." eScholarship@UMMS, 2000. http://escholarship.umassmed.edu/gsbs_diss/191.
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The human immunodeficiency virus type-1 matrix (HIV-1 MA) is best described as a multi-functional, structural protein. However, the multitude of functional activities ascribed to this viral component is not nearly as interesting as are its seemingly paradoxical and opposing roles during the viral life cycle.
At the time of virus infection, HIV-1 MA remains associated with the reverse transcription complex, in which viral nucleic acids are synthesized, and facilitates its translocation to the host cell nucleus (Bukrinsky, Sharova et al. 1992; Bukrinsky, Sharova et al. 1993). This activity of MA has been proposed to form the basis for the infection of non-dividing cells (Bukrinsky, Haggerty et al. 1993). An interaction between the C-terminally phosphorylated form of MA and HIV-1 integrase, an integral component of the complex, was initially proposed to mediate this association (Gallay, Swingler et al. 1995; Gallay, Swingler et al. 1995). However, conditions which promote dissociation of integrase from the reverse transcription complex do not reduce MA association (Miller, Farnet et al. 1997). The possibility of a direct interaction between MA and the viral genome is discussed in Chapter III.
The nucleophilic nature of HIV-1 MA is paradoxical with its reported activity in targeting the viral precursor proteins to the cytoplasmic membrane (Krausslich and Welker 1996), during the particle production phase of the viral life cycle. Furthermore, MA when expressed in the absence of other viral proteins exhibits a cytoplasmic localization (Fouchier, Meyer et al. 1997); a result which does not support a nuclear translocation role for this protein. The work presented here resolves this seemingly controversial issue.
We demonstrate that MA exhibits a strong nuclear export activity. This newly discovered activity is designed to effectively counteract the protein's innate nucleophilic nature, thus maintaining a cytoplasmic localization. The nuclear export function of MA is sensitive to changes within the conformation of the protein as C- and N-terminal deletions, as well as point mutations in the protein, abolish the activity. Furthermore, the export activity is mediated by the Crm1 NES receptor (Fornerod, Ohno et al. 1997; Fukuda, Asano et al. 1997; Ossareh-Nazari, Bachelerie et al. 1997) despite the lack of a leucine-rich export signal within the matrix coding region. Therefore, the interaction between matrix protein and Crm1 is most likely to be mediated by another, perhaps cellular, protein. Any changes in matrix structure may lead to the disruption of this protein-protein interaction. We discuss a model implicating a phosphorylation event in the inactivation of this nuclear export signal.
An even more fascinating issue regards the role of this nuclear export activity, during the viral life cycle, and is detailed in Chapter II. In short, mutations in MA which impair its nuclear export activity result in nuclear accumulation of the precursor Gag polyprotein (Pr55) and the nucleocapsid-associated viral genomic RNA. As a result, non-infectious virions deficient in genomic viral RNA are produced. Therefore, drugs designed to block this export activity can undermine the carefully orchestrated course of events during HIV replication and can shut down the growth of the virus.
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Nowak, Piotr. "Aspects of immune activation in HIV-1 infection /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-190-6/.
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Johnson, Jacklyn. "Properties of HIV-1 env and human seminal fluid that determine virus inhibition by antibodies and microbicides." Diss., University of Iowa, 2019. https://ir.uiowa.edu/etd/6966.
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Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection that leads to acquired immunodeficiency syndrome (AIDS). Approximately 36 million people worldwide are living with HIV-1, which is commonly acquired through sexual contact. Antiviral therapies control disease progression, but do not eliminate this virus from the host. Thus, global efforts are focused on developing vaccines that prevent HIV-1 transmission. Such vaccines are based on eliciting the production of protective antibodies that target the envelope glycoproteins (Envs) of this virus. Unfortunately, HIV-1 immunization trials have shown limited efficacy. A better understanding of the antibody-mediated inactivation process is needed to improve vaccine strategies. In this work we describe two novel factors that contribute to HIV-1 inactivation. First, we show that structural stability of the Env protein determines its sensitivity to vaccine-elicited antibodies. Different interactions within Env contribute to its stability. Perturbation of the Env-stabilizing interactions by physical and chemical treatments enhances sensitivity of HIV-1 to antibodies. Second, we found that the chemical composition of the transmission medium affects Env inhibition by antibodies and other inhibitory agents. Semen is the most common vehicle for HIV-1 transmission. This medium contains high concentrations of the sugar fructose. We found that semen fructose competitively blocks binding of antiviral agents that target sugar residues on Env. Together, this work advances our understanding of the mechanism that underlies HIV-1 inactivation by vaccine-elicited antibodies and provides novel strategies to enhance their potency.
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Chaudhuri, Rittik. "The mechanism of HIV-1 Nef-mediated downregulation of CD4." Thesis, University of Cambridge, 2010. https://www.repository.cam.ac.uk/handle/1810/224775.
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Nef, an accessory protein of HIV-1, is a critical determinant of viral pathogenicity. The pathogenic effects of Nef are in large part dependent on its ability to decrease the amount of CD4 on the surface of infected cells. Early studies suggested that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein-trafficking machinery. However, the specific sorting pathway that Nef uses to modulate CD4 expression remained uncertain. According to one model, Nef was thought to interfere with the transport of newly synthesized CD4 from the TGN to the cell-surface. Another model claimed that Nef facilitated the removal of CD4 from the plasma membrane. The primary goal of this thesis was to determine which of these models was correct. To accomplish this objective, a novel Nef-CD4 system was developed in Drosophila S2 cells. Nef was not only able to downregulate human CD4 in S2 cells, but it did so in a manner that was phenotypically indistinguishable from its activity in human cells. An RNAi screen targeting protein-trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP-2 complex in the Nef-mediated downregulation of CD4. In contrast, depletion of the related AP-1 and AP-3 complexes, which direct transport from the TGN and endosomes, had no effect. The requirement for AP-2 was subsequently confirmed in a human cell line. Yeast three-hybrid and GST pull-down assays were then used to demonstrate a robust, direct interaction between Nef and AP-2. This interaction was found to depend on a [D/E]xxxL[L/I]-type dileucine motif, located in the C-terminal loop of Nef, that is essential for CD4 downregulation. While mapping the binding site of AP-2 on Nef, a second determinant of interaction in the C-terminal loop was identified. Mutation of this motif, which conforms to a consensus [D/E]D diacidic sequence, prevented Nef from binding to AP-2 and down-regulating CD4. However, the same mutations did not affect the ability of Nef to interact with either AP-1 or AP-3, providing further evidence that these complexes are not required for the modulation of CD4 expression. Additional experiments indicated that the Nef diacidic motif most likely binds to a basic patch on AP-2 α-adaptin that is not present in the homologous AP-1 γ and AP-3 δ subunits. As with the Nef diluecine and diacidic motifs, the α-adaptin basic patch was shown to be necessary for CD4 downregulation. Moreover, all three of these motifs were needed for the cooperative assembly of a CD4-Nef-AP-2 tripartite complex, which was observed here for the first time using a yeast four-hybrid system. The data in this thesis uniformly support an endocytic model of Nef-mediated CD4 downregulation. Indeed, there is now strong evidence that Nef simultaneously binds CD4 and AP-2, thereby connecting the receptor to the cellular endocytic machinery and promoting its rapid internalization from the plasma membrane. In addition, the identification of novel motifs required for this process has provided new insights on endocytosis, and may facilitate the development of pharmacological inhibitors of Nef function.
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Kim, Adonia Lee. "The Role of Adaptor Protein Complex-3 Delta-Mediated HIV-1 Gag Trafficking in HIV-1 Replication: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/612.
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The process of HIV-1 particle production is a multi-step process directed by the viral structural protein Gag. As Gag is the only viral protein required to form virus-like particles, it presents a viable target for anti-viral therapeutics of which there are currently none. Although the functions of Gag during the particle assembly process have been well characterized, one of the least known parts of the assembly process is how Gag is targeted to the site of virus assembly.
Two main virus assembly sites have been identified in cells that support HIV-1 replication: the plasma membrane or multivesicular bodies (MVBs). However the mechanism by which Gag is targeted to either of these sites remains unknown. The δ subunit of Adaptor Protein Complex 3 has previously been identified as a cellular co-factor for HIV-1 Gag and was reported to mediate Gag trafficking to MVBs, providing a mechanism for Gag targeting to this assembly site. Additionally, AP-3δ was reported to be required for HIV-1 production, suggesting that Gag to MVB targeting is also required for HIV-1 production.
The work presented in this thesis further investigates the role of AP-3δ in Gag trafficking to MVBs and its role in HIV-1 production in previously unexplored host environments. Through the use of RNA interference-mediated depletion of AP-3δ, we determined that AP-3δ is dispensible for virus replication in infected HeLa cells, chronically infected HeLa-LAV cells and infected primary human monocyte-derived macrophages. We concomitantly disrupted AP-3 function by disrupting its association with membranes and observed no effect on virus production. Collectively, these results demonstrate that AP-3δ is not required for HIV-1 replication. However, AP-3δ was demonstrated to be required for Gag targeting to MVBs thus presenting a new model for the function of AP-3δ in the context of HIV-1 replication.
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Warncke, Malte L. [Verfasser], and Hans-Willi [Akademischer Betreuer] Mittrücker. "Generation of bispecific antibodies against the human immunodeficiency virus (HIV) envelope protein / Malte L. Warncke. Betreuer: Hans-Willi Mittrücker." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2013. http://d-nb.info/1037822560/34.
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Romani, Bizhan. "Mutagenesis and functional studies of the HIV-1 vpr gene and Vpr protein obtained from South African virus strains." Thesis, Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/6702.
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Thesis (PhD)--University of Stellenbosch, 2011.ENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of host cellular and other viral proteins. Vpr exerts several functions such as induction of apoptosis, induction of cell cycle G2 arrest, modulation of gene expression, and suppression of immune activation. The functionality of subtype C Vpr, especially South African strains, has not been studied. The aim of this study was to describe the diversity of South African HIV-1 subtype C vpr genes and to investigate selected functions of these Vpr proteins. Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, and subtyped using phylogenetic analysis. Fragments containing natural mutations were cloned in mammalian expression vectors. A consensus subtype C vpr gene was constructed and site-directed mutagenesis was used to induce mutations in postions in which no natural mutations have been described. The functionality of all constructs was compared with the wild-type subtype B Vpr, by transfecting human 293T cell line to investigate subcellular localization, induction of apoptosis and cell cycle G2 arrest. The modulation of genes expressed in the induction of apoptosis using TaqMan Low density arrays (TLDA) was also investigated. Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4 strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conserved including motifs FPRPWL and TYGDTW, but the C-terminal was more variable. The following mutations were constructed using site-directed mutagenesis: P14I, W18C, Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vpr localized to the nucleus but the W18C mutation disturbed the nuclear localization of Vpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, was lower than that of subtype B Vpr. All the natural mutants of subtype C Vpr induced cell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycle G2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteins induced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells. However, an artificially designed Vpr protein containing the consensus sequences of subtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-C Vpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants of Vpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associated gene expression was similar for all constructs, indicated that apoptosis was efficiently induced through the intrinsic pathway by the mutants. Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display a similar ability for nuclear localization and apoptosis induction. The induction of cell cycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype C Vpr proteins. The natural mutations studied in the isolates did not disturb the functions of subtype C Vpr and in some cases even potentiated the protein to induce apoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded as defective, since enhanced functionality would be more indicative of an adaptive role. The increased potency of the mutated Vpr proteins suggests that Vpr may increase the pathogenicity of HIV-1 by adapting apoptotic enhancing mutations.
