Дисертації з теми "HIV-1 infectivity"

Human Immunodeficiency Virus Type 1 (HIV-1), the virus that causes Acquired Immunodeficiency Syndrome (AIDS), attacks the immune system leaving patients susceptible to opportunistic infections that eventually cause death. Highly Active Antiretroviral Therapy, HAART, is the current drug strategy used to combat HIV. It is a combination therapy that includes HIV-1 Reverse Transcriptase and HIV-1 Protease inhibitors. Drug resistant strains arise that evade current HAART treatments; therefore novel drugs are needed. HIV-1 regulatory proteins such as Tat, Rev, Nef, Vpr, Vpu, and Vif are attractive new drug targets. Of particular interest is the HIV-1 Vif protein and its cellular binding partner APOBEC3G. In the absence of HIV-1 Vif, APOBEC3G, a cytidine deaminase, is able to mutate the viral cDNA and render the virus noninfectious. HIV-1 Vif binds to APOBEC3G and targets it for proteosomal degradation through an interaction with a Cullin-RING ligase complex. Blocking the HIV-1 Vif APOBEC3G interaction would allow APOBEC3G to perform its antiviral function. An attractive strategy to target the HIV-1 Vif APOBEC3G interaction would be a structure-based one. To apply structure-based drug design approaches to HIV-1 Vif and APOBEC3G, I attempted to collect high resolution structural data on HIV-1 Vif and APOBEC3G. My attempts were unsuccessful because the milligram quantities of soluble protein required were not obtained. Therefore, in Chapter III I used chemical cross-linking and mass spectrometry to probe the structural topology of HIV-1 Vif obtaining low resolution structural data. Chemical cross-linking formed HIV-1 Vif multimers including dimers, trimers, and tetramers. Analysis of the cross-linked monomer revealed that HIV-1 Vif’s N-terminal domain is a well-folded, compact, globular domain, where as the C-teriminal domain is predicted to be disordered. In addition, disorder prediction programs predicted the C-terminal domain of HIV-1 Vif to be disordered. Upon oligomerization the C-terminal domain undergoes a disorder-to-order transition that not only facilitates oligomerization but may facilitate other protein-protein interactions. In addition, HIV-1 Vif oligomerization bring Lys34 and Glu134 in close proximity to each other likely creating one molecular surface forming a “hot spot” of biological activity. In Chapter IV I confirmed my low resolution structural data via peptide competition experiments where I identified peptides that can be used as scaffolds for future drug design. HIV-1 Vif oligomerization is concentration dependent. The HIV-1 Vif peptides Vif(29-43) and Vif(125-139) were able to disrupt HIV-1 Vif oligomerization, which confirms the low resolution structural data. HIV-1 Vif peptides Vif(25-39) and Vif(29-43) reduced the amount of APOBEC3G immobilized on the Protein A beads, reduced the amount of HIV-1 Vif interacting with APOBEC3G, or degraded APOBEC3G itself. These peptides could be used as scaffolds to design novel drugs that disrupt the function of HIV-1 Vif and or APOBEC3G. Therefore, low resolution structural data and peptide competition experiments were successful in identifying structurally important domains in HIV-1 Vif. They also provided insight into a possible mechanism for HIV-1 Vif function where a disorder-to-order transition facilitates HIV-1 Vif’s ability to interact with a diverse set of macromolecules. These data advance our structural understanding of HIV-1 Vif and they will facilitate future highresolution studies and novel drug designs.

Human Immunodeficiency Virus Type 1 (HIV-1), the virus that causes Acquired Immunodeficiency Syndrome (AIDS), attacks the immune system leaving patients susceptible to opportunistic infections that eventually cause death. Highly Active Antiretroviral Therapy, HAART, is the current drug strategy used to combat HIV. It is a combination therapy that includes HIV-1 Reverse Transcriptase and HIV-1 Protease inhibitors. Drug resistant strains arise that evade current HAART treatments; therefore novel drugs are needed. HIV-1 regulatory proteins such as Tat, Rev, Nef, Vpr, Vpu, and Vif are attractive new drug targets. Of particular interest is the HIV-1 Vif protein and its cellular binding partner APOBEC3G. In the absence of HIV-1 Vif, APOBEC3G, a cytidine deaminase, is able to mutate the viral cDNA and render the virus noninfectious. HIV-1 Vif binds to APOBEC3G and targets it for proteosomal degradation through an interaction with a Cullin-RING ligase complex. Blocking the HIV-1 Vif APOBEC3G interaction would allow APOBEC3G to perform its antiviral function. An attractive strategy to target the HIV-1 Vif APOBEC3G interaction would be a structure-based one. To apply structure-based drug design approaches to HIV-1 Vif and APOBEC3G, I attempted to collect high resolution structural data on HIV-1 Vif and APOBEC3G. My attempts were unsuccessful because the milligram quantities of soluble protein required were not obtained. Therefore, in Chapter III I used chemical cross-linking and mass spectrometry to probe the structural topology of HIV-1 Vif obtaining low resolution structural data. Chemical cross-linking formed HIV-1 Vif multimers including dimers, trimers, and tetramers. Analysis of the cross-linked monomer revealed that HIV-1 Vif’s N-terminal domain is a well-folded, compact, globular domain, where as the C-teriminal domain is predicted to be disordered. In addition, disorder prediction programs predicted the C-terminal domain of HIV-1 Vif to be disordered. Upon oligomerization the C-terminal domain undergoes a disorder-to-order transition that not only facilitates oligomerization but may facilitate other protein-protein interactions. In addition, HIV-1 Vif oligomerization bring Lys34 and Glu134 in close proximity to each other likely creating one molecular surface forming a “hot spot” of biological activity. In Chapter IV I confirmed my low resolution structural data via peptide competition experiments where I identified peptides that can be used as scaffolds for future drug design. HIV-1 Vif oligomerization is concentration dependent. The HIV-1 Vif peptides Vif(29-43) and Vif(125-139) were able to disrupt HIV-1 Vif oligomerization, which confirms the low resolution structural data. HIV-1 Vif peptides Vif(25-39) and Vif(29-43) reduced the amount of APOBEC3G immobilized on the Protein A beads, reduced the amount of HIV-1 Vif interacting with APOBEC3G, or degraded APOBEC3G itself. These peptides could be used as scaffolds to design novel drugs that disrupt the function of HIV-1 Vif and or APOBEC3G. Therefore, low resolution structural data and peptide competition experiments were successful in identifying structurally important domains in HIV-1 Vif. They also provided insight into a possible mechanism for HIV-1 Vif function where a disorder-to-order transition facilitates HIV-1 Vif’s ability to interact with a diverse set of macromolecules. These data advance our structural understanding of HIV-1 Vif and they will facilitate future highresolution studies and novel drug designs.

Nef is an HIV -1 accessory protein with a fundamental role for virus replication in vivo and for the development of AIDS. Among its several activities, Nef is essential for full HIV-1 infectivity, a function highly prominent in lymphoid cells. So far, the mechanism by which Nef promotes HIV-1 infectivity has remained elusive. Over the course of 3 years, my PhD research activity has led to the identification of the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as potent inhibitors of HIV-1 infectivity counteracted by the viral protein Nef [Rosa et al., 2015]. SERINC5 is predominantly localized on the plasma membrane where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef relocalizes SERINC5 to an endosomal compartment preventing its incorporation into HIV-1 particles. The ability to counteract SERINC5 is conserved in Nef proteins encoded by different primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus (MLV). These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 in the interaction of the host with retroviral pathogens. Remarkably, SERINC5 potently inhibits HIV-1 even in the presence of Nef in a dose-dependent manner, suggesting that this cellular factor might be exploited as an anti-HIV-1 therapeutic gene.

Transmitted founders (TF) might carry motifs that provide a phenotypic advantage that enables human immunodeficiency virus type-1 (HIV-1) to overcome immune barriers within the female genital tract. One study compared over 5000 subtype B TF and mismatched chronic infection envelope (env) sequences and identified two putative transmission motifs: Histidine at position 12 of the signal peptide (His12) and a potential N-glycan site (PNG) at position 413-415. Although, His12 was shown to be important for subtype B Env expression and viral infectivity, in our own sequence analysis subtype C variants did not carry the transmission motifs and the aim of this study was to determine whether His12 and PNG413 was important for subtype C Env expression, processing, function and viral replication. Mutagenesis of a subtype C Env clone indicated that His12 decreased pseudovirion (PSV) entry efficiency without influencing Env expression, secretion and cleavage with no changes in the N-glycosylation profile. This suggested that His12 had a fitness cost and was thus selected against. However, His12 significantly enhanced the entry efficiency of infectious molecular clones (IMCs), suggesting that it might be beneficial for in vivo replication. The variation between the PSV and IMC entry of TZM-bl cells could be due to differences in assay conditions. On the other hand deletion of PNG413 enhanced Env expression, secretion, cleavage and PSV and IMC entry efficiency of TZM-bl cells. This would suggest that subtype C TFs carrying a PNG at 413-413 would have lower viral replicative capacity due to poor expression and processing of Env. The benefit of this phenotype on HIV-1 subtype C transmission needs to be further investigated. Unfortunately, PSV and IMC entry of TZM-bl cells could not be confirmed by IMC replication in peripheral blood monocytes because the clones could not replicate to measurable levels in these cells over the culture period. Overall, this study has shown that amino acid residues at positions 12 and 415 do play a role in modulating Env processing and function however the actual mechanism by which these polymorphisms impact viral fitness most likely differ to that of subtype B, explaining why His12 is absent and PNG413 is present in subtype C TFs.