AFRIKAANSE OPSOMMING: Agtergrond: Die virus protein R (Vpr) van Menslike Immuungebrek Virus tipe 1 (MIV-1) is ‘n bykomstige protein wat met ‘n aantal sellulêre proteine van die gasheer en ander virus proteine in wisselwerking tree. Vpr het 'n invloed op verskeie funksies onder andere die induksie van apoptose, die induksie van selsiklus G2 staking, modulering van geen uitdrukking en onderdrukking van immuun aktivering. Die funksionaliteit van subtipe C Vpr, en veral die van Suid-Afrikaanse stamme, is nie beskryf nie. Die doelwit van die studie was om die diversiteit van Suid Afrikaanse MIV-1 subtipe C vpr gene te beskryf en ook om selektiewe funksies van die Vpr proteine te ondersoek Metodiek: Die MIV-1 vpr streek van 58 stamme is vermeerder, die DNA volgordes is bepaal en die stamme is gesubtipeer deur filogenetiese analise. Fragmente met natuurlike mutasies is in ekspressie vektore gekloon. ‘n Konsensus subtipe C Vpr geen is ontwerp en mutasies in posisies waar geen natuurlike mutasies beskryf is nie, is ontwerp deur mutagenese. Die funksionaliteit van die konstrukte is met die wilde tipe subtype B vergelyk deur 293T sellyn te transfekteer en te ondersoek vir subsellulêre lokalisering, induksie van apoptose, en G2 selsiklus stilstand. Die modulering van geen uitdrukking in die induksie van apoptose is deur TLDA ondersoek. Resultate: Filogenetiese analise het 54 stamme as HIV-1 subtipe C geklassifiseer en 4 stamme as subtype B. Die Vpr aminosuur volgordes was konstant insluitend die FPRPWL en TYGDTW motiewe, maar die C-terminaal was meer variëerbaar. Deur mutagenese is die volgende mutasies ontwerp: P14I, W18C, Y47N, Q65H and Q88S. Subtipe B en al die natuurlike mutante van subtipe C het in die selkern gelokaliseer, maar die W18C mutasie het die lokalisasie versteur. Die G2 selsiklus stilstand van alle mutante en konsensus C was laer as die van subtype B. Al die natuurlike subtipe C mutante het G2 selsiklus tot stilstand gebring in 54.0-66.3% van die selle, terwyl subtype B selsiklus tot stilstand gebring het in 71.5% van die selle. Subtipe B en die natuurlike Vpr mutante het apoptose op ‘n soortgelyke wyse geinduseer, wat wissel tussen 95.3-98.6% van getransfekteerde selle. Die protein met die kunsmatig ontwerpte konsensus C volgorde het egter ‘n verlaagde vermoë gehad om apoptose te induseer. Die konsensus subtipe C het apoptose in 82.0% van getransfekteerde selle geinduseer en die kunsmatige mutante in 88.4 – 96.2% van die selle. Die induksie van die apoptose verwante geen ekspressie deur die mutante was soortgelyk as die van konsensus C en subtipe B Vpr wat ’n aangeduiding is dat apoptose effektief veroorsaak is deur die intrinsieke roete. Gevolgtrekking: Hierdie studie het aangetoon dat kern lokalisering en apoptose op ‘n soortgelyke wyse by beide MIV-1 subtipe B en C Vpr plaasvind. Die induksie van selsiklus G2 stilstand deur MIV-1 subtipe B Vpr is egter meer robuust as baie van die subtipe C Vpr proteïene. Natuurlike mutasies in MIV-1 Vpr kan nie as gebrekkig beskou word nie, aangesien beter funksionaliteit 'n aanduiding is vandie aanpasbare rol. Die verhoogde krag van die gemuteerde Vpr proteïen dui daarop dat Vpr die patogenisiteit van MIV-1 kan verbeter deur die aanpassing van mutasies.
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Vaine, Michael. "Antibody Responses Elicited by DNA Prime-Protein Boost HIV Vaccines: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/462.
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The best known correlate of protection provided by vaccines is the presence of pathogen specific antibodies after immunization. However, against the Human Immunodeficiency Virus-1 (HIV-1) the mere presence of antibodies specific for the viral Envelope (Env) protein is not sufficient to provide protection. This necessitates in depth study of the humoral responses elicited during infection and by vaccination. While a significant amount of effort has been invested in studying the evolution of antibody responses to viral infection, only limited progress in understanding antibody responses elicited through vaccination has been made. In the studies described here, I attempt to rectify this deficiency by investigating how the quality of a humoral response is altered with the use of different immunization regimens, in particular a DNA prime-protein boost regimen, or with the use of different model HIV-1 Env gp120 immunogens. In a New Zealand White (NZW) rabbit model, we demonstrate that the broader neutralizing activity elicited with the DNA prime-protein boost regimen may be the result of the elicitation of a higher avidity antibody response and a unique profile of antibody specificities. Specifically, use of a DNA prime-protein boost regimen elicits antibodies targeted to the CD4 binding domain of the HIV-1 Env, a specificity that was not frequently observed when only protein based immunizations were administered.
We extended this analysis to sera from healthy human volunteers who participated in early phase HIV vaccine trials utilizing either a protein alone immunization regimen, a canarypox prime-protein boost immunization regimen, or a DNA prime-protein boost immunization regimen. Evaluation of sera from these trials demonstrated that the use of a DNA prime-protein boost regimen results in an antibody response with greater neutralization breadth characterized by an increased frequency and titer of antibodies targeted toward the CD4 binding site (CD4bs). In addition to this, the antibody response elicited by the DNA prime-protein boost regimen also exhibited the capability to mediate antibody dependent cell-mediated cytotoxicity (ADCC) activity as well as activation of the complement system.
Additionally, in an attempt to better understand the capabilities of antibodies elicited by a DNA prime-protein boost regimen, we generated gp120 specific monoclonal antibodies (mAbs) from a single DNA primed-protein boosted NZW rabbit. Analysis of mAbs produced from this animal revealed that use of this immunization regimen elicits an antibody repertoire with diverse epitope specificity and cross reactivity. Furthermore, these select mAbs are capable of neutralizing heterologous HIV isolates. Further application of mAb generation in rabbits may provide a valuable tool to study immunogenicity of different vaccines and immunization regimens.
Concurrently, while demonstrating that a DNA prime-protein boost regimen elicits a higher quality antibody response than that observed with other leading techniques, we also demonstrated that immunogen selection can play a vital role in the quality of the resulting antibody response. By immunizing with two closely related but phenotypically distinct model gp120 immunogens, known as B33 and LN40, we demonstrated that disparate gp120s have different intrinsic abilities to raise a heterologous neutralizing antibody response. Additionally, we showed that residues found within and flanking the b12 and CD4 binding sites play critical roles in modulating neutralizing activity of sera from animals immunized with LN40 gp120, indicating that the broader neutralizing activity seen with this immunogen may be due to differential elicitation of antibodies to this domain.
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Shandilya, Shivender. "Structural Studies of the Anti-HIV Human Protein APOBEC3G Catalytic Domain: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/562.
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HIV/AIDS is a disease of grave global importance with over 33 million people infected world-wide and nearly 2 million deaths each year. The rapid emergence of drug resistance, due to viral mutation, renders anti-retroviral drug candidates ineffective with alarming speed and regularity. Instead of targeting mutation prone viral proteins, an alternative approach is to target host proteins that interact with viral proteins and are critical for the HIV life-cycle. APOBEC3G is a host anti-HIV restriction factor that can exert tremendous negative pressure by hypermutating the viral genome and has the potential to be a promising candidate for anti-retroviral therapeutic research.
The work presented in this thesis is focused on investigating the A3G catalytic domain structure and implications of various observed structural features for biological function. High-resolution crystal structures of the A3G catalytic domain were solved using data from macromolecular X-ray crystallographic experiments, revealing a novel intermolecular zinc coordinating motif unique to A3G. Major intermolecular interfaces observed in the crystal structure were investigated for relevance to biochemical activity and biological function.
Co-crystallization with a small-molecule A3G inhibitor, discovered using high-throughput screening assays, revealed a cysteine residue near the active site that is critical for inhibition of catalytic activity by catechol moieties. The serendipitous discovery of covalent interactions between this inhibitor and a surface cysteine residue led to further biochemical experiments that revealed the other cysteine, near the active site, to be critical for inhibition.
Computational modeling was used to propose a steric-hinderance based mechanism of action that was supported by mutational experiments. Structures of other human APOBEC3 homologs were modeled using in-silico methods examined for similarities and differences with A3G catalytic domain crystal structures. Comparisons based on these homology models suggest putative structural features that may endow substrate specificity and other characteristics to the APOBEC3 family members.
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Costa, Matthew R. "FC Receptor-Mediated Activities of Env-Specific Monoclonal Antibodies Generated from Human Volunteers Receiving a DNA Prime-Protein Boost HIV Vaccine: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/866.
Full textAbstract:
Human immunodeficiency type 1 (HIV-1) is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years’ infection and as a result, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with antibody dependent cellular cytotoxicity (ADCC) activity. Previous studies in the Lu lab demonstrated that in an HIV-1 vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities along with multiple Fc mediated effector functions. I developed a protocol for the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from these DP6-001 vaccinees. By utilizing a labeled gp120 bait to isolate Env specific B cells, paired heavy and light chain immunoglobulin (Ig) genes were cloned and allowed for the production of monoclonal antibodies with specificity for gp120. By using this protocol, 13 isolated mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens, with mostly conformational epitopes and a few V3 and C5 specific mAbs. Equally cross-reactive Fc-mediated functional activities, including ADCC and antibody dependent cellular phagocytosis (ADCP), were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional antibodies by the DNA prime- protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV-1 vaccine design.
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Costa, Matthew R. "FC Receptor-Mediated Activities of Env-Specific Monoclonal Antibodies Generated from Human Volunteers Receiving a DNA Prime-Protein Boost HIV Vaccine: A Dissertation." eScholarship@UMMS, 2010. http://escholarship.umassmed.edu/gsbs_diss/866.
Full textAbstract:
Human immunodeficiency type 1 (HIV-1) is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years’ infection and as a result, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with antibody dependent cellular cytotoxicity (ADCC) activity. Previous studies in the Lu lab demonstrated that in an HIV-1 vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities along with multiple Fc mediated effector functions. I developed a protocol for the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from these DP6-001 vaccinees. By utilizing a labeled gp120 bait to isolate Env specific B cells, paired heavy and light chain immunoglobulin (Ig) genes were cloned and allowed for the production of monoclonal antibodies with specificity for gp120. By using this protocol, 13 isolated mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens, with mostly conformational epitopes and a few V3 and C5 specific mAbs. Equally cross-reactive Fc-mediated functional activities, including ADCC and antibody dependent cellular phagocytosis (ADCP), were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional antibodies by the DNA prime- protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV-1 vaccine design.
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Sercovich, Mark J. "Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr)-mediated cell cycle arrest : an analysis of current mechanistic models /." Download the thesis in PDF, 2006. http://www.lrc.usuhs.mil/dissertations/pdf/sercovich2006.pdf.
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Kulkarni, Amod Prakash. "Delivery of Vaccinia Virus Complement Control Protein (VCP) and Curcumin to the rodents' brain : implications in Alzheimer's disease and HIV dementia." Doctoral thesis, University of Cape Town, 2008. http://hdl.handle.net/11427/3286.
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Sánchez-Velar, Nuria. "The Human Rev Interacting Protein (hRIP) is Required for Rev Function and HIV-1 Replication: a Dissertation." eScholarship@UMMS, 2005. http://escholarship.umassmed.edu/gsbs_diss/312.
Full textAbstract:
Retroviruses have evolved sophisticated mechanisms to ensure timely export of incompletely spliced viral messenger ribonucleic acids (mRNAs) for gene expression and for viral packaging. For example, the Human Immunodeficiency Virus type 1 (HIV-1) encodes the Rev regulatory protein, a sequence-specific RNA-binding protein that is responsible for the cytoplasmic accumulation of intron-containing viral mRNAs.
The HIV-1 Rev protein contains an amino terminal (N-terminal) Arginine-Rich Motif (ARM) RNA-binding domain (RBD) and a carboxy terminal (C-terminal) leucine-rich activation domain which functions as a Nuclear Export Signal (NES). The Rev ARM interacts in a sequence-specific manner with a cis-acting viral RNA stem-loop structure, the Rev Responsive Element (RRE), located in all incompletely spliced viral mRNAs. This initial interaction is followed by the recruitment of additional Rev molecules to form a RiboNucleoProtein (RNP) complex involving the RRE and Rev molecules.
The cytoplasmic accumulation of the Rev:RRE RNP complex is dependent on the interaction of Rev with key cellular cofactors. Rev activation domain mutants exhibit a trans-dominant negative phenotype, suggesting that this domain of Rev interacts with cellular proteins required for Rev function. Biochemical and genetic studies have identified several cellular proteins that bind to the activation domain of Rev and are therefore candidate cofactors for Rev function. Amongst these is the human Rev Interacting Protein [hRIP, 79], which is also known as the Rev/Rex activation domain-binding protein [Rab, 18].
hRIP was identified in a yeast two-hybrid assay with the HIV-1 Rev and its functionally equivalent Human T-cell Leukemia Virus type-1 (HTLV-1) Rex protein as baits. The interaction between hRIP and HIV-1 Rev is dependent on a functional Rev NES, as predicted for a bona fide Rev cellular cofactor, and the Nucleoporin-like (Nup-like) repeats in the C-terminus of hRIP (18, 79]. Additional genetic studies indicated that the interaction between hRIP and Rev is indirect and is most likely mediated by the cellular export receptor CRM1 (Chromosomal Region Maintenance 1) [1, 153].