La dissémination du VIH-1 par contacts cellulaires est plus efficace que sa transmission par particules virales libres. Cependant, la nature du matériel infectieux transféré à la jonction reste très mal connue. Nos travaux révèlent que l'infectivité du VIH-1 associée aux lymphocytes T est majoritairement portée à la surface cellulaire dans un biofilm viral. Initialement décrit pour le rétrovirus lymphotrope HTLV-1 (Human T-cell Leukemia Virus type-1), le biofilm viral est une colonie extracellulaire de particules virales infectieuses enchâssées dans un cocon de matrice extracellulaire (MEC). Par un panel de techniques de microscopie, nous décrivons la présence de biofilms viraux à la surface de lymphocytes T infectés par le VIH-1 (lignées chroniquement infectées, lymphocytes CD4+ primaires infectés in vitro par des souches de laboratoire et des isolats primaires, lymphocytes de patients). Nous identifions certains éléments de la MEC enrichis dans le biofilm du VIH-1 et démontrons que certains de ces composants, modulés par l'infection, favorisent la transmission du VIH-1. En effet, le biofilm viral joue un rôle clé dans la transmission directe (entre lymphocytes T) et indirecte (trans-infection) du VIH-1. De plus, le biofilm du VIH-1 confère une infectivité accrue aux particules virales, avantage préservé en présence d'antirétroviraux et d'anticorps neutralisants.L'ensemble de notre travail identifie une nouvelle entité infectieuse cruciale pour la dissémination du VIH-1 par contacts cellulaires. Ce nouveau mécanisme de transmission, potentiellement généralisable à d'autres virus, sera désormais à prendre en compte dans l'élaboration de nouvelles stratégies thérapeutiques
HIV-1 cell-to-cell spread is thousands fold more efficient than cell-free infection; yet the nature of the infectious material transferred at the junction remains poorly documented. We found that HIV-1 T cell-associated infectivity mostly resides at the cell surface in a viral biofilm. Initially described for HTLV-1, a viral biofilm is defined as extracellular viral particles aggregated within a scaffold of extracellular matrix (ECM) components exposed at the surface of infected cells. Using a combination of microscopy techniques, we report the presence of HIV-1 biofilms at the surface of HIV-1 infected T cells (chronically infected T cells lines as well as primary CD4+ T cells infected in vitro with HIV-1 laboratory strains as well as primary isolates). Importantly, we show that CD4+ T cells isolated from HIV-1 patients produce a viral biofilm as well. We partially characterize the composition of HIV-1 biofilm in ECM components and unravel their contribution to HIV-1 transmission. We show that HIV-1 biofilm is transferred both between T cells and during dendritic-cell (DC)-mediated trans-infection and confers viral particles with an increased infectivity as compared to their cell-free counterparts. This increased infectivity is preserved in the presence of antiretroviral treatment and neutralizing antibodies. Our findings hence identify HIV-1 biofilm as a new infectious entity central for the efficiency of HIV-1 cell-to-cell transmission. This new mechanism of intercellular transmission might be shared by other viruses and will require appraisal in the design of future therapeutical strategies

The Human Immunodeficiency Virus (HIV) is able to infect CD4+ T cells as well as macrophages. Macrophage-tropism has been linked to determinants in the envelope of HIV. These determinants allow envelopes to exploit low levels of CD4 for infection. Macrophages are an important reservoir of virus, especially during chronic infection, and are likely responsible for the bulk of virus produced after CD4+T cells have declined. Viral factors that may impact the ability to infect macrophages are worth studying because this cell type is so important in infection. It was previously reported that the macrophage-tropic primary isolate AD8 was vpu-independent. The molecular clone YU-2, derived from brain tissue without culture, was also reported to be macrophage-tropic despite having a mutation in the vpu start codon. It was therefore possible that vpu-independent envelopes could evolve in vivo. To examine this possibility, I constructed chimeras containing wild type or defective vpu start codons, and gp160 sequences from AD8, YU-2 or SF162 (a vpu-dependent control). I also used full length AD8 and YU-2 with wild type or defective vpu start codons. I infected macrophages with equal amounts of virus, and measured viral output over two weeks. Viruses with defective vpu start codons were released to lower levels compared to their wild type vpucounterparts. In contrast to previous reports, the AD8 envelope is not vpu-independent for replication in macrophages. The YU-2 envelope is also not vpu-independent. Macrophage-tropic envelopes from late stages of infection can be sensitive to antibodies that bind the CD4 binding site on gp120, implying that macrophage-tropic envelopes have more exposed CD4 binding sites. Neutralizing antibodies may act as modulators of macrophage-tropism over the course of infection. Using chimeras containing gp120 sequences derived from the PBMC of four HIV+patients, I examined the capacity for envelopes to infect macrophages. Three patients (MM1, 4, and 8) had macrophage-tropic envelopes before and after developing autologous neutralizing antibodies. Three patients (MM1, 4, and 23) developed heterologous antibodies against IIIB, an easily neutralized T-cell line adapted strain of HIV-1. This data indicates that macrophage-tropism in these patients is not modulated by the presence of neutralizing antibodies. The macrophage-tropism of envelopes tends to segregate depending on the tissue origin of the virus. Envelopes from two separate tissues from the same patient exhibit very different infectivity characteristics. The B33 envelope, from brain tissue, is very infectious and is macrophage-tropic, while the LN40 envelope, from lymph node tissue, is weakly infectious and is not macrophage-tropic. Replacing the entire gp41 of LN40 with that of B33 restores some infectivity to LN40. The cytoplasmic domain of gp41 contains many motifs important for assembly and infectivity. To examine which motifs are responsible for the weak infectivity of LN40, I made chimeras of gp41, as well as point mutations in gp41. The LN40 chimera containing the entire gp41 of B33 restored the most infectivity. Point mutations in the palmitoylation site, Pr55gagbinding region, and dileucine motif at the C-terminus also restored infectivity when combined. Determinants in the gp41 cytoplasmic domain are responsible for the weak infectivity of LN40; however, it is possible that there are contributing determinants in gp120, such as the ability to use low levels of CD4. Here, I examined how changes in the vpu and env genes of HIV-1 can impact infectivity, especially infectivity of macrophages. Changes that adversely impact the virus’ ability to infect macrophages may also impact the overall course of disease. However, the data here show that retaining the ability to infect, and replicate in, macrophages give HIV an advantage. I speculate that retaining the ability to infect macrophages gives the virus a reservoir for later in disease, when CD4+ T cells have been depleted, as well as way of avoiding neutralizing antibodies. This work further defines the importance of macrophages in HIV-1 infectivity and disease.

La protéine Nef des virus de l’immunodéficience humaine (VIH-1 et VIH-2) joue un rôle essentiel dans la physiopathologie de l’infection et induction du SIDA. La capacité de Nef à perturber le trafic intracellulaire de protéines membranaires, et notamment du récepteur CD4, circulant entre les compartiments de la voie d’endocytose pourrait rendre compte de son importance comme facteur de virulence au cours de l’infection naturelle. Les mécanismes responsables des perturbations de la voie d’endocytose induites par Nef au cours de l’infection ne sont pas totalement élucidés, mais il est admis qu’elles résultent d’interactions avec les complexes adaptateurs (AP) associés à la clathrine et participant au transport vésiculaire entre les différents compartiments de la voie d’endocytose. Notre objectif était de déterminer les mécanismes par lesquels Nef influe positivement sur le pouvoir infectieux du VIH-1 en interagissant avec la machinerie cellulaire de la voie d’endocytose. Notre programme s’est organisé autour de deux axes principaux: le premier a consisté à étudier l’implication respective des différents types de complexes AP (AP-1, -2 et -3) sur les perturbations du fonctionnement de la voie d’endocytose induites par Nef en analysant son impact sur le niveau d’expression de surface de CD4; le deuxième axe a consisté à évaluer l’impact de l’interaction de Nef avec les complexes AP sur les capacités infectieuses des particules virales. Le rôle respectif des différents complexes AP dans ces fonctions de Nef a donc été étudié après déplétion de l’expression des complexes AP-1, AP-2 et AP-3 par une approche d’ARN interférence. Les résultats obtenus montrent que contrairement à certaines données de la littérature, la déplétion des complexes AP de la voie d’endocytose ne semble pas avoir un impact majeur sur la capacité de Nef à moduler l’expression de surface de CD4, même si une légère diminution de l’activité de Nef a pu être révélée dans notre étude réalisée sur des cellules HeLa-CD4 transduites par les shRNA ciblant les complexes AP-2. Inversement, nos résultats confirment que la déplétion des complexes AP-1, AP-2 et AP-3 dans les cellules productrices des particules virales se traduit par une diminution importante des propriétés infectieuses de ces particules sur lesquelles l’impact positif de Nef n’est plus alors capable de se manifester. En conclusion, ce travail a donc permis de montrer que les complexes AP de la voie d’endocytose sont indispensables pour que Nef puisse exercer son rôle positif sur le pouvoir infectieux du VIH-1. Il est maintenant important de confirmer ces résultats en analysant le rôle fonctionnel des complexes AP sur les activités de Nef dans les cibles cellulaires naturelles du VIH-1, lymphocytes et macrophages
Nef protein of human immunodeficiency virus (HIV-1 and HIV-2) plays an essential role in the pathophysiology of infection and induction of AIDS. The ability of Nef to disrupt intracellular trafficking of membrane proteins, including the CD4 receptor, moving between the compartments of the endocytic pathway could account for its importance as a virulence factor during natural infection. The mechanisms responsible for disruption of the endocytic pathway induced by Nef during infection are not fully understood, but it is accepted that they arise from interactions with adaptor complexes (AP) associated with clathrin and participant in vesicular transport between the different compartments of the endocytic pathway. Our objective was to determine the mechanisms by which Nef positively affects the infectivity of HIV-1 by interacting with the cellular machinery of the endocytic pathway. Our program has been organized around two main axes: the first was to investigate the respective involvement of different types of complexes (AP-1, -2 and -3) on the Nef induced disruption of the endocytic pathway by analyzing its impact on the level of surface expression of CD4; the second axis was to evaluate the impact of the interaction of Nef with AP complexes on the infectious capacity of the viral particles. The respective roles of the different AP complexes in these functions of Nef has been studied after depletion of the expression of complex AP-1, AP-2 and AP-3 by RNA interference approach. The results show that, contrary to some literature data, depletion of AP complex endocytic pathway does not appear to have a major impact on the ability of Nef to modulate the surface expression of CD4, although a slight decreased activity of Nef could be revealed in our study on HeLa-CD4 cells transduced with the shRNA targeting complex AP-2. Conversely, our results confirm that the depletion of complex AP-1, AP-2 and AP-3 in the cells producing viral particles resulted in a significant decrease in infectious properties of these particles on which the positive impact of Nef is no longer able to manifest. In conclusion, this work has shown that complex AP of endocytic pathway are essential for Nef to exercise its positive role in the infectivity of HIV-1. It is now important to confirm these findings by analyzing the functional role of AP complexes on the activities of Nef in the natural cellular targets of HIV-1, lymphocytes and macrophages