A role for hRIP in Rev function or HIV-1 replication has remained elusive. The goal of this dissertation was to determine whether hRIP is required for Rev function and HIV-1 replication. We used two approaches, a dominant-negative mutant and RNA interference (RNAi), to ablate hRIP activity and analyzed Rev function and HIV-1 replication using standard assays.
The results of this dissertation demonstrate that hRIP is required for Rev function and HIV-1 replication. We show that Rev function is inhibited upon ablation of hRIP activity by either a trans-dominant negative mutant or RNAL Furthermore, we find that depletion of endogenous hRIP by RNAi results in the loss of viral replication in human cell lines and primary human macrophages. Unexpectedly, in the absence of functional hRIP, RRE-containing viral RNAs accumulate in the nuclear periphery where hRIP is localized. Comparable ablation of hRIP activity did not affect the intracellular localization or trafficking of a variety of proteins or cellular poly (A+ mRNA, suggesting that the inhibition of Rev-directed RNA export is specific.
In conclusion, the results of this dissertation demonstrate that hRIP is involved in the movement of Rev-directed RNAs from the nuclear periphery to the cytoplasm. Therefore, hRIP is required for Rev function and HIV-1 replication. The hRIP protein is not essential for the maintenance of cell viability and thus might represent a novel target for the development of antiviral agents for HIV-1.
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Chitongo, Rumbidzai. "Investigating the structural effect of Raltegravir resistance associated mutations on the South African HIV-1 Integrase subtype C protein structure." University of the Western Cape, 2020. http://hdl.handle.net/11394/8075.
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>Magister Scientiae - MScBackground and Aims Human Immunodeficiency Virus (HIV) type 1 group M subtype C (HIV-1C) accounts for nearly half of global HIV-1 infections, with South Africa (SA) being one of the countries with the highest infection burden. In recent years, SA has made great strides in tackling its HIV epidemic, resulting in the country being recognized globally as the one sub-Saharan country with the largest combination antiretroviral therapy (cART) programme. Regardless of the potency of cART, the efficacy of the treatment is limited and hampered by the emergence of drug resistance. The majority of research on HIV-1 infections, effect of antiretroviral (ARV) drugs and understanding resistance to ARV drugs has been extensively conducted, but mainly on HIV-1 subtype B (HIV-1B), with less information known about HIV-1C. HIV-1’s viral Integrase (IN) enzyme has become a viable target for highly specific cART, due to its importance in the infection and replication cycle of the virus. The lack of a complete HIV-1C IN protein structure has negatively impacted the progress on structural studies of nucleoprotein reaction intermediates. The mechanism of HIV-1 viral DNA’s integration has been studied extensively at biochemical and cellular levels, but not at a molecular level. This study aims to use in silico methods that involve molecular modeling and molecular dynamic (MD) simulations to prioritize mutations that could affect HIV-1C IN binding to DNA and the IN strand-transfer inhibitor (INSTI) dolutegravir (DTG). The purpose is to help tailor more effective personalized treatment options for patients living with HIV in SA. This study will in part use patient derived sequence data to identify mutations and model them into the protein structure to understand their impact on the HIV-1C IN protein structure folding and dynamics. Methods Our sample cohort consisted of 11 sample sequences derived from SA HIV-1 treatmentexperienced patients who were being treated with the INSTI raltegravir (RAL). The sequences were submitted to the Stanford HIV resistance database (HIVdb) to screen for any new/novel variants resulting from possible RAL failure. Some of these new variants were analyzed to analyse their effect, if any, on the binding of DTG to the HIV-1C IN protein. Additionally, an HIV-1C IN consensus sequence constructed from SA’s HIV-1 infected population was used to model a complete three-dimensional wild type (WT) HIV-1C IN homology model. All samples were sequenced by our collaborators at the Division of Medical Virology, Stellenbosch University together with the National Health Laboratory Services (NHLS), SA. The HIV-1CZA WT-IN protein enzyme was predicted using SWISS-MODEL, and the quality of the resulting model validated. Various analyses were conducted in order to study and assess the effect of the selected new variants on the protein structure and binding of DTG to the IN protein. The mutation Cutoff Scanning Matrix (mCSM) program was used to predict protein stability after mutation, while PyMol helped to study any changes in polar contact activity before and after mutation. PyMol was also used to generate four mutant HIV-1C IN complex structures and these structures together with the WT IN were subjected to production MD simulations for 150 nanoseconds (ns). Trajectory analyses of the MD simulations were also conducted and reported. Results A total of 21 new variants were detected in our sample cohort, from which only six were chosen for further analyses within the study. A homology model of HIV-1C IN was successfully constructed and validated. The structural quality assessment indicated high reliability of the HIV-1C IN tetrameric structure, with more than 90.0% confidence in modelled regions. Of the six selected variants, only one (S119P) was calculated to be slightly stabilizing to the protein structure, with the other five found to be destabilizing to the IN protein structure. Variant S119P showed a loss in polar contacts that could destabilize the protein structure, while variant Y143R, resulted in the gain of polar contacts which could reduce flexibility of the 140’s region affecting drug binding. Similarly, mutant systems P3 (S119P, Y143R) and P4 (V150A, M154I) showed reduced hydrogen bond formation and the weakest non-bonded pairwise interaction energy. These two systems, P3 and P4, also showed significantly reduced to none polar contacts between DTG, magnesium (MG) ions and the IN protein, compared to the WT IN and P2 mutant IN systems. Interestingly, the WT structure and systems P1 (I113V) and P2 (L63I, V75M, Y143R) showed the highest non-bonded interaction energy, compared to systems P3 and P4. This was further supported by the polar interaction analyses of simulation clusters from the WT IN and mutant IN system P2 (L63I, V75M, Y143R), which were the only protein structures that formed polar contacts with DTG, MG ions and DDE motif residues, while P1 only made contacts with DNA and IN residues. Conclusion Findings from this study leads to a conclusion that double mutants (S119P, Y143R) and (V150A, M154I) may result in a reduction in the efficacy of DTG, especially when in combination. Furthermore, variants identified in systems P1 and P2 may still allow for effective DTG binding to IN and outcompete viral DNA for host DNA to prevent strand transfer. To the best of our knowledge, this is the first study that uses the consensus WT HIV1C IN sequence to build an accurate 3D homology model to understand the effect of less frequently detected/reported variants on DTG binding in a South African context. https://etd.
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Oyeyemi, Oyebode. "Modelling HIV-1 interaction with the host system." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/modelling-hiv1-interaction-with-the-host-system(41095e34-78dd-4b75-bd25-9695a4cc768f).html.
Full textAbstract:
Human immunodeficiency virus (HIV-1) is the pathogenic agent of HIV infection thatprecedes the total breakdown of cellular immunity, a condition known as acquiredimmunodeficiency syndrome (AIDS). The pandemic nature of the disease has promptedintense research into its biology. Already, much is known about HIV-1 infection, lifecycle,and progression to aids. Systems biology enables the combination of complex data fromthese studies into a framework where their effect on the various levels of cellularorganization (i.e. Pathways, cells, tissues, organs and the whole body) could be studied insilico. In this thesis, first, we reviewed our knowledge of the HIV-1 Human InteractionDatabase. We examined its contents and identified processes that HIV-1 was not previouslyknown to interact with. Then, we attempted an in silico dynamic model of HIV-1 interaction. We built a model of HIV-1 interaction with the CD4 T cell activation pathway comprised of137 nodes (16 HIV-1, 121 human) and 336 interactions. The model reproduced expectedpatterns of T cell activation. Using interaction graph properties, we identified 26 host cellfactors, including MAPK1&3, Ikkb-Ikky-Ikka and PKA, which contribute to the net activationor inhibition of viral proteins. By following a logical Boolean formalism, we identified 9 hostcell factors essential to the functions of viral proteins in the activation pathway. This wasthe first attempt to model dynamic viral-host interaction relationships. Then, we organize HIV-1 interacting host genes into modules to represent cellular processesneeded by the virus. We combined HIV-1 interactions with host gene GO annotations toclassify host genes according to these needed cellular processes. We obtained 201 modulesand found the same set of viral proteins do not interact with host genes having similarmodules suggesting intelligence in its co-ordination of host processes. This work is one of agrowing list that explores coordination of HIV-1 interactions. But more importantly, it would bebeneficial to functionally downsize the large dynamic HIV-1 interaction network. Finally, in our discussion, we discuss our results and suggest possible ways in which our workon dynamic models could be improved. This work is opening up a new field of systems virologythat studies the effect of viruses on the host in terms of its temporal and spatial aspects.
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Lê-Bury, Gabrielle. "Infection des macrophages par le VIH-1 : facteurs moléculaires impliqués dans la production virale et dans le développement de bactéries opportunistes The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking Pronounced stealth phenotype and differential pyroptosis induction by invasive Salmonella Typhimurium revealed by coinfection of human macrophages with HIV Role of Solute Carriers in efficient HIV-1 production by human macrophages." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCB094.