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2012
A Síndrome da Imunodeficiência Adquirida (SIDA) representa hoje um dos principais problemas de Saúde Pública a nível mundial, tendo contraído a doença mais de 60 milhões de pessoas em todo o mundo desde a sua descoberta em 1981, um terço das quais faleceram subsequentemente. Apesar dos grandes progressos realizados no tratamento da SIDA, especialmente desde a introdução do regime terapêutico HAART em 1996, as alternativas terapêuticas actuamente disponíveis não permitem erradicar completamente o VIH-1 do organismo, o que resulta em toxicidade a longo prazo e leva eventualmente à emergência de estirpes de VIH-1 resistentes à terapêutica disponivel. Estes problemas levam à procura e desenvolvimento de novos fármacos activos contra o VIH, nomeadamente contra as estirpes de VIH resistentes aos fármacos actualmente utilizados. Entre os novos fármacos actualmente disponíveis encontra-se uma nova classe, os inibidores de entrada. O processo de fusão do VIH-1 é mediado pelas proteínas Env (gp120 e gp41) e inicia-se através da ligação da gp120 ao receptor celular CD4. A interacção gp120-CD4 dá origem a uma alteração conformacional na loop V3 da gp120 que expõe o epitopo de ligação ao receptor de quimiocinas, o que por sua vez induz alterações conformacionais na gp41 que culminam na conformação activa do péptido de fusão (conformação fusogénica). Portanto, a interação gp120-CD4 é fundamental para o processo de fusão vírus-célula. Igualmente, uma descoberta recente identificou na gp120 uma região conformacionalmente invariável que se sobrepõe parcialmente ao local de ligação ao CD4, esta região corresponde à zona de ligação do anticorpo b12 e possui a particularidade que se encontrar constitutivamente exposta no domínio exterior da gp120. Esta região encontra-se envolvida na ligação metaestável ao CD4, antes de ocorrer o rearanjo necessário para a existência de uma ligação estável. Este é por conseguinte um local de vulnerabilidade, relacionado com um requisito funcional para existência de uma associação eficiente com o CD4. iv O anticorpo b12 é um dos poucos anticorpos monoclonais humanos conhecidos que pode eficientemente neutralizar uma ampla gama de isolados primários de VIH-1 in vitro e pode proteger contra o desafio viral in vivo. O objetivo deste trabalho foi a avaliação da sequência alvo do anticorpo neutralizante b12 ou o epítopo de ligação ao CD4 fora do contexto da glicoproteína gp120. Para tal procedeu-se à construção de anticorpos de domínio, por meio de “grafting” da sequência alvo do anticorpo neutralizante b12 ou o epítopo de ligação ao CD4 num dos CDR’s, CDR1 ou CDR3 de um anticorpo de coelho de único domínio VL,.altamente estável. O potencial das construções VLB12 e VLCD4 foi testado para ligação ao CD4 por ELISA e por Citometria de Fluxo. A Citometria de Fluxo foi efectuada recorrendo a células HEK293T, HeLa-P4 e Jurkat, ambas as construções apresentam ligação ao receptor CD4, e o VLB12 CDR1, em particular, e parece ter uma ligação ao CD4 de elevada afinidade. A partir dos resultados de Citometria de Fluxo e dos rendimentos obtidos para as purificações as construções VLB12 CDR1 e VLCD4 CDR3 foram selecionados para análise posterior. Pretendeu-se também caracterizar o local de ligação das construções VLB12 CDR1 e VLCD4 CDR3 no receptor CD4, nomeadamente identificar qual o domínio do CD4 ao qual se ligam os anticorpos VLB12 e VLCD4. Para tal, realizaram-se ensaios de ELISA utilizando como antigénios que o receptor celular humano CD4 na sua forma solúvel, quer uma construção em que os domínios 1 e 2 do CD4 humano foram colocados em fusão com uma IgG2 humana na qual as regiões variáveis das cadeias leves e das cadeias pesadas foram substituidas pelos domínios 1 e 2 do CD4 humano dando origem a uma proteína de fusão designada CD4-IgG2. Dos ensaios de ELISA foi possivel concluir que ambos os anticorpos VLB12 e VLCD4 se ligam ou ao domínio 1 ou ao domínio 2 do CD4 humano. Para identificar especificamente a qual dos domínios se ligavam o VLB12 e o VLCD4, foram realizadas experiências em que células HEK293T foram transfectadas com CD4 humano (hCD4), CD4 de murganho (mCD4) ou com CD4 humano em que o domínio 1 foi substituido pelo domínio 1 de murganho (hCD4mD1). Estas células transfectadas com os diferentes receptores celulares CD4 foram submetidas a ensaios de Citometria de Fluxo para avaliação da ligação dos anticorpos VLB12 e VLCD4 aos diferentes receptores celulares CD4 e concluiu-se que o VLB12 se liga ao domínio 2 e que o VLCD4 se liga ao domínio v 1 do CD4 humano. Realizaram-se também ensaios de ELISa em que se verificou que os epitopos reconhecidos pelo VLB12 e pelo VLCD4 são ambos conformacionais. A fim de avaliar a capacidade do VLB12 e do VLCD4 na inibição da infecção por VIH-1, foram realizados ensaios de inibição com o clone molecular NL4-3 do VIH-1 (subtipo B) e com os subtipos J e H de isolados primários de VIH-1. Os ensaios de inibição foram realizados em células TZM-bl num ensaio de um ciclo único de infecção. O VLB12 foi capaz de inibir o VIH-1NL4-3 com um IC50 = 3,89 µM e o VLCD4 foi capaz de inibir o HIV-1NL4-3 com um IC50 = 6,57 µM. Realizaram-se igualmente ensaios de inibição com o T20 para o qual se obteve um IC50 = 0,0694 µM.Quanto aos isolados primários, o VLB12 foi capaz de inibir o VIH-1 subtipo H com um IC50 = 3,97 µM e o VIH-1 subtipo J com um IC50 = 3,86 µM, mas o VLCD4 não foi capaz de inibir os subtipo J e H de isolados primários de VIH-1. Para o T20 obteve-se um IC50 = 1,33 nanoM para o subtipo H do VIH-1 e para o subtipo J do VIH-1 obteve-se um IC50 = 0,42 nanoM. Estes resultados indicam que o VLB12 apresenta um amplo potencial de inibição ao contrário do VLCD4 que não foi capaz de inibir qualquer um dos isolados primários dos subtipo não B. Devido à elevada afinidade de ligação ao receptor CD4 apresentada pelo VLB12, uma das abordagens desta Tese teve por objectivo avaliar o potencial de um novo sistema de entrega de moleculas para terapia génica em que se utilizaram nanopartículas revestidas com VLB12 para avaliar a entrega de biomoléculas a células que expressam o receptor CD4. Foram testados dois tipos diferentes de formulações de nanopartículas, de quitosano e de PEI, para a entrega do plasmídeo FUGW-dsRed em células Jurkat. Ambas as formulações de nanopartículas foram bem sucedidos na entrega do plasmídeo FUGW-dsRed às células Jurkat, conforme determinado por imunofluorescência. A formulação de nanopartículas de quitosano provou globalmente ser mais eficaz, devido à reduzida toxicidade celular. A quantidade ideal de VLB12 adsorvido às nanopartículas deu origem a um aumento de 16% da população dsREd positiva vi fluorescente com um direccionamento das nanopartículas para o receptor alvo (CD4) devido à presença do VLB12. Numa abordagem distintadas anteriores, pretendeu-se avaliar as propriedades antigénicas do VLB12 para utilização numa formulação de vacina. Deste modo, a imunidade das mucosas foi testada utilizando o VLB12 encapsulado em nanopartículas de quitosano, por comparação com a via subcutânea, utilizando como adjuvante alumínio e realizando as mesmas formulações sem adjuvante. A resposta de IgG total específico nas amostras de soro foi avaliada por ELISA utilizando como antigénio o VLB12. Foram também avaliadas as respostas Th1 ou Th2 através do racio dos antigénios específicos IgG1-IgG2 nas amostras de soro. Para avaliar o potencial antigénico do VLB12 como vacina, foram realizados ensaios de inibição de VIH-1 usando os soros dos diferentes grupos de murganhos, aos quais foi administrado cada uma das formulações. Os ensaios de inibição foram realizados em células TZM-bl, com uma MOI de 0,1, num ensaio de um ciclo único de infecção. Apesar de os grupos vacinados apresentarem títulos de IgG específico elevados, nenhum dos soros obtidos apresentou efeito inibitório sobre o VIH-1NL4-3. Confirmou-se por ELISA que os soros obtidos não reconheciam a gp120 o que indica que a framework do VL é altamente imunogénica, não tendo sido obtida uma proporção de anticorpos dirigidos contra a região alvo b12 suficiente para se obter um efeito inibitório nos ensaios de inibição do VIH-1NL4-3. O anticorpo b12 é um dos poucos anticorpos monoclonais humanos conhecidos que pode eficientemente neutralizar uma ampla gama de isolados primários de VIH-1 in vitro e pode proteger contra o desafio viral in vivo. O objetivo deste trabalho foi a avaliação da sequência alvo do anticorpo neutralizante b12 ou o epítopo de ligação ao CD4 fora do contexto da glicoproteína gp120. Para tal procedeu-se à construção de anticorpos de domínio, por meio de “grafting” da sequência alvo do anticorpo neutralizante b12 ou o epítopo de ligação ao CD4 num dos CDR’s, CDR1 ou CDR3 de um anticorpo de coelho de único domínio VL,.altamente estável. O potencial das construções VLB12 e VLCD4 foi testado para ligação ao CD4 por ELISA e por Citometria de Fluxo. A Citometria de Fluxo foi efectuada recorrendo a células HEK293T, HeLa-P4 e Jurkat, ambas as construções apresentam ligação ao receptor CD4, e o VLB12 CDR1, em particular, e parece ter uma ligação ao CD4 de elevada afinidade. A partir dos resultados de Citometria de Fluxo e dos rendimentos obtidos para as purificações as construções VLB12 CDR1 e VLCD4 CDR3 foram selecionados para análise posterior. Pretendeu-se também caracterizar o local de ligação das construções VLB12 CDR1 e VLCD4 CDR3 no receptor CD4, nomeadamente identificar qual o domínio do CD4 ao qual se ligam os anticorpos VLB12 e VLCD4. Para tal, realizaram-se ensaios de ELISA utilizando como antigénios que o receptor celular humano CD4 na sua forma solúvel, quer uma construção em que os domínios 1 e 2 do CD4 humano foram colocados em fusão com uma IgG2 humana na qual as regiões variáveis das cadeias leves e das cadeias pesadas foram substituidas pelos domínios 1 e 2 do CD4 humano dando origem a uma proteína de fusão designada CD4-IgG2. Dos ensaios de ELISA foi possivel concluir que ambos os anticorpos VLB12 e VLCD4 se ligam ou ao domínio 1 ou ao domínio 2 do CD4 humano. Para identificar especificamente a qual dos domínios se ligavam o VLB12 e o VLCD4, foram realizadas experiências em que células HEK293T foram transfectadas com CD4 humano (hCD4), CD4 de murganho (mCD4) ou com CD4 humano em que o domínio 1 foi substituido pelo domínio 1 de murganho (hCD4mD1). Estas células transfectadas com os diferentes receptores celulares CD4 foram submetidas a ensaios de Citometria de Fluxo para avaliação da ligação dos anticorpos VLB12 e VLCD4 aos diferentes receptores celulares CD4 e concluiu-se que o VLB12 se liga ao domínio 2 e que o VLCD4 se liga ao domínio v 1 do CD4 humano. Realizaram-se também ensaios de ELISa em que se verificou que os epitopos reconhecidos pelo VLB12 e pelo VLCD4 são ambos conformacionais. A fim de avaliar a capacidade do VLB12 e do VLCD4 na inibição da infecção por VIH-1, foram realizados ensaios de inibição com o clone molecular NL4-3 do VIH-1 (subtipo B) e com os subtipos J e H de isolados primários de VIH-1. Os ensaios de inibição foram realizados em células TZM-bl num ensaio de um ciclo único de infecção. O VLB12 foi capaz de inibir o VIH-1NL4-3 com um IC50 = 3,89 µM e o VLCD4 foi capaz de inibir o HIV-1NL4-3 com um IC50 = 6,57 µM. Realizaram-se igualmente ensaios de inibição com o T20 para o qual se obteve um IC50 = 0,0694 µM.Quanto aos isolados primários, o VLB12 foi capaz de inibir o VIH-1 subtipo H com um IC50 = 3,97 µM e o VIH-1 subtipo J com um IC50 = 3,86 µM, mas o VLCD4 não foi capaz de inibir os subtipo J e H de isolados primários de VIH-1. Para o T20 obteve-se um IC50 = 1,33 nanoM para o subtipo H do VIH-1 e para o subtipo J do VIH-1 obteve-se um IC50 = 0,42 nanoM. Estes resultados indicam que o VLB12 apresenta um amplo potencial de inibição ao contrário do VLCD4 que não foi capaz de inibir qualquer um dos isolados primários dos subtipo não B. Devido à elevada afinidade de ligação ao receptor CD4 apresentada pelo VLB12, uma das abordagens desta Tese teve por objectivo avaliar o potencial de um novo sistema de entrega de moleculas para terapia génica em que se utilizaram nanopartículas revestidas com VLB12 para avaliar a entrega de biomoléculas a células que expressam o receptor CD4. Foram testados dois tipos diferentes de formulações de nanopartículas, de quitosano e de PEI, para a entrega do plasmídeo FUGW-dsRed em células Jurkat. Ambas as formulações de nanopartículas foram bem sucedidos na entrega do plasmídeo FUGW-dsRed às células Jurkat, conforme determinado por imunofluorescência. A formulação de nanopartículas de quitosano provou globalmente ser mais eficaz, devido à reduzida toxicidade celular. A quantidade ideal de VLB12 adsorvido às nanopartículas deu origem a um aumento de 16% da população dsREd positiva vi fluorescente com um direccionamento das nanopartículas para o receptor alvo (CD4) devido à presença do VLB12. Numa abordagem distintadas anteriores, pretendeu-se avaliar as propriedades antigénicas do VLB12 para utilização numa formulação de vacina. Deste modo, a imunidade das mucosas foi testada utilizando o VLB12 encapsulado em nanopartículas de quitosano, por comparação com a via subcutânea, utilizando como adjuvante alumínio e realizando as mesmas formulações sem adjuvante. A resposta de IgG total específico nas amostras de soro foi avaliada por ELISA utilizando como antigénio o VLB12. Foram também avaliadas as respostas Th1 ou Th2 através do racio dos antigénios específicos IgG1-IgG2 nas amostras de soro. Para avaliar o potencial antigénico do VLB12 como vacina, foram realizados ensaios de inibição de VIH-1 usando os soros dos diferentes grupos de murganhos, aos quais foi administrado cada uma das formulações. Os ensaios de inibição foram realizados em células TZM-bl, com uma MOI de 0,1, num ensaio de um ciclo único de infecção. Apesar de os grupos vacinados apresentarem títulos de IgG específico elevados, nenhum dos soros obtidos apresentou efeito inibitório sobre o VIH-1NL4-3. Confirmou-se por ELISA que os soros obtidos não reconheciam a gp120 o que indica que a framework do VL é altamente imunogénica, não tendo sido obtida uma proporção de anticorpos dirigidos contra a região alvo b12 suficiente para se obter um efeito inibitório nos ensaios de inibição do VIH-1NL4-3.
The HIV-1 entry process is mediated by the Env proteins, and begins with the binding of gp120 to the CD4 cellular receptor. A recent discovery has identified in gp120 a conformationally invariant surface that overlaps a distinct subset of the CD4-binding site, which is a binding site for the b12 natural antibody. The goal of this work was to evaluate both the b12 target sequence and the CD4 binding epitope outside of the gp120 context and to evaluate the potential for CD4 binding of these two epitopes individually. This was done through grafting of the b12 target sequence or CD4 binding epitope into either CDR1 or CDR3 of a highly stable VL antibody. The potential of the VLB12 and VLCD4 constructs was tested for CD4 binding by ELISA assay and Flow Citometry analysis; both graftings present binding to the CD4 receptor and VLB12, in particular. In order to evaluate the VLB12 and VLCD4 ability to inhibit HIV-1 infection, inhibition assays were performed with the HIV‐1NL4‐3 subtype B molecular clone and with HIV‐1 subtypes J and H primary isolates. VLB12 was able to inhibit HIV‐1NL4‐3 with an IC50 = 3,89 µM and VLCD4 was able to inhibit HIV‐1NL4‐3 with an IC50 = 6,57 µM. As for the HIV‐1 primary isolates, VLB12 was able to inhibit subtype H with an IC50 = 3,97 µM and subtype J with an IC50 = 3,86 µM but VLCD4 was not able to inhibit either subtype J or subtype H. These results indicate that VLB12 presents potentially a broad inhibition spectrum as opposed to VLCD4 that was not able to inhibit either of the non subtype B primary isolates. Due to the VLB12 high binding affinity to the CD4 receptor the potential of VLB12 coated nanoparticles as a new therapeutic delivery system was evaluated. Chitosan and PEI nanoparticle formulations were tested, for the delivery of FUGW-dsRed plasmid to Jurkat cells. Both nanoparticle formulations were successful in delivery of specific CD4 targeted FUGW-dsRed as determined by immunofluorescence. Chitosan nanoparticle formulation proved to be more effective due to the lower cell toxicity. The optimal amount of VLB12 adsorbed to the nanoparticles gave a 16 % positive dsREd fluorescent population with specific VLB12 targeting. viii To evaluate the vaccine antigen properties, of VLB12 the mucosal immunity was tested by using encapsulated VLB12 in nanoparticles of chitosan in comparison with subcutaneous routes with aluminium adjuvant and without adjuvant. Th1 or Th2 responses were evaluated by the ratio of antigen-specific IgG2a-IgG1 in the serum samples. HIV-1 neutralization assays using serum from different groups to evaluate the VLB12 as potential vaccine antigen were performed but the serum presented no inhibitory effect on HIV-1NL4-3.
Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/28211/2006)