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Le Virus de l'Immunodéficience Humaine de type 1 (VIH-1) infecte les macrophages. Contrairement aux lymphocytes T CD4+, les macrophages résistent aux effets cytotoxiques du virus et représentent un réservoir pour ce pathogène. Dans ces cellules, le virus est produit et stocké dans un compartiment intracellulaire spécifique appelé VCC (Virus-Containing Compartment). Ce compartiment à pH neutre, transitoirement connecté à la membrane plasmique, reste cependant très peu caractérisé. Par ailleurs, le VIH-1 induit une perturbation des fonctions des macrophages, permettant ainsi le développement de bactéries opportunistes, telles que des souches particulières de Salmonella Typhimurium. Nous avons étudié en particulier des souches de Salmonella Typhimurium invasives non typhiques qui se sont développées, chez des patients séropositifs. Les objectifs de ma thèse ont donc été d'étudier, dans les macrophages primaires humains, les facteurs moléculaires impliqués dans la production du VIH-1 et le développement de souches invasives de Salmonella Typhimurium. Dans un premier temps, j'ai contribué à étudier les effets de l'infection par le VIH-1 sur les fonctions des macrophages. Leur fonction majeure est la phagocytose qui est un mécanisme de défense contre les pathogènes permettant leur internalisation et leur dégradation. Il avait déjà été montré au laboratoire que l'étape d'internalisation était en partie inhibée par le facteur de virulence Nef dans les macrophages infectés. Dans ce travail, nous avons montré que l'infection de ces cellules par le VIH-1 inhibe également la maturation des phagosomes, mais via la protéine virale Vpr. Nous avons aussi mis en évidence que le VIH-1 conduit le macrophage dans en état de pré-activation, mais empêche la cellule de répondre à un stimulus ultérieur comme une surinfection bactérienne. Dans un second temps, j'ai participé à l'étude des coinfections entre VIH-1 et les bactéries Salmonella Typhimurium invasives, qui ont émergé avec l'infection par le virus, en comparaison avec des souches de référence. Ce travail nous a permis de montrer que les bactéries, pour leur survie, n'exploitent pas le compartiment viral dans les macrophages co-infectés. J'ai ensuite observé que la souche invasive de Salmonella Typhimurium induit moins de mort cellulaire par pyroptose qu'une souche de référence. J'ai alors déterminé les voies de signalisation en amont de cette mort cellulaire qui est associée à un mécanisme inflammatoire. Ainsi, j'ai mis en évidence que la souche invasive de Salmonella détourne les mécanismes de pyroptose et survit mieux dans les macrophages, ce qui pourrait expliquer la dissémination observée chez les patients. Enfin, j'ai initié l'étude de nouveaux facteurs cellulaires impliqués dans la production virale par les macrophages. À la suite d'une analyse transcriptomique sur des macrophages primaires humains infectés ou non par le VIH-1, nous avons identifié un nombre important de transporteurs membranaires appelés SLC (Solute Carrier) dont l'expression est modulée par l'infection. Après la sélection de candidats, j'ai pu mettre en évidence que certains de ces SLC étaient importants pour la production virale par les macrophages. En conclusion, l'ensemble de ces travaux contribue à définir comment le VIH-1 infecte les macrophages et diminue leurs fonctions d'activation et de clairance, et comment se développent des bactéries opportunistes pathogènesHuman Immunodeficiency Virus type 1 (HIV-1) infects macrophages. In contrast to CD4+ T cells, macrophages are resistant to the cytotoxic effects of the virus and represent a reservoir for the pathogen. In these cells, the new virions are produced and stored in a specific intracellular compartment called Virus-Containing Compartment (VCC). This non-acidic compartment, transiently connected to the plasma membrane, remains poorly characterized. In addition, HIV-1 induces an alteration of macrophage function, allowing the development of opportunistic bacteria, such as specific strains of Salmonella Typhimurium. In particular, we studied invasive non-typhoidal Salmonella Typhimurium (iNTS) strains that developed in HIV-positive patients. The aims of my thesis have been to identify the molecular factors involved in the production of HIV-1 in primary human macrophages and to study the development of the invasive strains of Salmonella Typhimurium. First, I participed in studying the effects of HIV-1 infection on macrophage function. Their main role is phagocytosis, which is a defense mechanism enabling internalization and degradation of pathogens. It has previously been shown in the host laboratory that in HIV-1 infected macrophages, the internalization step is partially inhibited by the virulence factor Nef. In this work, we have shown that the infection of these cells by HIV-1 also inhibits the maturation of phagosomes, in this case, via the viral protein Vpr. Further, we have demonstrated that HIV-1 leads to a pre-activation state of the macrophage, while preventing the cell from responding to subsequent stimuli, such as bacterial superinfection. Secondly, I studied the coinfections between HIV-1 and an invasive strain of Salmonella Typhimurium that was compared to reference strains. This work demonstrated that bacteria do not hijack the viral compartment for their survival in co-infected macrophages. Additionally, the invasive strain of Salmonella Typhimurium was observed to induce less cell death by pyroptosis than a reference strain. The signaling pathways upstream of this cell death were determined to be associated with an inflammatory mechanism. Hence, it was demonstrated that the invasive strain of Salmonella hijacks the mechanism of pyroptosis to survive in macrophages. This may explain the dissemination observed in patients. Finally, a study of new cellular factors involved in viral production in macrophages was conducted. Following a transcriptomic analysis of human primary macrophages infected, or not, with HIV-1, we identified a large number of membrane transporters called SLC (Solute Carrier) whose expression was modulated by the infection. After selecting some of the candidates for further study, I have demonstrated that some of these SLCs are important for viral production in macrophages. In conclusion, this work contributes to defining how HIV-1 infects macrophages and disturbs their activation and clearance functions, and how opportunistic pathogenic bacteria develop
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Roes, Claas Verfasser], Dieter [Akademischer Betreuer] [Willbold, and Bernd [Akademischer Betreuer] König. "Structural investigation of the interaction of the human BST2 with the HIV-1 Virus protein U / Claas Roes. Gutachter: Dieter Willbold ; Bernd König." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2015. http://d-nb.info/1069985880/34.
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Lee, Se Il. "Statistical thermodynamics of virus assembly." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/33900.
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Experiments show that MgSO4 salt has a non-monotonic effect as a function of MgSO4 concentration
on the ejection of DNA from bacteriophage lambda.
There is a concentration, N0, at which the minimum amount of DNA is ejected.
At lower or higher concentrations, more DNA is ejected. We propose that this non-monotonic behavior
is due to the overcharging of DNA at high concentration of Mg⁺² counterions.
As the Mg⁺² concentration increases from zero, the net charge of ejected DNA changes its sign from negative to positive.
N0 corresponds to the concentration at which DNA is neutral.
Our theory fits experimental data well.
The DNA-DNA electrostatic attraction is found to be -0.004 kBT/nucleotide.
Simulations of DNA-DNA interaction of a hexagonal DNA bundle support our theory.
They also show the non-monotonic DNA-DNA interaction and reentrant behavior of DNA condensation by divalent counterions.
Three problems in understanding the capsid assembly for a retrovirus are studied:
First, the way in which the viral membrane affects the structure of in vivo assembled HIV-1 capsid is studied.
We show that conical and cylindrical capsids have similar energy at high surface tension of the viral membrane,
which leads to the various shapes of HIV-1 capsids. Secondly, the problem of RNA genome packaging inside spherical viruses
is studied using RNA condensation theory. For weak adsorption strength of capsid protein, most RNA genomes are located at the center
of the capsid. For strong adsorption strength, RNA genomes peak near the capsid surface and the amount of RNA packaged is proportional to the capsid area instead its volume. Theory fits experimental data reasonably well.
Thirdly, the condensation of RNA molecules by nucleocapsid (NC) protein is studied.
The interaction between RNA molecules and NC proteins is important for the reverse transcription of viral RNA which relates to the viral infectivity.
For strong adsorption strength of the NC protein, there is a screening effect by RNA molecules around a single NC protein.
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Yandrapalli, Naresh. "Role of HIV-1 Gag protein multimerization in the generation of nanodomains in lipid membranes." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT097/document.
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La polyprotéine Gag du VIH-1 qui contient quatre principaux domaines (Matrix (MA), capside (CA), nucléocapside (NC), et P6) est l’orchestrateur privilégié de l'assemblage du virus HIV-1, assemblage qui a lieu pendant la phase tardive de la réplication. Il est bien connu que Gag interagit avec les lipides de la membrane plasmique de la cellule hôte et s’auto-assemble sur le feuillet interne de cette dernière afin de générer de nouvelles particules virales. Le bourgeonnement de ces particules virales hors de la cellule hôte est décrit comme étant dépendant de la machinerie cellulaire ESCRT. Différentes études structurales, fonctionnelles ainsi que des simulations de dynamique gros grain ont montré que la liaison de Gag à la membrane est médiée par une interaction duale. Une spécifique de nature éléctrostatique, qui associe une région hautement basique (HBR) du domaine MA de Gag au lipide acide,phosphatidyl inositol biphosphate (PI(4,5)P2) du feuillet interne de la membrane plasmique. Une de type hydrophobe, qui consiste en l’insertion du myristate de Gag dans la membrane plasmique. Savoir si Gag reconnait spécifiquement des domaines lipidiques pré-existants de type « rafts » ou si, au contraire, Gag tri ses lipides et les réorganise latéralement afin d’optimiser sa multimérisation et son bourgeonnement est une question à la fois fondamentale et d’actualité en virologie.Durant ma thèse, j’ai vérifié l’existence de la seconde hypothèse en utilisant des membranes modèles contenant du PI (4,5) P2 marqué de façon fluorescente et différent mutants et produits de la protéine Gag non-myristoylée. Ces expériences ont montré de fortes affinités de ces protéines pour les membranes contenant du PI (4,5) P2. S’appuyant sur les propriétés d’auto-extinction de fluorescence du marqueur choisit et à l’aide des différents variants de la protéine Gag, j'ai pu montré que la multimérisation de Gag génère l’existence de nanodomaines contenant du PI (4, 5) P2 et du cholestérol, la sphingomyéline étant au contraire exclue de ces domaines. En marquant la protéine Gag par un autre fluorophore, j’ai pu montrer par microscopie optique sur des vésicules lipidiques géantes (GUVs) que la protéine Gag partitionnait préférablement dans des microdomaines lipidiques de type liquide désordonnés (Ld). Par la suite, j’ai testé la capacité de la protéine Gag d’induire la formation de vésicules sur des membranes modèles (Bicouches supportés et GUVs) contenant du PI(4,5) P2 et de la phosphatidyl sérine (PS). En utilisant une microbalance à cristal de quartz (QCM-D) et des techniques de microscopie de fluorescence, j’ai suivi l'auto-assemblage de Gag dans le temps et ai montré que la protéine Gag était suffisante pour générer une courbure de la membrane et libérer des vésicules lipidiques. Grâce à différents produits de maturation de cette protéine, j’ai montré que la présence des domaines MA et CA est suffisante pour produire ces vésicules.L’ensemble de ces résultats suggèrent que la liaison et la multimérisation de la protéine Gag ne se produit pas dans des domaines lipidiques préexistants de type « raft », mais, au contraire, que la liaison et multimérisation de la protéine Gag génère l’existence de domaines lipidiques enrichis en PI (4,5) P2 et en cholestérol. La générescence de ces domaines lipidiques pourrait participer à la courbure de la membrane plasmique nécessaire au bourgeonnement du virusGag polyprotein of HIV-1 is made of four main domains Matrix (MA), Capsid (CA), Nucleocapsid (NC), and P6 and is the prime orchestrator of virus assembly that occurs during the late phase of replication. It is well known that Gag interacts with host cell lipids and self-assemble along the inner-leaflet of the plasma membrane in order to generate virus like particles (VLPs). Budding of these VLPs out of the living cell is described to be ESCRT dependent. Structural, functional and simulation based studies has shown that Gag membrane binding is mediated by a bipartite interaction. One specific electrostatic interaction, between the highly basic region (HBR) of its MA domain and the host cell acidic lipid phosphatidyl inositol bisphophate (PI(4,5)P2), plus a hydrophobic interaction through Gag’s myristate insertion in the plasma membrane. It is still an opened question whether Gag would specifically recognize pre-existing lipid domains such as rafts to optimize its multimerization or, on the contrary, would reorganize lipids during its multimerization. During my Ph.D. I explored the second hypothesis using purified myr(-) Gag protein and model membranes containing fluorescently labelled PI(4,5)P2.Bonding experiments have shown strong affinities of these purified proteins towards PI(4,5)P2 containing lipid bilayers. Using PI(4,5)P2 fluorescence self-quenching properties, I found that multimerization Gag generates PI(4,5)P2/Cholesterol enriched nanoclusters. On the opposite, sphingomyelin was excluded from these nanoclusters. In addition to this, using a fluorescently labelled myr(-) Gag, I have observed its preferable partitioning into lipid disordered (Ld) phases of giant unilamellar vesicles (GUVs). Further, possibility of whether HIV-1 Gag alone, as a minimal system, can induce the formation of vesicles on PI(4,5)P2/PS containing supported lipid bilayers (SLBs) & GUVs was tested. Using quartz crystal microbalance (QCM-D) and fluorescence microscopy techniques, I monitored the self-assembly of HIV-1 Gag with time and found that Gag was sufficient to generate membrane curvature and vesicle release. Moreover, using mutants of this protein, I found that having MA and CA domain is enough for Gag to produce vesicle like structures. Taken together, these results suggest that binding and multimerization of Gag protein does not occur in pre-existing lipid domains (such as “rafts”) but this multimerization is more likely to induce PI(4,5)P2/Cholesterol nanoclusters. This nanophase separation could locally play a role in the membrane curvature needed for the budding of the virus
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Hinton, Michael. "Cellular Mechanisms by which Alcohol Promotes HIV Protease Inhibitor-induced Hepatotoxicity." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/6089.
Full textAbstract:
CELLULAR MECHANISMS BY WHICH ALCOHOL PROMOTES HIV PROTEASE INHIBITOR-INDUCED HEPATOTOXICITY
Michael Hinton, B.S.
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2019
Major Director: Huiping Zhou
Professor, Department of Microbiology and Immunology
The development of highly-active-antiretroviral therapy(HAART) has allowed management of HIV and extended the lives of those infected. Alcohol abuse, which is very common in HIV-1 infected patients, is one of the most important co-morbid risk factors for liver injury and has been associated with the occurrence of serious metabolic syndrome and subsequent discontinuation of HAART in HIV patients. We have identified endoplasmic reticulum (ER) stress-induced proapoptotic factor CCAAT-element-binding protein homologous protein (CHOP) as an important mechanism underlying HIV PI-induced inflammation and hepatic lipotoxicity. However, little is known about the mechanistic pathways by which alcohol promotes HIV PI-induced hepatic lipotoxicity. The aim of this study was to determine if inhibition of CHOP expression prevents alcohol- and HIV PI-induced apoptosis and dysregulation of lipid metabolism. We demonstrated that co-administration of alcohol and HIV PIs induced unfolded protein response (UPR) activation, ER stress, and CHOP upregulation in rodent hepatocytes. Both alcohol and HIV PI-induced lipid accumulation and apoptosis were significantly reduced in CHOP-/- hepatocytes. Also, CHOP-/- hepatocytes treated with alcohol and HIV PIs showed inflammation.. Activation of the ER stress-induced proapoptotic factor CHOP is a key cellular mechanism underlying alcohol and HIV PI-induced hepatotoxicity. CHOP expression is key for alcohol and HIV PI-induced dysregulation of key genes involved in lipid metabolism in hepatocytes. Limitations of the study include the usage of global CHOP-/- in lieu of tissue-specific conditional knockout mouse models, nonobservance of the effects of alcohol and HIV PIs on extra-hepatic tissues, and incomplete investigation of the interplay of hepatocytes and resident macrophages.
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Chen, Yuxin. "Characterization of Envelope-Specific Antibody Response Elicited by HIV-1 Vaccines: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/760.
Full textAbstract:
Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function.
First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens.
Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells.
Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies.
Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.
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Chen, Yuxin. "Characterization of Envelope-Specific Antibody Response Elicited by HIV-1 Vaccines: A Dissertation." eScholarship@UMMS, 2001. http://escholarship.umassmed.edu/gsbs_diss/760.
Full textAbstract:
Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function.
First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens.
Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells.
Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies.
Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.
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Chougui, Ghina. "Antagonism of HUSH restriction by lentiviral Vpx and Vpr proteins HIV-2/SIV viral protein X counteracts HUSH repressor complex." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCB080.
Full textAbstract:
Les rétrovirus VIH-1 et 2 responsables du SIDA, bien que très similaires sur le plan de leur organisation génomique, diffèrent par leurs protéines auxiliaires. Ces dernières inactivent des facteurs cellulaires antiviraux et permettent ainsi l'établissement d'un environnement cellulaire favorable à la réplication virale. Vpx, une protéine auxiliaire spécifique du VIH-2, est connue pour sa capacité à augmenter l'infection virale, une activité longtemps reliée à son unique faculté à contrecarrer SAMHD1, un facteur de restriction actif à l'étape de transcription inverse. Cependant, plusieurs éléments de la littérature suggèrent que Vpx confère un avantage au virus indépendamment de SAMHD1. Nous avons donc étudié la possibilité d'une cible supplémentaire inactivée par Vpx et, à partir d'un crible protéomique, nous avons identifié le complexe HUSH (HUman Silencing Hub). Composé de TASOR, MPP8 et Periphilin, le complexe HUSH est impliqué dans le control épigénétique de transgènes intégrés ainsi que des éléments transposables Line-1. Nous avons montré la capacité de Vpx à lier le complexe HUSH et à induire sa dégradation par le protéasome grâce au détournement de l'adaptateur d'ubiquitine ligase DCAF-1 et ce, de façon indépendante de SAMHD1. De ce fait, Vpx est capable de réactiver des provirus latents du VIH, contrairement à des mutants de Vpx incapables d'induire une dégradation de HUSH. Bien que l'antagonisme du complexe HUSH humain ne soit pas conservé au sein de toutes les lignées lentivirales, y compris le VIH-1, il est une caractéristique de certains Vpr des VIS des singes verts africains ainsi que du VIS divergent du singe de l'Hoest. Le caractère ancien de cette fonction post-intégrative insoupçonnée des gènes vpx/vpr, ainsi que la spécificité d'espèces observée, sont deux critères favorables au statut de facteur de restriction pour le complexe HUSH. Dressant ainsi le contrôle épigénétique comme barrière de l'immunité intrinsèque, nécessaire au maintien de l'intégrité du génome cellulaireHIV-1 and 2 are both responsible for AIDS, though similar, these two retroviruses harbour different sets of auxiliary proteins. Through the inactivation of cellular antiviral factors, these auxiliary proteins allow the establishment of a favourable environment for viral replication. Vpx, an HIV-2 only auxiliary protein, is known for its ability to increase viral infection, which was long linked to its sole capacity to counteract SAMHD1, a restriction factor active at the reverse transcription step. However, several lines of evidence suggested a SAMHD1-independent advantage of Vpx. We therefore investigated the possibility of an additional Vpx target and through a proteomic screen, we identified the HUman Silencing Hub (HUSH) complex. HUSH complex, including: TASOR, MPP8 and Periphelin, was reported to epigenetically silence integrated transgenes and recently Line-1 transposable elements. Here, we show that Vpx binds the HUSH complex and induces its proteasomal degradation through the hijacking of the DCAF-1 ubiquitin ligase adaptor, independently from SAMHD1-antagonism. As a consequence, Vpx is able to reactivate HIV latent proviruses, unlike Vpx mutants unable to induce HUSH degradation. Although antagonism of human HUSH complex is not conserved among all lentiviral lineages including HIV-1, it is a feature of Vpr from SIVs of African green monkeys and from the divergent SIV of l'Hoest's monkey, arguing in favor of an ancient lentiviral species-specific vpx/vpr gene function. Altogether, our results highlight an unexpected post-integration activity of Vpx and suggest HUSH complex as a restriction factor. They also support the idea of epigenetic control as an intrinsic immunity barrier maintaining the integrity of the cellular genome
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Hérate, Cécile. "Contribution de la forme nucléaire de l'uracile DNA glycosylase aux étapes précoces du cycle de réplication du virus de l'immunodéficience humaine de type 1." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05T017/document.
Full textAbstract:
La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primairesThe HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells
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Nadeem, Muhammad Faisal. "Chaperone mechanism of the HIV-1 Gag and its promotion by the RPL7 host protein." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ025.
Full textAbstract:
La protéine multidomaine Pr55Gag de VIH-1 joue un rôle crucial dans les étapes finales de la réplication virale, notamment lors de la reconnaissance et la sélection de l’ARN génomique ainsi que lors de la production de nouvelles particules virales. Outre son rôle structural, Pr55Gag chaperonne aussi les séquences d’acides nucléiques, une propriété cruciale pour la dimérisation de l’ARN génomique et l’hybridation de l’amorce ARNt à l’ARN génomique. Des partenaires cellulaires comme la protéine ribosomale RPL7 sont supposées être recrutées par Pr55Gag afin d’augmenter son potentiel chaperon. Afin d’étudier le mécanisme d’hybridation des acides nucléiques par Gag et RPL7, nous avons examiné leur effet sur la réaction d’hybridation entre dTAR, la version ADN de l’élément de transactivation virale et sa séquence complémentaire cTAR. Nos résultats révèlent que Gag et RPL7 présentent des mécanismes différents pour promouvoir l’hybridation cTAR/dTAR. Utilisés de concert, RPL7 peut aider Gag à chaperonner des séquences stables de l’ARN génomique que Gag seule pourrait difficilement chaperonner. Ce renforcement par RPL7 de l’activité chaperonne de Gag jouerait un rôle critique dans l’assemblage du virusThe multidomain Pr55 Gag protein of HIV-1 plays a crucial role during late stages of viral replication, notably for the recognition and selection of genomic RNA as well as for the production of new viral particles. In addition to its structural role, Pr55 Gag also chaperones nucleic acid sequences, a property which is crucial for genomic RNA dimerization and annealing of the primer tRNA to the genomic RNA. Cellular partners like ribosomal protein RPL7 are thought to be recruited by Pr55 Gag to enhance its chaperoning potential. To investigate the nucleic acid annealing mechanism of Gag and RPL7, we examined their effect on the annealing reaction between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence taken as relevant model HIV-1 sequences. Our data show that Gag and RPL7 exhibit different mechanisms for promoting the cTAR/dTAR annealing. When used together, RPL7 can help Gag to chaperone stable sequences of the genomic RNA that Gag would hardly be able to chaperone alone. This RPL7-driven boost in Gag chaperone activity is thought to be critical in the viral assembly process
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Mittal, Seema. "Role of Protein Flexibility in Function, Resistance Pathways and Substrate Recognition Specificity in HIV-1 Protease: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/573.
Full textAbstract:
In the 30 years since the Center for Disease Control's Morbidity and Mortality Weekly Report published the first mention of what later was determined to be AIDS (Acquired immunodeficiency syndrome) and HIV (Human immunodeficiency virus) recognized as the causative pathogen, much has been done to understand this disease’s pathogenesis, development of drugs and emergence of drug resistance under selective drug therapy. Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, have helped stabilize the HIV prevalence at extraordinarily high levels. Despite the recent stabilization of this global epidemic, its dimensions remain staggering with estimated (33-36 million) people living with HIV-AIDS in 2007 alone. This is because the available drugs against AIDS provide treatment for infected individuals, but HIV evolves rapidly under drug pressure and develops resistant strains, rendering the therapy ineffective. Therefore, a better understanding underlying the molecular mechanisms of viral infection and evolution is required to tackle drug resistance and develop improved drugs and treatment regimens.
HIV-1 protease is an important target for developing anti-HIV drugs. However, resistant mutations rapidly emerge within the active site of the protease and greatly reduce its affinity for the protease inhibitors. Frequently, these active site drug resistant mutations co-occur with secondary/ non-active site/ associated or compensatory mutations distal to the active site. The role of these accessory mutations is often suggested to be in maintaining viral fitness and stability of protease. Many of the non-active site drug resistant mutations are clustered in the hydrophobic core in each monomer of the protease. Molecular dynamic simulation studies suggest that the hydrophobic core residues facilitate the conformational changes that occur in protease upon ligand binding. There is a complex interdependence and interplay between the inherent adaptability, drug resistant mutations and substrate recognition by the protease. Protease is inherently dynamic and has wide substrate specificity. The PI (protease inhibitor) resistant mutations, perhaps, modulate this dynamics and bring about changes in molecular recognition, such that, in resistant proteases, the substrates are recognized specifically over the PIs for the same binding site. In this thesis research, I have investigated these three complementary phenomena in concert.
Chapter II examines the importance of hydrophobic core dynamics in modulating protease function. The hydrophobic core in the WT protease is intrinsically flexible and undergoes conformational changes required for protease to bind its substrates. This study investigated if dynamics is important for protease function by engineering restricted vs. flexible hydrophobic core region in each monomer of the protease, using disulfide chemistry. Under oxidizing conditions, disulfide bond established cross-link at the interface of putative moving domains in each monomer, thereby, restricting motion in this region. Upon reduction of the disulfide bond, the constraining influence was reversed and flexibility returned to near WT. The disulfide cross-linked protease showed significant loss of function when tested in functional cleavage assay. Two protease variants (G16C/L38C) and (R14C/E65C) were engineered and examined for changes in structure and enzymatic activity under oxidizing and reducing conditions. (R14C/E65C) was engineered as an internal control variant, such that cysteines were engineered between putative non-moving domains. Structurally, both the variants were very similar with no structural perturbations under oxidizing or reducing conditions. While significant loss in function was observed for (G16C/L38C) only under oxidizing conditions, (R14C/E65C) did not show any loss of function under oxidizing or reduced conditions, as expected. Successful regain of function for cross-linked (G16C/L38C) was obtained upon reversible reduction of the disulfide bond. Taken together, these data demonstrate that the hydrophobic core dynamics modulates protease function and support the hypothesis that the distal drug resistant mutations, possibly causing drug resistance by modulating hydrophobic core dynamics via long range structural perturbations. Since protease recognizes and cleaves more than 10 substrates at different rates, our further interest is to investigate if there is a differential loss of activity for some specific substrates over the others, and whether the order of polypeptide cleavage is somehow affected by restricted core mobility. In order to better answer these questions it is essential to understand: what determines the substrate binding specificity in protease? A two-pronged approach was applied to address this question as described in chapter III and IV respectively.
In chapter III, I investigated the determinants of substrate specificity in HIV-1 protease by using computational positive design and engineered specificity-designed asymmetric protease (Pr3, A28S/D30F/G48R) that would preferentially bind to one of its natural substrates, RT-RH over two other substrates, p2-NC and CA-p2, respectively. The designed protease was expressed, purified and analyzed for changes in structure and function relative to WT. Kinetic studies on Pr3 showed that the specificity of Pr3 for RT-RH was increased significantly compared to the wild-type (WT), as predicted by the positive design. ITC (Isothermal Titration Calorimetry) studies confirmed the kinetic data on RT-RH. Crystal structural of substrate complexes of WT protease and Pr3 variant with RT-RH, CA-p2 and p2-NC were further obtained and analyzed. The structural analysis, however, only partially confirmed to the positive design due to the inherent structural pliability of the protease. Overall, this study supports the positive computational design approach as an invaluable tool in facilitating our understanding of complex proteins such as HIV 1 protease and also proposes the integration of internal protein flexibility in the design algorithms to make the in-silico designs more robust and dependable.