The field of host-retrovirus interactions is complex and wide since multiple cellular proteins can act as positive or negative factors for viral infections or pathogenesis. In this work, we investigated the role of some cellular proteins on HIV-1 infectivity. The first examined factor is HLA-C and its potential role in AIDS progression. The analysis of the HLA-C genotype of 96 HIV-1 positive patients unveils the statistically significant association between some HLA-C variants, less stably bound to β2microglobulin/peptide complex, and a more rapid AIDS progression (p-value = 0.0143). The second considered aspect is the role of cellular proteins on HIV-associated neurocognitive disorders development (HAND). Finding new biomarkers and genetic factors linked to HIV associated neurocognitive disorders was the aim of this second study. The analyzed factors include HLA-C, Apolipoprotein E, β2microglobulin, and Neurofilament Light Chain (NFL). A cohort of 32 patients was selected for this study and the subjects were classified according to their neurocognitive status. HLA-C and Apolipoprotein E genotypes were determined through an Allele Specific PCR approach, while β2microglobulin and Neurofilament Light Chain plasma levels were quantified. Unfortunately, an association between the analyzed genetic factors/biomarkers and HAND development could not be determined due to the poor patient’s recruitment caused by the COVID-19 pandemic. The third analyzed factor is ACOT8, a thioesterase discovered as an HIV-1 Nef protein-interacting partner. To unravel ACOT8 involvement in HIV-1 infectivity pseudotyped viruses were produced using Hek293T wild type or ACOT8 knock-out cell lines. The infection step was performed using TZM-bl wild type or TZM-bl ACOT8 knock-out cell lines. This approach revealed a probable ACOT8 role on virus production and infection that will be further analyzed.

"October, 2004"
Bibliography: leaves 118-154.
xii, 154 leaves : ill. (some col.), plates (col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Sciences, Discipline of Microbiology and Immunology, 2005