Chapter IV probed the substrate specificity determining factors in HIV-1protease system by focusing on the substrate sequences. Previous studies have demonstrated that three N-terminal residues immediate to the scissile bond (P1-P3) are important in determining recognition specificity. This work investigated the structural basis of substrate binding to the protease. Catalytically active WT protease was crystallized with decameric polypeptides corresponding to five of the natural cleavage sites of protease. The structural analyses of these complexes revealed distinct P side product bound in all the structures, demonstrating the higher binding affinity of N terminal substrate for protease.
This thesis research successfully establishes that intrinsic hydrophobic core flexibility modulates function in HIV-1 protease and proposes a potential mechanism to explain the role of non-active site mutations in conferring drug resistance in protease. Additionally, the work on specificity designed and N terminal product bound protease complexes advances our understanding of substrate recognition in HIV protease.
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Dai, Weiwei. "SERINC5: Its Sensitivity to Nef and Restriction of HIV-1." eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/984.
Full textAbstract:
The accessory protein Nef of human immunodeficiency virus type 1 (HIV-1) has long been known to enhance the infectivity of HIV-1 progeny virions. The multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 were recently identified as novel antiviral proteins that restrict HIV-1 infectivity. Nef enhances HIV-1 infectivity by removing SERINCs from the plasma membrane, which prevents their incorporation into progeny HIV-1 virions. To exploit this potent intrinsic antiretroviral factor for potential therapy development, it is critical to explore the determinants in SERINC5 that govern its downregulation by Nef and its restriction on HIV-1 infectivity. Here I report that the ability to inhibit HIV-1 infectivity is conserved among vertebrate SERINC5 proteins, whereas the sensitivity to downregulation by Nef is not. However, a Nef-resistant SERINC5 became Nef-sensitive when its intracellular loop 4 (ICL4) was replaced by that of Nef-sensitive human SERINC5. Conversely, human SERINC5 became resistant to Nef when its ICL4 was replaced by that of a Nef-resistant SERINC5. In general, ICL4 regions from SERINCs that exhibited resistance to a given Nef conferred resistance to the same Nef when transferred to a sensitive SERINC, and vice versa. I demonstrate that human SERINC5 can be modified to restrict HIV-1 infectivity even in the presence of Nef. Moreover, by generating chimeras between SERINC5 and SERINC2, which does not exhibit antiretroviral activity, I demonstrate that SERINC5’s inhibitory function, unlike the sensitivity to Nef, requires the participation of more than one region. Helix 4 and extracellular loop 5 (ECL5) of SERINC5 are both required for the potent restriction of HIV-1 infectivity. In contrast, a large amino-terminal portion of SERINC5 is not required for its antiretroviral activity of SERINC5. The determinants in ECL5 disperse throughout the loop. Furthermore, the ECL5 of SERINC5 is a hotspot region that determines the Env-dependent antiretroviral activity of SERINC5.
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Yakoob, Hena. "Influence of the Anti-HIV drug Elvitegravir on Chlamydial Development and the Characterization of Chlamydial Polymorphic Membrane Protein Expression in Herpes Simplex Virus (HSV)/C. trachomatis Co-infected Cells." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/honors/259.
Full textAbstract:
Chlamydia trachomatis is the most common bacterial agent of sexually transmitted infections worldwide and a common co-infection in AIDS patients. Chlamydial genital tract infections are often asymptomatic; therefore many infections go untreated and result in complications like chronic inflammation, ectopic pregnancy, and pelvic inflammatory disease. Chlamydia share a unique developmental cycle and under stress, can enter a state known as persistence, in which the bacteria are noninfectious but still viable. Removal of the stressor allows the chlamydiae to re-enter and complete the developmental cycle. Exposure to low-dose quinolones can cause the chlamydiae to enter persistence and halt the developmental cycle. Notably, 1 in 20 people living with HIV/AIDS also suffers from chlamydial infections. Since the anti-HIV drug Elvitegravir (EVG) is a quinolone derivative, we hypothesized that EVG exposure would inhibit chlamydial development. To ascertain whether EVG affects chlamydial development, HeLa cells were infected with C. trachomatis or C. muridarum and then either mock treated or treated with EVG. The percent infectivity and production of infectious progeny were determined by immunofluorescence assay and chlamydial titer assay, respectively. Transmission electron microscopy (TEM) was used to examine chlamydial morphology and determine whether EVG caused Chlamydia to become persistent. Though percent infectivity and chlamydial morphology were similar between treated and untreated Chlamydia-infected cells, the production of infectious progeny was significantly decreased in EVG-exposed Chlamydia-infected cells. These data indicate that EVG is not a persistence-inducer, but does inhibit chlamydial development in vitro. In other studies, we tested chlamydial polymorphic membrane protein (PMP) expression in chlamydia/HSV co-infected cells by immunofluorescence staining. Since penicillin-induced persistence decreases the expression of some chlamydial PMPs, we hypothesized that expression of PMP-A and PMP-B would be decreased by HSV-induced persistence. The results indicated that there was no significant difference in expression of PMP-A or PMP-B in co-infected versus C. trachomatis singly-infected cells. These data suggest that PMP expression is not a good indicator of chlamydial persistence when induced by HSV.
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Buglione-Corbett, Rachel. "Adjuvant-Specific Serum Cytokine Profiles in the Context of a DNA Prime-Protein Boost HIV-1 Vaccine: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/666.
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In recent years, heterologous prime-boost vaccination constructs have emerged as a promising strategy to generate broad and protective immunity against a variety of pathogens. The utility of DNA vaccination in priming the immune system, in particular, has improved the immunogenicity of vaccines against difficult pathogens such as HIV-1. In addition, many vaccine formulations include an adjuvant to augment immune responses. However, the mechanisms and profiles of many adjuvants remain largely unknown, particularly in the context of such combination immunization approaches.
My thesis research studied the effects of several adjuvants, QS-21, aluminum hydroxide, MPL, and ISCOMATRIX™ adjuvant in the context of a previously described pentavalent HIV-1 Env DNA prime-protein boost vaccine, DP6-001. In a murine model, we quantified HIV antigen-specific humoral and T cell responses, as well as pro-inflammatory serum cytokine and chemokines, both shortly after immunization and at the termination of studies. Our data indicates that each candidate adjuvant generates a unique pattern of biomarkers as well as improved immunogenicity in the context of the DP6-001 DNA prime-protein boost vaccine.
Additionally, we examined the impact of several innate signaling pathways on the adaptive immunity raised by DP6-001 and adjuvants, as well as on the unique serum cytokine profiles. These studies provide valuable information in selection of an adjuvant for inclusion in future prime-boost strategies, with the goal of enhancing immunogenicity while minimizing reactogenicity. Furthermore, these studies provided insight about the utility of different current adjuvants in a prime-boost formulation, and the unique immune environment induced by DNA priming.
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Buglione-Corbett, Rachel. "Adjuvant-Specific Serum Cytokine Profiles in the Context of a DNA Prime-Protein Boost HIV-1 Vaccine: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/666.
Full textAbstract:
In recent years, heterologous prime-boost vaccination constructs have emerged as a promising strategy to generate broad and protective immunity against a variety of pathogens. The utility of DNA vaccination in priming the immune system, in particular, has improved the immunogenicity of vaccines against difficult pathogens such as HIV-1. In addition, many vaccine formulations include an adjuvant to augment immune responses. However, the mechanisms and profiles of many adjuvants remain largely unknown, particularly in the context of such combination immunization approaches.
My thesis research studied the effects of several adjuvants, QS-21, aluminum hydroxide, MPL, and ISCOMATRIX™ adjuvant in the context of a previously described pentavalent HIV-1 Env DNA prime-protein boost vaccine, DP6-001. In a murine model, we quantified HIV antigen-specific humoral and T cell responses, as well as pro-inflammatory serum cytokine and chemokines, both shortly after immunization and at the termination of studies. Our data indicates that each candidate adjuvant generates a unique pattern of biomarkers as well as improved immunogenicity in the context of the DP6-001 DNA prime-protein boost vaccine.
Additionally, we examined the impact of several innate signaling pathways on the adaptive immunity raised by DP6-001 and adjuvants, as well as on the unique serum cytokine profiles. These studies provide valuable information in selection of an adjuvant for inclusion in future prime-boost strategies, with the goal of enhancing immunogenicity while minimizing reactogenicity. Furthermore, these studies provided insight about the utility of different current adjuvants in a prime-boost formulation, and the unique immune environment induced by DNA priming.
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Santerre, Maryline. "Étude de l'action sur l'épissage de protéines nucléaires se liant à la région de l'ARN du virus VIH-1 contenant le site d'épissage A7 et role de ces protéines sur d'autres sites accepteurs d'épissage de VIH-1." Thesis, Nancy 1, 2010. http://www.theses.fr/2010NAN10115/document.
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L'épissage est une étape clef de la multiplication du VIH-1. Par utilisation de 4 sites donneurs et 8 sites accepteurs d'épissage, plus de 40 ARNm différents sont produits. Une approche protéomique nous a permis d'identifier de nouvelles protéines interagissant avec la région de l'ARN viral contenant le site A7. Nous avons démontré l'interaction directe avec l'ARN viral de 5 des protéines identifiées (nucléoline, hnRNP A1/B, hnRNP H et hnRNP K). Nous avons montré que hnRNP K a plusieurs sites de fixation dans la région du site A7 et que hnRNP A1et hnRNP K se lient de façon coopérative. Nous avons montré un effet inhibiteur de hnRNP K sur l'épissage au site A7. Comme la protéine hnRNP A1 est un régulateur négatif de plusieurs sites accepteurs d'épissage (A1, A2, A3, A7), nous avons testé si la protéine hnRNP K pouvait renforcer l'inhibition à ces sites. En fait, hnRNP K active l'épissage in vitro des introns entre le site donneur D1 et les sites accepteurs A1, A2 et A3. Nous avons montré que la protéine hnRNP K renforce fortement l'activité de ASF/SF2 au site A2, ce qui indique que selon le contexte, la protéine hnRNP K peut être activatrice ou inhibitrice de l'épissage du VIH-1. J'ai observé de plus que la surexpression de la protéine hnRNP K dans des cellules HeLa, transfectées avec le plasmide p PSP contenant le virus VIH-1 dépourvu de ses capacités d'encapsidation, produit un changement très marqué de l'épissage alternatif de l'ARN PSP, ce qui confirme la forte influence de hnRNP K sur l'épissage alternatif du VIH-1. L'augmentation de la concentration cellulaire de hnRNP K dans les cellules HeLa conduit aussi à une diminution de la protéine virale Nef. La protéine hnRNP K intervient donc non seulement dans la régulation du site A7, mais aussi dans celle de la majorité des sites d'épissage régulés de l'ARN du VIH. L'action de cette protéine sur plusieurs des sites d'épissage montre que la protéine hnRNP K est probablement un régulateur général de l'épissage de VIH-1HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 transcripts in HeLa cell nuclear extracts by affinity chromatography to identify new associated proteins. We showed that, in addition to the well known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. We demonstrated that hnRNP K has multiple binding sites in the vicinity of site A7 and that binds cooperatively to hnRNP A1 to the A7 RNA region and limits the A7 utilization in vitro. As hnRNP A1 is a negative regulator of several HIV-1 splicing sites (A1, A2, A3), we tested whether hnRNP K may also reinforce hnRNP A1 inhibition at these sites. Surprisingly, hnRNP K activated in vitro splicing of the D1-A1, D1-A2 and D1-A3 introns. Interestingly, hnRNP K was found to reinforce strongly the ASF/SF2 activity at site A2, which indicates that depending on the splicing site hnRNP K can be a splicing activator or inhibitor. To test how hnRNP K influences the relative utilization of HIV-1 splicing sites in cellulo, we used plasmid p PSP containing all the HIV-1 splicing sites and tested the effect of over-expression in HeLa cells on alternative splicing of the PSP RNA. Doubling the amount of hnRNP K in HeLa cells led to a drastic change of the PSP RNA alternative splicing, which confirms the strong influence of hnRNP K on alternative splicing. Moreover, increase of cellular concentration of hnRNP K strongly decrease the viral Nef protein production. hnRNP K protein affects A7 splicing regulation but also regulates the majority of regulated splicing sites of HIV. By extension of the study of hnRNP K effect to other HIV-1 splicing sites, we discovered that hnRNP K is a general regulator of HIV-1 splicing
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Basta, Beata. "Nouvelles molécules antivirales ciblant la protéine de la nucléocapside du virus VIH-1." Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00868465.
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Étant donnée la séquence hautement conservée de la NC et son rôle crucial dans le cycle viral de VIH-1, les molécules inhibant la NC sont susceptibles d'agir comme complément aux thérapies anti-rétrovirales à haute activité (HAART) basées sur des médicaments ciblant les enzymes virales, Des médicaments anti-NC sont ainsi susceptibles d'entraîner un maintien de l'inhibition de la réplication d'un large panel d'isolats VIH-1 incluant des lignées virales résistantes aux médicaments ciblant les enzymes virales. Récemment, dans le cadre du consortium Européen TRIoH, de nouvelles stratégies visant à cibler spécifiquement les propriétés chaperonnes de la NC sur les acides nucléiques ont été développées. Selon une stratégie protégée par un brevet soumis, une série de peptides a été conçue afin d'agir comme compétiteurs de la NC et pouyant ainsi inhiber la réplication du virus. Au sein de cette série, plusieurs peptides ont montré une inhibition efficace des propriétés de déstabilisation des acides nucléiques par la NC. Quatre de ces peptides ont été testés en milieu cellulaire et trois d'entre eux ont montré qu'ils pouvaient inhiber efficacement la réplication du HIV-1 dans les lymphocytes. Dans ce contexte, un premier objectif de cette thèse fût de caractériser avec précision les propriétés de ces peptides. En outre, un objectif supplémentaire fût de caractériser le mécanisme moléculaire vis-à-vis de la NC de petites molécules anti-virales développées par les groupes de D. Daelemans (Leuven) et M. Botta (Sienne).
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Akbay, Burkitkan. "Regulation of the Akt/mTORC1 Pathway by HIV Transcriptional Activator Tat in B Cells Modulation of mTORC1 Signaling Pathway by HIV-1 Production of Stable Cell Lines on the Basis of the Cultured RPMI 8866 B-Cells with Constant and Inducible Expression of the Human Immunodeficiency Virus Tat Protein HIV-1 Tat Activates Akt/mTORC1 Pathway and AICDA Expression by Downregulating Its Transcriptional Inhibitors in B Cells." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL026.
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Les lymphomes agressifs à cellules B sont la principale cause de décès chez les personnes infectées par le VIH-1, bien que les cellules B ne soient pas ciblées par le virus. Les mécanismes exacts du développement de ces lymphomes ne sont pas connus. Des études antérieures de notre équipe ont révélé que le HIV-1 Tat peut pénétrer les cellules B, où il peut induire la production de ROS, endommager l'ADN et augmenter les chances de translocations oncogènes spécifiques au lymphome de Burkitt. En outre, dans de nombreuses cellules immunitaires, le VIH-1 et ses protéines (par exemple Tat) peuvent réguler la voie Akt/mTORC1, un intégrateur central de nombreux signaux intra et extracellulaires, y compris l'infection virale et les dommages à l'ADN. Cependant, aucune étude n'a examiné la régulation de la voie Akt/mTORC1 par Tat dans les cellules B. J'ai testé dans cette thèse l'hypothèse selon laquelle Tat pourrait produire des effets oncogènes dans les cellules B en modulant la voie de signalisation Akt/mTORC1 et en régulant l'expression des gènes impliqués dans la lymphomagenèse. J'ai découvert que HIV-1 Tat activait la voie de signalisation Akt/mTORC1, ce qui entraîne une activation aberrante de l'AICDA (cytidine désaminase induite par l'activation) en raison de l'inhibition des répresseurs transcriptionnels c-Myb et E2F8 de l'AICDA. Ces perturbations peuvent finalement conduire à une instabilité génomique accrue et à une prolifération qui pourrait provoquer des malignités des cellules BAggressive B cell lymphomas are the main cause of death in HIV-1 infected individuals, although B cells are not targeted by the virus. The exact mechanisms of the development of these lymphomas are not known. Previous studies of our team revealed that HIV-1 Tat can penetrate B cells, where it can induce ROS production, DNA damage and increase the chances of the oncogenic translocations specific for Burkitt lymphoma. In addition in many immune cells HIV-1 and its proteins (e.g. Tat) can regulate Akt/mTORC1 pathway, a central integrator of many intra and extracellular signals including viral infection and DNA damage. However, no studies have examined the regulation of Akt/mTORC1 pathway by Tat in B cells. In this thesis I have tested the hypothesis that HIV-1 Tat might produce oncogenic effects in B cells by modulating Akt/mTORC1 signaling pathway and regulating expression of genes involved in lymphomagenesis. I found that HIV-1 Tat activated Akt/mTORC1 signaling pathway, which leads to aberrant activation of AICDA (activation induced cytidine deaminase) due to inhibition of AICDA transcriptional repressors c-Myb and E2F8. These perturbations may ultimately lead to an increased genomic instability and proliferation that might cause B cell malignancies
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Weiss, Eric R. "Investigating the Roles of NEDD4.2s and Nef in the Release and Replication of HIV-1: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/641.
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Replication of HIV-1 requires the assembly and release of mature and infectious viral particles. In order to accomplish this goal, HIV-1 has evolved multiple methods to interact with the host cell. HIV-1 recruits the host cell ESCRT machinery to facilitate the release of nascent viral particles from the host cell membrane. Recruitment of these cellular factors is dependent on the presence of short motifs in Gag referred to as Late-domains. Deletion or mutation of these domains results in substantial decrease in the release of infectious virions. However, previously published work has indicated that over-expression of the E3 ubiquitin ligase, NEDD4.2s is able to robustly rescue release of otherwise budding-defective HIV-1 particles. This rescue is specific to the NEDD4.2s isoform as related E3 ubiquitin ligases display no ability to rescue particle release. In addition, rescue of particle release is dependent on the presence of the partial C2 domain and a catalytically active HECT domain of NEDD4.2s. Here I provide evidence supporting the hypothesis that a partial C2 domain of NEDD4.2s constitutes a Gag interacting module capable of targeting the HECT domains of other E3 ubiquitin ligases to HIV-1 Gag. Also, by generating chimeras between HECT domains shown to form poly-ubiquitin chains linked through either K48 or K63 of ubiquitin, I demonstrate that the ability of NEDD4.2s to catalyze the formation of K63-polyubiquitin chains is required for its stimulation of HIV-1 L-domain mutant particle release. In addition, I present findings from on-going research into the role of the HIV-1 accessory protein Nef during viral replication using the culture T-cell line, MOLT3. My current findings indicate that downregulation of CD4 from the host cell membrane does not solely account for the dramatic dependence of HIV-1 replication on Nef expression in this system. In addition, I present evidence indicating that Nef proteins from diverse HIV-1 Groups and strains are capable of enhancing HIV-1 replication in this system. Analysis of a range of mutations in Nef known to impact interaction with cellular proteins suggest that the observed replication enhancement requires Nef targeting to the host cell membrane and may also require the ability to interact with select Src-kinases. Lastly, we find that the ability of Nef to enhance replication in this system is separate from any increase in viral particle infectivity, in agreement with current literature.
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Gonzalez, Daniel. "Les "phosphate binding protein" : entre import du phosphate et inhibition de la transcription virale." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4019.
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Les « phosphate binding protein » (PBP) constituent une famille de protéines présentes de manière ubiquitaire chez les bactéries et plus marginalement chez les Eucaryotes. Impliquées dans l'import du phosphate extracellulaire chez les bactéries, les PBPs présentent un site de fixation du phosphate très bien caractérisé avec, notamment, une liaison hydrogène particulière nommée «low barrier hydrogen bond» (LBHB). Cette LBHB est impliquée dans la discrimination entre le phosphate et des anions proches chez les PBPs. Bien que cette discrimination semble nécessiter une haute conservation du site de fixation du phosphate, dans la nature différentes configurations sont observées. Au cours de ce travail, nous nous sommes intéressés à la PBP d'un organisme pathogène, C.perfringens qui présente un site de fixation alternatif. Avec, entre autre, une perte de la LBHB, cette PBP présente la plus faible capacité de discrimination testée à ce jour. Cette faible capacité de discrimination pourrait être liée au biotope de la bactérie ou bien à un phénomène d'adaptation fonctionnelle. D'autre part, certaines PBPs présentent des propriétés d'inhibition du VIH via l'étape de la transcription virale. Cependant, ces protéines sont particulièrement difficiles à produire en système hétérologue limitant l'étude fonctionnelle. Afin de lever ce verrou technique, nous avons développé une nouvelle méthodologie basée sur la phylogénie en vue de solubiliser notre modèle d'étude (HPBP). Nous avons obtenu un variant soluble de HPBP qui conserve ses activités antivirales permettant de débloquer les études fonctionnellesThe "phosphate binding protein" constitutes a family of proteins ubiquitously found in Prokaryotes but also more sparsely distributed in Eukaryotes. Involved in phosphate import, PBPs exhibits a well-characterized phosphate binding site with a peculiar hydrogen bond called "low barrier hydrogen bond" (LBHB). This LBHB is involved in the unique discrimination properties of PBPs, capable of discriminating phosphate from other similar anions such as arsenate of sulfate. Albeit this high discriminating property needs a high conservation of the phosphate binding pocket, different configurations are observed in nature. Herein, we have been interested in a PBP from a human pathogen, Clostridium perfringens, which presents an alternative phosphate binding site. Exhibiting a loss of the LBHB, C.perfringens PBP is the least discriminating PBP isolated so far. This weak discrimination property might be related to the environment of C.perfringens or to a functional adaptation of the PBP. On the other hand, PBPs issued from eukaryotic tissues exhibit HIV inhibition properties via a step not yet targeted in current therapies, i.e. the transcription. However, these proteins are difficult to obtain from human tissues and their expression in heterologous system remains impossible. We have developed a new methodology based on phylogeny in order to solubilise our study model, HPBP. Thus, we have obtained a soluble variant of HPBP which conserves the HIV-inhibiting properties. This unique tool both allow to unlock functional studies and lead to a better understanding on how PBPs are capable of inhibiting HIV
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Fernández, Arauzo Leticia. "Péptidos sintéticos del GB virus C. Aplicación en el diagnóstico de infección y en el diseño de potenciales agentes terapéuticos contra el VIH-1." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/482076.
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El GB virus C (GBV-C), también conocido como virus de la hepatitis G, es un virus humano no patogénico. Su infección es bastante frecuente, encontrándose incluso en personas sanas. Su prevalencia es mucho mayor en individuos considerados de riesgo, ya que comparte las vías de transmisión con otros virus, como por ejemplo con el virus de la hepatitis B, el virus de la hepatitis C o el virus de la inmunodeficiencia humana (VIH).
Numerosos estudios asocian la coinfección del GBV-C y el VIH con una menor progresión de la enfermedad y con una mayor supervivencia de los pacientes una vez que el SIDA se ha desarrollado. Sin embargo, el mecanismo de acción aún no se ha determinado. De este modo, el estudio de la interacción entre ambos virus podría dar lugar a nuevos agentes terapéuticos para el tratamiento del SIDA. Por otro lado, en cuanto al diagnóstico, actualmente no existe ningún sistema comercial para detectar marcadores específicos de la infección causada por el GBV-C.
De esta manera, en esta tesis se ha estudiado la capacidad de diversas moléculas peptídicas de regiones de la proteína de envoltura E2 y de la proteína no estructural NS4a del GBV-C para inhibir la entrada del VIH-1 a la célula, con el fin de conocer su potencial aplicación como agentes terapéuticos contra dicho virus. Asimismo, se ha estudiado la utilidad de dichas construcciones para desarrollar nuevos sistemas de diagnóstico de la infección causada por el GBV-C, empleando el ensayo inmunoenzimático de ELISA y la técnica de microarrays.
En primer lugar, se ha realizado un estudio detallado de la proteína E2 del GVB-C mediante la preparación de microarrays peptídicos con 124 secuencias lineales de dicha proteína. Para ello, se han empleado sueros procedentes de pacientes infectados por el VIH-1, de los cuales se conocía la presencia o ausencia de anticuerpos anti-E2 del GBV-C, y sueros de personas voluntarias sanas. Este ensayo ha permitido identificar dominios potencialmente antigénicos de la proteína E2 del GBV-C.