Introduction HLA-C plays a crucial role in the progression of HIV-1 infection. Host genetic HLA-C variants, appear to be associated with a different ability to control HIV-1 infection. A higher HLA-C expression is associated with a better activation of cytotoxic T lymphocytes (CTLs) and of Killer Immunoglobulin like receptors (KIR) on NK-cells, which lead to a better HIV-1 infection control. Vice-versa, a lower HLA-C expression leads to a rapid progression toward AIDS. In addition, different HLA-C alleles present different binding stabilities to β2microglobulin (β2m)/peptide. Noteworthy, some HLA-C highly expressed/protective alleles are also stably bound to β2m/peptide, while some low expressed/non-protective variants present an unstable bond to β2m/peptide. Finally virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. Experiments In the present work, it was first characterized the association between HLA-C and HIV-1 Env. We investigated if HIV-1 infection involves HLA-C free chains or the heterotrimeric complex, and to this purpose the A3.01 cell line and its HIV-1-infected counterpart ACH-2, as well as PM1 cells, were used as in vitro infection model. HEK-293T β2m negative cells, generated using CRISPR/Cas9 system, were used to produce HIV-1 pseudoviruses and to test their infectivity. Then, the proportion between HLA-C associated to β2m and HLA-C presents as free chains on the cell surface was characterized on PBMC from healthy donors, bearing both Stable or Unstable HLA-C alleles. In addition, PBMC were tested for their ability to support HIV-1 infection in vitro. Results HIV-1 infection induces the appearance of HLA-C free chains on the surface of infected cells, which may be responsible for the increased HIV-1 infectivity. HIV-1 Env-pseudotyped viruses produced in the absence of β2m, thus lacking HLA-C on their envelope, were less infectious than those produced in the presence of β2m. By analysing PBMC from healthy donors, differences in HLA-C heterotrimers stability and HLA-C expression levels were found. Finally, it was reported that R5 HIV-1 virions produced by PBMC having Unstable HLA-C alleles were more infectious than those produced by PBMC having the Stable variants. Conclusions The outcome of HIV-1 infection might depend both on the HLA-C surface expression levels and on HLA-C/β2m/peptide binding stability. According to this model, PBMC carrying low expressed/Unstable HLA-C alleles have a high proportion of HLA-C free chains on their surface that raises viral infectivity and, at the same time, a low proportion of HLA-C heterotrimeric complexes which leads to a poor control of HIV-1 infection, and thus to a rapid progression toward AIDS.
È noto che alcuni polimorfismi del sistema HLA giocano un ruolo cruciale nell’eziopatogenesi e nella prognosi di numerose malattie infettive, fra le quali l’AIDS (Sindrome da Immunodeficienza Acquisita). Recenti studi hanno evidenziato una forte correlazione fra i livelli di espressione di HLA-C e il controllo della replicazione del virus dell’immunodeficienza umana (HIV-1). Alti livelli di espressione sono stati correlati con un miglior controllo dell’infezione, mentre bassi livelli sono stati associati con una progressione più rapida della malattia. Inoltre, è noto che la molecola HLA-C, presente sull’envelope di HIV-1, in associazione con la glicoproteina Env, è in grado di aumentarne l’infettività. Il ruolo protettivo di alti livelli di espressione di HLA-C sembra essere in contraddizione con il ruolo dell’ HLA-C stesso nell’aumentare l’infettività virale quando incorporato nel virione. Ciò potrebbe essere dovuto alla presenza di diverse conformazioni dell’ HLA-C. È infatti noto che diverse varianti alleliche dell’HLA-C presentano una diversa stabilità di legame con la β2 microglobulina (β2m) e il peptide. In particolare, l’HLA-C può presentarsi associato alla β2m e al peptide, costituendo un complesso che svolge un ruolo chiave nell’attivazione del sistema immunitario, oppure come free chain, dissociato dal complesso. I primi risultati di questo lavoro hanno dimostrato che la proteina Env di HIV-1 è in grado di associarsi all’HLA-C quando presente nella conformazione di free chain. L’ipotesi testata nello studio prevede l’esistenza di un’associazione fra la suscettibilità all’infezione da HIV-1 e le diverse varianti alleliche di HLA-C che possono essere preferibilmente presenti o come complesso trimerico o come free chain. Individui con varianti di HLA-C aventi una forte stabilità come trimero completo mostrerebbero una maggiore immunità contro HIV-1 e una ridotta infettività virale, mentre soggetti con varianti dell’HLA-C che facilmente si dissociano dalla β2m e dal peptide mostrerebbero una ridotta risposta immunitaria nei confronti di HIV-1 e la produzione di virioni maggiormente infettivi. Nel suo complesso, questo studio fornisce nuove informazioni che potrebbero rivelarsi utili per la progettazione di nuove strategie vaccinali e approcci terapeutici contro HIV-1.

Genetic variation plays an important role in determining an individual’s susceptibility to infectious disease. PSIP1 encodes LEDGF/p75, which stably associates with the core domain of HIV-1 integrase via a highly-conserved integrase binding domain (IBD) located in its C-terminal. Through this interaction, the protein tethers HIV-1 IN to chromosomes at sites corresponding to regions of high LEDGF/p75-mediated transcription. Genetic variation within PSIP1 was identified and characterized in black South Africans to establish whether variation in this influences an individual’s susceptibility to HIV infection. PCR assays were designed to amplify regions within the upstream non-coding region, IBD and DNA-binding domains of the gene and selected polymorphisms were then genotyped using allele-specific PCR, RFLP-PCR and Pyrosequencing™ assays. Three insertion-deletion (indel) and eight single nucleotide polymorphisms (SNP) where identified through sequencing. Four of the SNPs had been recorded previously, while the seven other polymorphisms had not and appear to be unique to our population. Differences in allelic and genotypic frequencies where found between the various ethnic groups represented in this study, which were reflected in the underlying haplotype structure within this gene, suggesting that genetic substructure exists within the black South African population. Differences in allele and genotype frequencies were also seen between HIV+ individuals and the general population. Thus variation within PSIP1 may influence an individual’s susceptibility to HIV-1 infectivity and/or rate of disease progression.

Die vorliegende Arbeit thematisiert das computergestützte strukturbasierte Design auf dem Gebiet der HIV-1-Integrase und des Macrophage Infectivity Potentiator (MIP) von Legionella pneumophila. Die durchgeführten Studien geben wertvolle Aufschlüsse über den Wirk-mechanismus einer bekannten Integrase-Inhibitorenklasse and zeigt darüber hinaus einen neuartigen Ansatz zur Integrase-Inhibition auf. Im Falle des MIP-Enzyms konnten zwei niedermolekulare Inhibitoren ermittelt werden. Die Integrase-Studien ergaben wertvolle Informationen im Hinblick auf das Design neuer Inhibitoren. Docking-Experimente konnten die Hypothese weiter untermauern, nach der die Klasse der Diketosäure-Inhibitoren nicht als freie Liganden, sondern als Metallion-Komplexe an das aktive Zentrum der Integrase binden. Die Ergebnisse dieser Studie helfen dabei, das Verständnis über den Wirkmechanismus dieser wichtigen Klasse von Integrase-Inhibitoren weiter zu vertiefen. Um der Entwicklung von Integrase-Inhibitoren einen neuen Impuls zu geben, wurde eine neue Strategie zur Inhibition dargelegt: Anstatt an das aktive Zentrum soll eine neue Inhibitor-Klasse an das Dimerisierungs-Interface eines Integrase-Monomers binden, die katalytisch notwendige Dimerisierung verhindern und somit die enzymatische Aktivität stören. Das Hauptproblem hierbei bestand in den fehlenden Strukturdaten des freien Monomers. Hierzu wurden Molekulardynamik-Simulationen durchgeführt, um nähere strukturelle Informationen zu erhalten. Momentaufnahmen unterschiedlicher Konformationen dienten als Input-Strukturen für eine Docking-Studie mit dem peptidischen Inhibitor YFLLKL, um dessen Bindemodus aufzuklären. Hierbei zeigte sich, dass dieser Ligand an eine Interface-Konformation bindet, die durch eine Y-förmige Bindestelle charakterisiert ist. Im nächsten Schritt sollte diese Protein-Konformation mit kleinen, nicht-peptidischen Molekülen adressiert werden. Die erste Strategie bestand darin, ein Pharmakophor-Modell zu erstellen, das zur Suche nach Molekülen mit einer guten Komplementarität zur Y-förmigen Bindetasche geeignet ist. Das folgende virtuelle Screening ergab zehn Verbindungen, die eine gute Komplementarität und günstige hydrophobe Wechselwirkungen aufwiesen. Leider zeigte keine der Verbindungen eine reproduzierbare Aktivität im Integrase-Assay. Hierbei verbleiben jedoch gewisse Zweifel, da in dem Assay die Zugabe von BSA vorgeschrieben war, das möglicherweise die hydrophoben Inhibitor-Kandidaten gebunden hat. Die erwähnte erste Strategie wurde überdacht: In einem zweiten Ansatz galt die Hauptaufmerksamkeit der Absättigung von wasserstoffbrückenbildenden Resten. Diese waren zuvor von den eher hydrophoben Verbindungen nicht optimal abgesättigt worden. Zwei Pharmakophor-Modelle wurden erstellt und in einem virtuellen Screening eingesetzt: Docking-Studien der Hits zeigten jedoch, dass nach wie vor viele wasserstoffbrückenbildende Reste des Proteins nicht vom Liganden abgesättigt wurden. Nach abschließender eingehender Betrachtung der Bindemoden der verbliebenen Moleküle aus dem virtuellen Screening konnten nur acht für weitere Testungen ausgewählt werden (Ergebnisse der experimentellen Testung durch Kooperationspartner stehen noch aus). Diese geringe „Ausbeute“ an geeigneten Verbindungen für das Integrase-Dimerisierungsinterface zeigt, wie schwer dieses Target zu adressieren ist: Das Interface weist eine schnell wechselnde Abfolge von basischen, sauren und hydrophoben Resten auf. Im Gegensatz zu anderen Protein-Protein-Interfaces zeigt das Integrase-Interface keine „aufgeräumte“ Bindetasche mit klar voneinander getrennten hydrophoben und hydrophilen Bereichen. Für das zweite Enzym, MIP, konnten mit Hilfe des strukturbasierten Designs zwei niedermolekulare Inhibitoren gefunden werden. Beide Verbindungen führten zu einer deutlichen Abnahme der katalytischen Aktivität. Soweit bekannt, sind bisher keinerlei niedermolekulare MIP-Inhibitoren veröffentlicht. Der Vergleich von MIP mit der humanen PPIase FKBP12 zeigte eine größtenteils ähnliche Tasche, die jedoch einen entscheidenden Unterschied aufweist, nämlich in der Orientierung des Restes Tyr109. Die detaillierte Betrachtung der Strukturdaten beider Enzyme konnte schließlich eine Erklärung liefern, warum ein ketoacyl-substituiertes Pipecolinderivat nicht an MIP bindet, ein sulfonsubstituiertes Pipecolinderivat hingegen das Enzym inhibiert. Die Erkenntnisse über das Inhibitoren-Design für Legionella-MIP können auch auf andere Organismen (z.B. Trypanosomen) übertragen werden, bei denen ebenfalls (homologes) MIP ein Pathogenitätsfaktor ist
In this thesis, computational structure-based design approaches were employed to target the HIV-1 integrase and the macrophage infectivity potentiator (MIP) of Legionella pneumophila. The thesis yields valuable information about the mechanism of action of a known class of integrase inhibitors and a novel approach towards enzyme inhibition, which still is mainly unaddressed in current integrase research. For the MIP enzyme, two small-molecule MIP inhibitors were discovered. The computational studies of HIV-1 integrase have provided valuable information for IN inhibitor design. Docking experiments supported the hypothesis that the well-known diketo acid inhibitors enter the IN active site not as free ligands, but rather as metal complexes. These results help to reveal the mechanism of action of this important class of IN inhibitors.To give an impulse for the development of a novel class of inhibitors, a new strategy towards IN inhibition was introduced: An alternative binding site, the dimerization interface of an IN catalytic core domain monomer, was explored for inhibitor design. The lack of structural data of the free monomer was overcome by extensive MD studies. Snapshots derived from the MD simulation were used as protein input structures in a docking study with the inhibitory peptide YFLLKL to reveal its potential binding mode. The docking procedure showed that the peptidic ligand binds to a dimerization interface conformation which shows a Y-shaped binding site.. The next step was to address this protein conformation with small, non-peptidic molecules. The first strategy towards finding small-molecule interface binders was to create a pharmacophore model with hydrophobic features and shape constraints, aiming to find molecules with a good complementarity to the Y-shaped dimerization interface. Virtual screening yielded a total of 10 compounds, which all displayed good shape complementarity and favorable hydrophobic interactions. Unfortunately, none of the compounds showed a reproducible inhibitory activity in biological assays. Some doubts remain about the validity of the assay results: The use of BSA was critical, since it is not unlikely that BSA “intercepted” the hydrophobic candidate compounds. The first strategy towards finding small-molecule dimerization inhibitors was reconsidered: In the second approach, the satisfaction of hydrogen bonding residues at the dimerization interface, was of major interest. Two pharmacophore models were employed, which retrieved several hundred hit molecules. However, docking of these molecules showed that still many hydrogen bonding groups of the protein remained unaddressed by the ligands. Eventually, after visual inspection, only eight molecules were selected as candidate compounds for further testing (results pending). This small “yield” underlines the difficulties in finding interface binders: The IN dimerization interface is a peculiar target with frequently alternating basic, acidic, and hydrophobic residues. It is not a well-ordered binding site with continuous hydrophobic areas and distinct hydrogen bond donors / acceptors. Other protein-protein interfaces show such well-ordered binding sites. Accordingly, the peculiarity of the IN dimerization interface, in addition to the delicate task of disrupting protein-protein interactions at all, makes the development of IN dimerization inhibitors very challenging. For MIP, the studies revealed two experimentally validated MIP inhibitors, which significantly reduce MIP enzymatic activity. To our knowledge, no small-molecule MIP inhibitor has been reported in the literature so far. A detailed analysis of the available structural data of MIP and a comparison to the human PPIase counterpart, FKBP12, pointed out a conformational diversity among the MIP structures and a crucial difference between the two PPIases, which could be traced to mainly one residue (Tyr109). The detailed comparison of FKBP12 and MIP complex structures made it possible to give an explanation, why a ketoacyl-substituted pipecoline derivative most probably does not bind to MIP, but a sulfone-substituted pipecoline derivative does bind to MIP. Knowledge of Legionella MIP inhibitors could be transferred also to other organisms (e.g. trypanosoms), where homologous MIP proteins are also pathological factors

Le VIH-1 a développé plusieurs mécanismes menant à la dégradation de son récepteur cellulaire, la molécule CD4, dans le but d’augmenter la relâche de particules virales infectieuses et d’éviter que la cellule soit surinfectée. L’un de ces mécanismes est la dégradation, induite par la protéine virale Vpu, du CD4 nouvellement synthétisé au niveau du réticulum endoplasmique (RE). Vpu doit lier CD4 et recruter l’ubiquitine ligase cellulaire SCFβ-TrCP, via sa liaison à β-TrCP, afin de dégrader CD4. Puisque CD4 doit être retenu au RE pour permettre à Vpu d’induire sa dégradation via le système ubiquitine-protéasome, il a été suggéré que ce processus implique un mécanisme semblable à une voie cellulaire de dégradation des protéines mal-repliées appelée ERAD (« endoplasmic reticulum-associated degradation »). La dégradation par ERAD implique généralement la dislocation des protéines du RE vers le cytoplasme afin de permettre leur poly-ubiquitination et leur dégradation par le protéasome. Nous avons démontré que Vpu induit la poly-ubiquitination de CD4 dans des cellules humaines. Nos résultats suggèrent aussi que CD4 doit subir une dislocation afin d’être dégradé par le protéasome en présence de Vpu. De plus, un mutant transdominant négatif de l’ATPase p97, qui est impliquée dans la dislocation des substrats ERAD, inhibe complètement la dégradation de CD4 par Vpu. Enfin, nos résultats ont montré que l’ubiquitination sur des résidus accepteurs de l’ubiquitine (lysines) de la queue cytoplasmique de CD4 n’était pas essentielle, mais que la mutation des lysines ralentit le processus de dégradation de CD4. Ce résultat suggère que l’ubiquitination de la queue cytosolique de CD4 pourrait représenter un événement important dans le processus de dégradation induit par Vpu. L’attachement de l’ubiquitine a généralement lieu sur les lysines de la protéine ciblée. Toutefois, l’ubiquitination sur des résidus non-lysine (sérine, thréonine et cystéine) a aussi été démontrée. Nous avons démontré que la mutation de tous les sites potentiels d’ubiquitination cytoplasmiques de CD4 (K, C, S et T) inhibe la dégradation par Vpu. De plus, la présence de cystéines dans la queue cytoplasmique apparaît suffisante pour rendre CD4 sensible à Vpu en absence de lysine, sérine et thréonine. Afin d’expliquer ces résultats, nous proposons un modèle dans lequel l’ubiquitination de la queue cytosolique de CD4 serait nécessaire à sa dégradation et où les sites d’ubiquitination de CD4 seraient sélectionnés de façon non spécifique par l’ubiquitine ligase recrutée par Vpu. Enfin, nous avons observé que la co-expression d’une protéine Vpu incapable de recruter β-TrCP (Vpu S52,56/D) semble stabiliser le CD4 qui est retenu au RE. De plus, d’autres mutants de Vpu qui semblent capables de recruter β-TrCP et CD4 sont toutefois incapables d’induire sa dégradation. Ces résultats suggèrent que l’association de Vpu à CD4 et β-TrCP est essentielle mais pas suffisante pour induire la dégradation de CD4. Par conséquent, ces résultats soulèvent la possibilité que Vpu puisse recruter d’autres facteurs cellulaires pour induire la dégradation de CD4. Les résultats présentés ont permis de mieux définir le mécanisme de dégradation de CD4 par Vpu dans des cellules humaines. De plus, ces résultats nous ont permis d’élaborer un modèle dans lequel l’ubiquitine ligase cellulaire SCFβ-TrCP démontre de la flexibilité dans le choix des résidus à ubiquitiner afin d’induire la dégradation de CD4. Enfin, ces études jettent un oeil nouveau sur le rôle de Vpu dans ce processus puisque nos résultats suggèrent que Vpu doive recruter d’autres partenaires cellulaires, mis à part β-TrCP, pour induire la dégradation de CD4.
HIV-1 has developed many mechanisms leading to the down-regulation of its cellular receptor, the CD4 molecule, in order to increase the release of infectious viral particles and to inhibit superinfection of the target cell. One of these mechanisms is the HIV-1 Vpu-mediated degradation of newly synthesized CD4 at the level of endoplasmic reticulum (ER). Vpu must interact with CD4 and recruit the cellular ubiquitin ligase SCFβ-TrCP, via its binding to β-TrCP, in order to induce CD4 degradation. Because CD4 has to be retained in the ER to allow Vpu to induce its degradation via the ubiquitin-proteasome system, it has been suggested that this process involves a mechanism reminiscent of a cellular degradation pathway involved in the proteolysis of unfolded proteins called ERAD (endoplasmic reticulum-associated degradation). The ERAD degradation usually involves the dislocation of proteins from the ER to the cytoplasm in order to induce their poly-ubiquitination and subsequent degradation by the proteasome. We demonstrated that Vpu induces the poly-ubiquitination of CD4 in human cells. Our results also suggest that CD4 has to be dislocated in order to be degraded by the proteasome in presence of Vpu. Furthermore, the expression of a transdominant negative mutant of the ATPase p97, that is involved in the dislocation of ERAD substrates, inhibits completely the Vpu-mediated CD4 degradation process. Finally, our results demonstrated that the ubiquitination of putative ubiquitin acceptor residues (lysines) in the cytosolic tail of CD4 is not essential but the mutation of these lysines slowed down the process of CD4 degradation induced by Vpu. This results suggests that ubiquitination of CD4 cytosolic tail could represent an important step during Vpu-mediated CD4 degradation. Ubiquitin is usually attached on lysine residues in the targetted protein. However, the ubiquitination on non-lysine residues (S, T and C) has also been demonstrated. We demonstrated that the mutation of all cytosolic potential ubiquitination sites (K, C, S and T) of CD4 abolishes Vpu-mediated degradation. In addition, the presence of cysteines in the cytosolic tail of CD4 appeared sufficient to render CD4 sensitive to Vpu in absence of lysine, serine or threonine. In order to explain these results, we propose a model in which CD4 cytosolic tail ubiquitination is necessary for its degradation and where ubiquitination sites are selected non specifically by the ubiquitin ligase recruited by Vpu. Finally, we observed that co-expression of a phosphorylation mutant of Vpu unable to interact with β-TrCP (Vpu S52,56/D) appears to stabilize ER-retained CD4 molecules. In addition, other Vpu mutants seem able to recruit β-TrCP and CD4 without inducing CD4 degradation. These results suggest that Vpu association with CD4 and β-TrCP is essential but not sufficient for CD4 degradation. Consequently, these results raised the possibility that other cellular factors could be recruited by Vpu in order to induce CD4 degradation. The results presented here allowed us to better define the mechanism underlying Vpu-mediated CD4 degradation. In addition, these results allowed us to elaborate a model in which the ubiquitin ligase SCFβ-TrCP show some flexibility in the choice of ubiquitination sites in order to induce CD4 degradation. Finally, theses studies shed a new light on the role of Vpu in the CD4 degradation process because our results suggest that Vpu could recruit, in addition of β-TrCP, other cellular partners in order to induce CD4 degradation.

HIV-1, per compiere il suo ciclo infettivo, si serve dell’associazione con numerose proteine cellulari. In questo lavoro mi sono occupata di studiare l’interazione tra proteine di HIV-1 e proteine cellulari, in particolare tra Env e HLA-C e tra Nef e la tioesterasi umana (ACOT8). Studi precedenti hanno dimostrato come la presenza di HLA-C sui virioni di HIV-1 ne aumenti l’infettività; è stata inoltre riportata una correlazione tra i livelli di espressione di HLA-C e la replicazione virale. In questo studio abbiamo dimostrato che la presenza di HIV-1 aumenta la quantità di molecole HLA-C open conformer (non associate alla β2-microglobulina) sulla membrana di cellule infettate; che la proteina Env è in grado di ripristinare la presenza di HLA-C sulla superficie di cellule negative per la β2m; che HLA-C associa alternativamente o con la β2m o con Env; che Env può sostituire la β2m nel trasporto di molecole HLA-C open conformer sulla membrana cellulare; che pseudovirus di HIV-1 sono meno infettivi in presenza di β2m, suggerendo un meccanismo competitivo tra Env e β2m per il legame a HLA-C. In nostri dati suggeriscono quindi che l’aumento di infettività di HIV-1 in presenza di HLA-C è strettamente legato alla disponibilità di molecole open conformer, dimostrando come la stabilità del legame tra HLA-C e laβ2m sia un fattore discriminante per l’associazione con Env. Pertanto le differenze osservate tra i livelli di espressione in membrana tra i diversi alleli di HLA-C può essere dovuta ad una diversa affinità per il legame alla β2m. Di conseguenza la maggiore o minore stabilità di associazione con la β2m o con Env, può conferire alle diverse varianti alleliche di HLA-C una propensione per agire come molecole immuno-competenti o come molecole accessorie per HIV-1. La proteina Nef di HIV-1 interagisce con diverse proteine cellulari, tra cui ACOT8. Tale interazione può modulare la composizione dei raft lipidici durante l’infezione virale. Attualmente le regioni di Nef coinvolte nell’associazione con ACOT8 sono state identificate, ma, a causa della mancanza di dati strutturali per ACOT8, non vi è alcuna informazione riguardante le regioni di ACOT8 importanti per l’interazione con Nef. In questo lavoro, mediante l’utilizzo di metodi di predizione bioinformatica, è stata modellizzata la struttura di ACOT8 e, attraverso procedure di docking, è stata riscontrata un’alta complementarietà di carica tra le superfici di Nef e ACOT8, che ha permesso l’identificazione degli aminoacidi di ACOT8 possibilmente coinvolti nell’interazione. Tali dati sono poi stati validati attraverso esperimenti in vitro. Sono stati preparati diversi mutanti di delezione per ACOT8 e, mediante immunofluorescenza e saggi di co-immunoprecipitazione, abbiamo dimostrato come la mutazione p∆PK (K91S) sia sufficiente per compromettere l’associazione con Nef, suggerendo il coinvolgimento della Lys91 in tale interazione. Inoltre anche le regioni ∆PAK (R45-F55) e ∆PK (R86-P93) sono risultate essere importanti per il legame a Nef, indicando che più aminoacidi possono essere coinvolti nell’associazione tra le due proteine. Al contrario, in seguito alla delezione p∆PAK (K52), l’interazione con Nef è stata mantenuta, indicando come tale aminoacido non sia invece coinvolto. Tali osservazioni aprono la strada per il possibile sviluppo di molecole inibitorie volte a ridurre la funzionalità di ACOT8 e quindi comprometterne l’interazione con Nef, riducendo così l’infettività di HIV-1.
HIV-1 relies on several host cell proteins to carry out its infective cycle. In this work I investigated the interactions between HIV-1 proteins and human proteins, in particular between Env and HLA-C and between Nef and the thioesterase 8 (ACOT8). HLA-C presence on HIV-1 virions increases viral infectivity; in addition a correlation between HLA-C expression levels and HIV-1 replication control has been reported. In this study we demonstrated that: HIV-1 presence increases the amount of HLA-C open conformers (not bound to β2m) on infected cells membrane; HIV-1 Env relocates HLA-C on the cell surface of β2m-negative cells; HLA-C alternatively associates with Env or with β2m; Env replaces β2m in the transport of HLA-C open conformers to the cell surface; HIV-1 pseudoviruses are less infectious in the presence of β2m, suggesting a competitive mechanism in HLA-C binding to Env or β2m. Taken together, our data suggest that the enhanced infectivity conferred to HIV-1 by HLA-C specifically involves HLA-C molecules as open conformers. In addition this work draws attention to HLA-C stability as a discriminatory factor for the association with Env. Differences observed between HLA-C alleles may be the consequence of different binding affinities to β2m, which may account for distinct surface expression levels of HLA-C allelic variants. Differences in binding affinity to β2m or to alternative ligands such as HIV-1 Env may confer to allelic variants of HLA-C the ability to preferentially act either as a conventional immune-competent molecule or as an accessory molecule involved in HIV-1 infectivity. HIV-1 Nef interacts with several cellular proteins, among which ACOT8. This interaction may modulate lipid composition in membrane rafts during HIV-1 infection. Currently, the regions involved in the interaction have been experimentally characterized on Nef but not on ACOT8. The lack of structural information for ACOT8 hampers a deep characterization of the putative interaction regions. In this work we modelled, through in silico predictions, the ACOT8 structure in order to identify the aminoacids putatively involved in the interaction with Nef. Our data demonstrated a high charge complementarity between Nef and ACOT8 surface, which allowed the identification of the ACOT8 putative contact points involved in the interaction. The predictions were then validated by in vitro assays. Several ACOT8 deletion mutants were prepared. Through immunofluorescence and co-immunoprecipitation assays we demonstrated that the ACOT8 p∆PK mutation (K91S) is sufficient to abrogate the association with Nef, indicating that K91 plays a fundamental role for this interaction. In addition also the ACOT8 ∆PAK deletion (R45- F55), as well as the ∆PK deletion (R86-P93) resulted to be determinant for the Nef association, suggesting that other residues might be involved. No effects on Nef binding were observed upon ACOT8 p∆PAK deletion (K52) indicating that this aminoacid is not significantly involved in the interaction. Our findings open the way to further investigations for the designing of new inhibitory molecules that interfere with the Nef activity and thus reduce HIV-1 infectivity.