Teniendo en cuenta los resultados obtenidos y estudios previos realizados en el grupo de investigación, se ha llevado a cabo la síntesis de moléculas peptídicas derivadas del dominio N-terminal de la proteína E2 del GBV-C. Por un lado, se ha realizado la síntesis en fase sólida siguiendo una estrategia Fmoc/tBu, de péptidos lineales y de MAPs tetraméricos de tipo lineal homogéneo (con cuatro secuencias peptídicas iguales) y de tipo lineal heterogéneo (con secuencias peptídicas iguales dos a dos). También se han sintetizado péptidos cíclicos en solución mediante la reacción de transtioesterificación intramolecular conocida como Ligación Química Nativa. Por último, se ha llevado a cabo la formación de MAPs tetraméricos de tipo cíclico mediante ligación quimioselectiva en solución. Todas las moléculas sintetizadas se han caracterizado por HPLC, UPLC y espectrometría de masas (ESI y MALDI-TOF) y se han purificado por HPLC semipreparativo.
Posteriormente, se han realizado ensayos de fusión celular con el fin de evaluar la actividad anti-VIH-1 de las moléculas peptídicas que derivan del dominio N-terminal de la proteína E2 del GBV-C. Las líneas celulares empleadas fueron la Hela-env y la TZM-bl. De este estudio pudo concluirse que las construcciones de tipo cíclico tienen mayor tendencia hacia una mayor inhibición de la entrada del VIH-1 en la célula.
Se ha estudiado la capacidad para detectar anticuerpos anti-GBV-C de todas las secuencias y construcciones sintéticas mediante el ensayo inmunoenzimático de ELISA con sueros procedentes de pacientes con hepatitis C crónica, de pacientes sometidos a hemodiálisis, infectados con VIH-1 y sueros de donantes voluntarios sanos. Los resultados obtenidos demostraron la potencial utilidad diagnóstica de la región E2(7-26) del GBV-C.
Por último, con un panel de sueros de pacientes infectados por el VIH-1, del que no se tenía información sobre la presencia o ausencia de anticuerpos anti-E2 del GBV-C, se ha evaluado el valor diagnóstico de los dominios peptídicos identificados mediante microarrays. La combinación de las secuencias peptídicas ensayadas ha permitido establecer una reactividad del 47% en cuanto a la detección de anticuerpos anti-E2 del GBV-C en personas infectadas por el VIH-1. Además, esta tecnología ha miniaturizado el ensayo inmunoenzimático de ELISA y ha demostrado la utilidad de los péptidos sintéticos como potenciales antígenos para el desarrollo de un sistema de diagnóstico de la infección del GBV-C.GB virus C (GBV-C) (also formerly known as hepatitis G virus) is a non-pathogenic human virus. Its infection is more frequently in groups considered as high risk because it has similar routes of transmissions with other viruses such as hepatitis B virus, hepatitis C virus or human immunodeficiency virus (HIV). However, it is detected even in healthy people. There is a strong evidence for GBV-C association with ameliorated course of human immunodeficiency virus (HIV) disease, although its mechanism of action is yet to be determined. Thus, the study of the interaction between these viruses could give new therapeutic agents to HIV treatment. At present, there are no commercial systems to detect specific markers of GBV-C infection. In this thesis, the synthesis of branched peptide molecules was carried out by conjugating regions of the envelope protein E2 and the structural protein NS4 of the GBV-C virus. Afterwards, their antigenic capacity was evaluated. The ability of the synthesized molecules to inhibit cell fusion mediated by HIV-1 envelope protein was also studied in order to select potential inhibitors of virus entry into the cell. First of all, a detailed study of the E2 protein of GVB-C was carried out by preparing peptide microarrays with 124 linear sequences of this protein, using sera from patients infected with HIV-1 and healthy volunteers. Thus, potentially antigenic domains of the E2 protein of GBV-C were identified. After carrying out these assays and studying the accessibility profile of the potentially antigenic domain E2 (7-26), solid phase synthesis of linear, cyclic and branched peptides was carried out following an Fmoc/tBu strategy. Peptide constructions were characterized by HPLC, UPLC and mass spectrometry (ESI and MALDI-TOF) and purified by semipreparative HPLC. Subsequently, cell fusion assays were performed in order to evaluate the anti-HIV-1 activity of the peptide molecules derived from the N-terminal domain of the E2 protein of GBV-C. From this study, it could be concluded that the cyclic type constructions have a greater tendency towards a greater inhibition of HIV-1 entry in the cell. The ability to detect anti-GBV-C antibodies of all sequences and synthetic constructions were studied by the ELISA immunoenzymatic assay with sera from patients with chronic hepatitis C, patients undergoing hemodialysis, HIV-1 infected and sera from healthy volunteer donors. The results obtained demonstrated the potential diagnostic utility of the E2 region (7-26) of the GBV-C. Finally, the diagnostic value of the peptide domains identified by microarrays was evaluated with a panel of sera from patients infected with HIV-1. The combination of the peptide sequences studied allowed to establish a reactivity of 47% in the detection of anti-E2 antibodies of GBV-C in people infected with HIV-1. In addition, this technology has miniaturized the ELISA immunoenzymatic assay and it has demonstrated the usefulness of synthetic peptides as potential antigens for the development of a GBV-C infection diagnosis system.
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Khoury, Georges. "Étude des mécanismes moléculaires régulant l'expression de la protéine TAT du virus de l'immunodéficience humaine, au niveau de la production de ses ARN messagers et de leur traduction." Thesis, Université de Lorraine, 2012. http://www.theses.fr/2012LORR0251.
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La protéine Tat du VIH-1 est essentielle à la multiplication virale. Elle permet la transactivation de la transcription et, par ses propriétés apoptotiques, elle participe à la pathologie SIDA. D'où l'importance d'étudier les mécanismes régulant sa production. L'épissage alternatif de l'ARN du VIH-1, en particulier, l'utilisation des sites accepteurs d'épissage A3 et A7 est nécessaire pour la production des ARNm tat. L'utilisation du site A3 est fortement régulée par des éléments agissant en cis contenus dans une structure tige-boucle SLS3A3 située en aval du site A3. En purifiant les complexes RNP formés en extrait nucléaire sur un segment de l'ARN viral renfermant le site A3, et en analysant par spectrométrie de masse les protéines contenues dans ces complexes, nous avons pu mettre en évidence la fixation d'une protéine inhibitrice de l'utilisation du site A3, la protéine DAZAP1. Sur la base d'un ensemble de données antérieures du laboratoire et de nouvelles données que j'ai obtenues, nous avons montré que la protéine SRSF7, sans doute en synergie avec SRSF1, limite la fixation de DAZAP1 et active l'épissage au site A3. Nous avons aussi montré que la protéine virale Tat exerce un rétro-contrôle négatif au niveau de la production de l?ARNm tat, ceci en limitant l'activation du site A3 par SRSF7. La partie apicale de la structure tige-boucle SLS3A3 (motif B) est très conservée dans les souches de VIH-1. L'équipe d'E Guittet a déterminé sa structure 3D par RMN. La conformation de sa boucle terminale est caractéristique des structures tige-boucle reconnues par les protéines à domaines dsRBD (double stranded RNA Binding Domain). J'ai pu confirmer cette hypothèse en purifiant les complexes formés par le motif B en extrait nucléaire. Nous avons ainsi pu montrer que la protéine kinase PKR, qui joue chez l'Homme un rôle majeur dans la réponse à une infection virale, est un partenaire du motif B. Par utilisation de sondes chimiques de la structure 2D de l'ARN, j'ai pu montrer que la structure tige-boucle SLS3A3, contenant le codon d'initiation de l'ORF Tat, est présente dans l'ARNm tat1. Nous avons alors développé un système visant à étudier les mécanismes de régulation de l'initiation de la traduction de la protéine Tat. Par l'emploi d'une construction bicistronique, j'ai pu confirmer l'existence d'une activité IRES dans la région 5'UTRtat1 de l'ARNm tat1 et définir deux segments ayant cette activité. Des résultats préliminaires obtenus avec une construction bicistronique, nous ont permis de commencer à tester l'effet de différentes protéines SR et hnRNP sur l'activité de ces IRESHIV-1 Tat protein is essential for viral replication. It allows the transcription of full-length viral RNAs, and due to its apoptotic properties it contributes to the AIDS disease. Hence, it is important to study the mechanisms regulating its production. Alternative splicing of the HIV-1 RNA, in particular, the use of acceptor sites A3 and A7 is required for tat mRNA production. Splicing at site A3 is highly regulated by cis-acting elements contained in a stem-loop structure SLS3A3, located downstream from site A3. By purifying RNP complexes formed in nuclear extract on a segment of the viral RNA containing site A3 followed by mass spectrometry analysis, we were able to highlight the binding of a new inhibitory protein, DAZAP1. Based on a set of ancient laboratory data and new results that I obtained, we have shown that SRSF7 protein, probably in synergy with SRSF1, limits the binding of DAZAP1 and splicing activation at site A3. We also showed that the viral protein Tat exerts a negative feedback control on tat mRNA production by restricting splicing activation of site A3 by SRSF7. The apical part of the stem-loop structure SLS3A3 (B motif) is highly conserved among HIV-1 strains. E Guittet team determined its 3D structure by NMR. The conformation of this apical loop is characteristic of stem-loop structures recognized by dsRBD proteins (double-stranded RNA binding domain). I was able to confirm this hypothesis by purifying RNP complexes formed by the B motif in nuclear extract. Thus, we have shown that the RNA dependent protein kinase (PKR), which plays in humans a major role in response to viral infection, is a partner of the B motif. By using chemical probes specific of the 2D structure of RNAs, I showed that the stem-loop structure SLS3A3 that contains the initiation codon of Tat is present in tat1 mRNA. We then developed a system to study the mechanisms regulating the initiation of translation of Tat protein. By using a bicistronic construct, I was able to confirm the existence of IRES activity in the 5?UTRtat1 region of tat1 mRNA, and define two segments that contain this activity. Preliminary results obtained with a bicistronic construct allowed us to begin testing the effect of different SR and hnRNP proteins on the activity of the IRES
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Calao, Miriam. "Role of IkB kinase (IKK) complex post-translational modifications in NF-kB signaling and therapeutic applications for the treatment of HIV-1 infection." Doctoral thesis, Universite Libre de Bruxelles, 2009. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210318.
Full textAbstract:
Les facteurs de transcription de la famille Rel/NF-κB régulent l’expression d’un grand nombre de gènes impliqués dans les réponses immunitaires et inflammatoires ainsi que dans la régulation de la prolifération et de la survie cellulaire. Le caractère transitoire de l’activation de NF-κB est donc crucial pour poterger les cellules de l’autoxicité due à une trop forte expression des gènes cibles de ce facteur de transcription. Dans le cadre de notre thèse de doctorat, nous avons étudié les mécanismes moléculaires régulant la cinétique d’activation de NF-κB, en accordant une attention toute particulière au complexe kinase IKK, qui semble être le regulateur clef de l’activation de NF-κB. Nos résultats suggèrent que p300 pourrait réguler la durée d’activation des IKKs d’une part par acétylation directe, et d’autre part, indépendamment de son activité HAT, en stabilisant les IKKs et donc en prolongeant leur demie-vie et par conséquent leur activation.Certains virus utilisent la voie de signalisation NF-κB afin de promouvoir leur propre réplication. C’est le cas du virus HIV-1 (Human Immunodeficiency Virus type 1), qui contient dans son promoteur deux sites de liaison pour NF-κB. Notre laboratoire a précédemment montré que l’utilisation du TNFα en combinaison avec la TSA, active l’expression virale de manière synergique. L’administration combinée d’un activateur du facteur NF-κB et d’un inhibiteur de désacétylases pourrait, en présence d’une thérapie anti-HIV-1 efficace, être envisagée dans le but d’éliminer les cellules réservoirs infectées de manière latente. L’utilisation thérapeutique du TNFα ou de la TSA étant inenvisageable en raison de leur toxicité, nous avons étudié l’effet d’autres substances ayant un plus grand potentiel thérapeutique et nous avons apporté une preuve de principe du potentiel thérapeutique de la coadministration de plusieurs activateurs viraux (inhibiteurs de HDACs[HDACIs]+inducteurs de la voie NF-κB) pour réduire le pool des réservoirs cellulaires infectés de manière latente.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished