Literatura académica sobre el tema "NS4B protein"

ABSTRACTFlavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimatedKd(dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach.IMPORTANCEFlavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cells, in cells transiently expressing NS4A and NS4B proteins, orin vitrowith recombinant NS4A and NS4B proteins. We mapped the minimal regions required for the NS4A-NS4B interaction to be amino acids 40 to 76 of NS4A and amino acids 84 to 146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17 to 80 of NS4A and the NS4A amino acids that may participate in the NS4A-NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A-NS4B interaction, demonstrating the biological importance of the NS4A-NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of the NS4A-NS4B interaction could be pursued for flavivirus antiviral development.

ABSTRACT Flaviviruses are insect-borne, positive-strand RNA viruses that have been disseminated worldwide. Their genome is translated into a polyprotein, which is subsequently cleaved by a combination of viral and host proteases to produce three structural proteins and seven nonstructural proteins. The nonstructural protein NS4B of dengue 2 virus partially blocks activation of STAT1 and interferon-stimulated response element (ISRE) promoters in cells stimulated with interferon (IFN). We have found that this function of NS4B is conserved in West Nile and yellow fever viruses. Deletion analysis shows that that the first 125 amino acids of dengue virus NS4B are sufficient for inhibition of alpha/beta IFN (IFN-α/β) signaling. The cleavable signal peptide at the N terminus of NS4B, a peptide with a molecular weight of 2,000, is required for IFN antagonism but can be replaced by an unrelated signal peptide. Coexpression of dengue virus NS4A and NS4B together results in enhanced inhibition of ISRE promoter activation in response to IFN-α/β. In contrast, expression of the precursor NS4A/B fusion protein does not cause an inhibition of IFN signaling unless this product is cleaved by the viral peptidase NS2B/NS3, indicating that proper viral polyprotein processing is required for anti-interferon function.

ABSTRACT The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a “membranous web” (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.

ABSTRACT A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 4B (NS4B), a poorly characterized integral membrane protein, is thought to function as a scaffold for replication complex assembly; however, functional interactions with the other HCV nonstructural proteins within this complex have not been defined. We report that a Con1 chimeric subgenomic replicon containing the NS4B gene from the closely related H77 isolate is defective for RNA replication in a transient assay, suggesting that H77 NS4B is unable to productively interact with the Con1 replication machinery. The H77 NS4B sequences that proved detrimental for Con1 RNA replication resided in the predicted N- and C-terminal cytoplasmic domains as well as the central transmembrane region. Selection for Con1 derivatives that could utilize the entire H77 NS4B or hybrid Con1-H77 NS4B proteins yielded mutants containing single amino acid substitutions in NS3 and NS4A. The second-site mutations in NS3 partially restored the replication of Con1 chimeras containing the N-terminal or transmembrane domains of H77 NS4B. In contrast, the deleterious H77-specific sequences in the C terminus of NS4B, which mapped to a cluster of four amino acids, were completely suppressed by second-site substitutions in NS3. Collectively, these results provide the first evidence for a genetic interaction between NS4B and NS3 important for productive HCV RNA replication.

The aim of the study was to investigate immunogenic properties of mosaic recombinant proteins constructed on the data of hepatitis C virus NS4A and NS4B antigens. Four mosaic recombinant proteins, containing the T and B epitopes of the NS4A and NS4B antigens, were created by genetic engineering methods in the E. coli system. To enhance the immune response they were linked in different variations to the nucleotide sequences of murine interleukin-2 (IL-2), the Neisseria meningiditis lipopeptide, and the T helper epitope of the core protein of hepatitis C virus. The immunogenic properties of these recombinant proteins were analyzed by immunoblotting, ELISA and ELISpot using sera from immunized mice and patients infected with hepatitis C virus. Recombinant proteins specifically reacted with the sera of immunized mice and infected patients in immunoblotting. According to the ELISA data, the predominant formation of antibodies to NS4B was observed when mice were immunized with the recombinant proteins containing both antigens. Analysis of gamma-interferon production by T-lymphocytes upon contact with activated dendritic cells showed in ELISpot that the maximum production of this cytokine was detected when adjuvant components were located at the N- and C-ends of the recombinant protein. The highest level of gamma-interferon production during stimulation with this drug was detected in lymphocytes from the bone marrow and lymph nodes. The recombinant protein containing the T and B epitopes of NS4A and NS4B, murine IL-2 and the lipopeptide Neisseria meningiditis had the greatest immunostimulate effect among the four constructions. This recombinant protein formed nanoparticles of 100-120 nm in size.

ABSTRACT Like most positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits endoplasmic reticulum (ER)-to-Golgi protein traffic (K. V. Konan, T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard, J. Virol. 77:7843-7855). However, both NS4AB and NS4B can induce “membranous web” formation, first reported by Egger et al. (D. B Egger, R. Gosert, L. Bianchi, H. E. Blum, D. Moradpour, and K. Bienz, J. Virol. 76:5974-5984), which is also observed in HCV-infected cells (Y. Rouille, F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson, J. Virol. 80:2832-2841) and cells that bear a subgenomic NS5A-green fluorescent protein (GFP) replicon (D. Moradpour, M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice, J. Virol. 78:7400-7409). To determine the intracellular origin of the web, we examined NS4B colocalization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that, in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein NS4B. Furthermore, an immunoisolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild-type and constitutively active Rab5 proteins were associated with NS4B. Interestingly, expression of dominant-negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 small interfering RNAs into NS5A-GFP replicon cells resulted in a significant decrease in GFP fluorescence. Finally, Rab5 protein was found to coimmunoprecipitate with HCV NS4B. These studies suggest that EE proteins, including Rab5, may play a role in HCV genome replication or web formation.

Infections with viruses in the genus Flavivirus are a worldwide public health problem. These enveloped, positive sense single stranded RNA viruses use a small complement of only 10 encoded proteins and the RNA genome itself to remodel host cells to achieve conditions favoring viral replication. A consequence of the limited viral armamentarium is that each protein exerts multiple cellular effects, in addition to any direct role in viral replication. The viruses encode four non-structural (NS) small transmembrane proteins (NS2A, NS2B, NS4A and NS4B) which collectively remain rather poorly characterized. NS4A is a 16kDa membrane associated protein and recent studies have shown that this protein plays multiple roles, including in membrane remodeling, antagonism of the host cell interferon response, and in the induction of autophagy, in addition to playing a role in viral replication. Perhaps most importantly, NS4A has been implicated as playing a critical role in fetal developmental defects seen as a consequence of Zika virus infection during pregnancy. This review provides a comprehensive overview of the multiple roles of this small but pivotal protein in mediating the pathobiology of flaviviral infections.

The hepatitis C virus (HCV) NS3 protein contains a serine proteinase domain implicated in the maturation of the viral polyprotein. NS3 forms a stable heterodimer with NS4A, a viral memebrane protein that acts as an activator of the IMS3 proteinase. The three-dimensional structure of the NS3 proteinase complexed with an NS4A-derived peptide has been determined. The NS3 proteinase adopts a chymotrypsin-like fold. A β-strand contributed by NS4A is clamped between two β-strands within the N terminus of NS3. Consistent with the requirement for extraordinarily long peptide substrates (P6-P4’), the structure of the NS3 proteinase reveals a very long, solvent-exposed substrate-binding site. The primary specificity pocket of the enzyme is shallow and closed at its bottm by Phe-154, explaining the preference of the NS3 proteinase for cysteine residues in the substrate P, position. Another important feature of the NS3 proteinase is the presence of a tetrahedral zinc-binding site formed by residues Cys-97, Cys-99, Cys-145 and His-149. The zinc-binding site has a role in maintaining the structural stability and guiding the folding of the NS3 serine proteinase domain. Inhibition of the NS3 proteinase activity is regarded as a promising strategy to control the disease caused by HCV. Remarkably, the NS3 proteinase is susceptible to inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A/NS4B, NS4B/NS5A and NS5A/NS5B cleavage sites. The Ki values of the inhibitory products are lower than the Km values of the respective substrates and follow the order NS4A<NS5A<NS4B. Starting from the observation that the NS3 proteinase undergoes product inhibition, very potent, active site-directed inhibitors have been generated using a combinatorial peptide chemistry approach.

AbstractThe need to identify anti-Flaviviridaeagents has resulted in intensive biochemical study of recombinant nonstructural (NS) viral proteases; however, experimentation on viral protease-associated replication complexes in host cells is extremely challenging and therefore limited. It remains to be determined if membrane anchoring and/or association to replicase-membrane complexes of proteases, such as hepatitis C virus (HCV) NS3-4A, plays a regulatory role in the substrate selectivity of the protease. In this study, we examined trans-endoproteolytic cleavage activities of membrane-anchored and replicase-associated NS3-4A using an internally consistent set of membrane-anchored protein substrates mimicking all known HCV NS3-4A polyprotein cleavage sequences. Interestingly, we detected cleavage of substrates encoding for the NS4B/NS5A and NS5A/NS5B junctions, but not for the NS3/NS4A and NS4A/NS4B substrates. This stringent substrate recognition profile was also observed for the replicase-associated NS3-4A and is not genotype-specific. Our study also reveals that ER-anchoring of the substrate is critical for its cleavage by NS3-4A. Importantly, we demonstrate that in HCV-infected cells, the NS4B/NS5A substrate was cleaved efficiently. The unique ability of our membrane-anchored substrates to detect NS3-4A activity alone, in replication complexes, or within the course of infection, shows them to be powerful tools for drug discovery and for the study of HCV biology.

Dengue viruses (DENVs) are mosquito-borne flaviviruses, which are responsible for a wide range of illnesses, from a mild febrile illness to the more serious dengue haemorrhagic fever and dengue shock syndromes. The DENV NS4B is a highly hydrophobic integral ER membrane protein that has been reported to perturb type I interferon (IFN) signalling and has emerged as a major target for anti-viral strategies. The objective of this study was to determine the mechanism of action of two consecutive nucleotide mutations in the NS4B gene (nt 7020 and 7021) that result in the substitution of threonine 66 with alanine, which allow the virus to replicate persistently in mammalian and mosquito cells. Initially, a transient gene expression system was developed to analyse the effect of the mutations on the localisation of NS4B and its ability to perturb the IFN response and the unfolded protein response. The mutations were not found to significantly affect any of these processes. Novel cell lines were then produced that stably expressed the NS4B gene and its mutant counterpart in an inducible fashion. The cell lines were used to identify cellular proteins (DNAJA2, PFN2, FAF2, BAX and PMSG1) that potentially interact with the NS4B proteins using a high-throughput proteomic approach. Finally, HEK293T cell lines that were either stably persistently infected with a virus containing the NS4B mutations or contained a wild type DENV replicon or a replicon containing the NS4B mutations were established. Genome sequencing demonstrated that the NS4B mutations were more stable in a replicon than in the full viral genome. The effect of the NS4B mutations on host cell processes was investigated by transcriptomic analysis using RNAseq. The transcriptomic analysis revealed that canonical cellular pathways were affected by viral replication including IFN signaling, virus recognition, ubiquitination, apoptosis, and ER stress. Further analysis showed that the NS4B mutations affected the ability of the virus and a DENV replicon to effectively suppress IFN signaling and IFN sensitive gene expression. This study provides further insight into the role of the NS4B protein in the viral-host interaction and provides a mechanism to explain how a persistent DENV infection may occur.

Le virus Zika (ZIKV), historiquement limité à l'Afrique et à l'Asie, est devenu une préoccupation mondiale majeure, surtout après les épidémies récentes dans les Amériques associées à des malformations congénitales graves et des troubles neurologiques. Bien que la recherche se soit principalement concentrée sur le génotype asiatique/américain, des preuves croissantes montrent que les souches africaines du ZIKV pourraient également représenter une menace sévère, notamment en termes de pathogénicité fœtale. Cette thèse vise à améliorer notre compréhension des mécanismes moléculaires de la pathogénicité des souches contemporaines de ZIKV d'Afrique de l'Ouest, en mettant l'accent sur les protéines non structurales dans la réplication virale, l'évasion immunitaire et la réponse au stress des cellules hôtes.Pour cela, nous avons généré un clone moléculaire infectieux, GUINEA-18, à partir d'une souche de ZIKV (ZIKV-15555) isolée en Guinée en 2018, représentant une souche contemporaine de la lignée africaine. Nous avons comparé ce clone avec le clone bien caractérisé de la souche historique MR766 (MR766MC). Les propriétés de réplication des deux clones ont été examinées dans les lignées cellulaires VeroE6, A549 et HCM3. GUINEA-18 a montré un taux de réplication plus lent, une cytotoxicité réduite et une moindre capacité à activer le système immunitaire inné comparé à MR766MC, suggérant une interaction différente avec les cellules hôtes.Pour explorer ces différences, nous avons construit des virus chimériques en échangeant les régions codantes des protéines non structurales entre GUINEA-18 et MR766MC. Nos résultats ont souligné les rôles critiques des protéines NS1 à NS4B dans la réplication et la pathogénicité, avec NS4B étant clé pour GUINEA-18. Nous avons également découvert que GUINEA-18 inhibe efficacement l'assemblage des granules de stress cytoplasmiques (SGs) dans les cellules A549, un mécanisme de défense cellulaire. Cette inhibition dépend des protéines NS1 à NS4B, soulignant leur rôle dans l'évasion des défenses de l'hôte.La thèse examine aussi le rôle de NS1 dans la pathogénicité des souches contemporaines. L'alignement des séquences protéiques a révélé sept substitutions dans la protéine NS1 de GUINEA-18 par rapport à celle de MR766. Ces mutations montrent que NS1CWA est sécrétée plus efficacement et présente une localisation subcellulaire différente de NS1MR766, augmentant la réplication virale et la cytotoxicité tout en réduisant l'activation des réponses immunitaires. Un virus chimérique MR766 avec NS1CWA a montré ces traits pathogéniques renforcés, soulignant l'importance de NS1 dans la virulence des souches contemporaines du ZIKV d'Afrique de l'Ouest.En conclusion, cette thèse analyse les déterminants moléculaires de la réplication et de la pathogénicité des souches contemporaines du ZIKV d'Afrique de l'Ouest. Les recherches mettent en évidence les rôles critiques des protéines NS1 à NS4B, en particulier NS1 et NS4B. Les résultats soulèvent des questions sur les risques associés aux souches actuelles du ZIKV en Afrique subsaharienne et soulignent la nécessité de surveillance continue pour comprendre les implications pour la santé publique. Ce travail offre des perspectives pour les stratégies de gestion et de prévention des maladies associées au ZIKV, surtout dans les régions où la lignée africaine est prévalente
The Zika virus (ZIKV), historically confined to Africa and Asia, has become a significant global health concern, especially after recent outbreaks in the Americas linked to severe congenital malformations and neurological disorders. While much research has focused on the Asian/American ZIKV genotype, evidence suggests that African ZIKV strains might also pose a serious threat to public health, particularly regarding fetal pathogenicity. This thesis aims to enhance our understanding of the molecular mechanisms underlying the pathogenicity of contemporary ZIKV strains from West Africa, focusing on nonstructural proteins involved in viral replication, immune evasion, and the host cell stress response.To achieve this, we generated an infectious molecular clone, GUINEA-18, from a ZIKV strain (ZIKV-15555) isolated in Guinea in 2018. This clone represents a contemporary African ZIKV strain. We compared it with the infectious molecular clone of the historical African ZIKV strain MR766, designated MR766MC. The replication properties of both viral clones were examined in VeroE6, A549, and HCM3 cells. GUINEA-18 exhibited a slower replication rate, reduced cytotoxicity, and a lower ability to activate the host’s innate immune system compared to MR766MC, suggesting different interactions with host cells.To dissect these differences, we created chimeric viruses by swapping nonstructural protein-coding regions between GUINEA-18 and MR766MC. Results highlighted the critical roles of NS1 to NS4B proteins in replication efficiency and pathogenicity, with NS4B being crucial for GUINEA-18’s replication properties. GUINEA-18 also developed an efficient mechanism to inhibit the assembly of cytoplasmic stress granules (SGs) in A549 cells, a defense mechanism typically triggered by viral infection. The ability of GUINEA-18 to block SG formation depended on the NS1 to NS4B proteins, underscoring their role in evading host defenses.Further investigation into the NS1 protein revealed seven amino acid substitutions in GUINEA-18 compared to MR766. Functional analyses showed that the contemporary NS1 protein (NS1CWA) is secreted more efficiently and has a different subcellular localization than NS1 from MR766 (NS1MR766). This altered behavior of NS1CWA significantly enhances viral replication and cytotoxicity while reducing the activation of innate immune responses in infected cells. A chimeric MR766 virus containing NS1CWA demonstrated these enhanced traits, emphasizing NS1’s role in the virulence of contemporary West African ZIKV strains.In conclusion, this thesis provides a comprehensive analysis of the molecular determinants of replication and pathogenicity in contemporary West African ZIKV strains. The research underscores the crucial roles of NS1 to NS4B proteins, particularly NS1 and NS4B, in these processes. The findings raise questions about risks associated with circulating ZIKV strains in sub-Saharan Africa and highlight the need for ongoing surveillance and research to understand the public health implications. This work contributes valuable insights that could inform future strategies for managing and preventing ZIKV-associated diseases, especially in regions where the African lineage of the virus is prevalent

African horse sickness is an equid disease caused by African horse sickness virus (AHSV). AHSV produces seven structural proteins that form the virion and four non-structural proteins with various roles during replication. The first part of this study investigated the intracellular distribution and co-localisations of NS1 with other AHSV proteins to facilitate its eventual functional characterisation. Confocal microscopy revealed that NS1 formed small cytoplasmic foci early after infection that gradually converged into large fluorescent NS1 tubule bundles. Tubule bundles were more organised in AHSV-infected cells than in cells expressing NS1 alone, suggesting that tubule bundle formation requires the presence of other AHSV proteins or regulation of NS1 expression rates. NS1 occasionally co-localised with VP7 crystalline structures, independently of other AHSV proteins. However, when NS1-eGFP, a modified NS1 protein that contains enhanced green fluorescent protein (eGFP) near the C-terminus, was co-expressed with VP7, co-localisation between these proteins occurred in most co-infected cells. It is not clear how the addition of eGFP to NS1 induces this co-localisation and further investigation will be required to determine the function of NS1 during viral replication. The second part of the study focused on characterising the novel non-structural AHSV protein NS4. The NS4 open reading frame (ORF) occurs on segment 9, overlapping the VP6 ORF in a different reading frame. In silico analysis of segment 9 nucleotide and NS4 predicted amino acid sequences revealed a large amount of variation between serotypes, and two main types of NS4 were identified based on these analyses. These proteins differed in length and amino acid sequence and were named NS4-I and NS4-II. Immunoblotting confirmed that AHSV NS4 is translated in AHSV infected insect and mammalian cells, and also in Sf9 insect cells infected with recombinant baculoviruses that overexpress the genome segment 9 proteins, VP6 and NS4. Confocal microscopy showed that NS4 localised to both the cytoplasm and nucleus, but not the nucleolus, in AHSV-infected cells and recombinant baculovirus infected Sf9 cells. Nucleic acid protection assays using bacterially expressed purified NS4 showed that both types of NS4 bind dsDNA, but not dsRNA. This was the first study to focus on AHSV NS4. Future work will focus on determining the role of non-structural proteins in viral pathogenesis, and will involve the use of a reverse genetics system for AHSV.
Dissertation (MSc)--University of Pretoria, 2013.
Genetics
MSc
Unrestricted

African horse sickness is one of the most deadly infectious diseases of horses. The disease is caused by African horse sickness virus (AHSV), an arbovirus transmitted by culicoides midges. AHSV is classified within the genus Orbivirus, family Reovirdae. The AHSV genome is composed by ten segments of double stranded RNA (dsRNA) encoding seven structural and at least four non-structural (NS) proteins. AHSV shares structural and functional similarities with Bluetongue virus, the ‘prototype’ species of the genus Orbivirus. An alternative open reading frame (ORF), overlapping the main ORF encoding the VP6, has been identified in segment 9 in both AHSV and BTV. This additional ORF encodes the non-structural protein NS4. The BTV NS4 localises in the nucleoli of the infected cells. NS4 is an interferon antagonist and a determinant of virus virulence. In this thesis, I aimed to characterise the AHSV NS4. Unlike the BTV NS4, the AHSV NS4 are relatively variable mong different virus strains. I have divided these proteins into four different subtypes: NS4I, NS4-IIα, -IIβ, and IIγ based upon their sequence similarity and on the presence of N-terminal or C-terminal truncations. In contrast to BTV, all four of these NS4 types localise in the cytoplasm of either transfected or infected cells. In addition, in transient transfection assays all the NS4 types show the ability to hamper gene expression, with NS4-IIβ being the most efficient. In order to further understand the biological significance of the AHSV NS4 we used reverse genetics to generate viruses expressing the four types of NS4 (AHSV-NS4-I, AHSV-NS4-IIα, AHSV-NS4-IIβ, AHSV-NS4-IIγ) and the corresponding NS4 deletion mutants (AHSV-ΔNS4-I etc.). Deletion of NS4 did not affect virus replication kinetics in either KC cells or interferon incompetent cells such as the BSR cell line. Similarly, both AHSV-NS4-IIβ and the corresponding ΔNS4 mutant showed similar replication kinetics in the interferon competent E. Derm cell line and in primary horse endothelial cells. In contrast, AHSV-NS4-I, AHSV-NS4-IIα, and AHSV-NS4-IIγ replicated more efficiently than the corresponding ΔNS4 viruses in these horse cells. Interestingly, the defects in replication of the NS4 viruses were removed after treatment with an inhibitor of the JAK/STAT pathway. Indeed, we observed that primary horse cells infected with the NS4 mutants released higher levels of type I interferon (IFN) than cells infected with the corresponding NS4 expressing viruses. In addition, we found the NS4 to be a determinant of virus virulence in vivo in NIH-Swiss mice infected with the viruses described above. Collectively, the data described in this thesis suggest that the NS4 is one of the proteins used by AHSV to modulate the IFN response.

Philosophiae Doctor - PhD
Hepatitis C was first recognized as a transfusion-associated liver disease not caused by hepatitis A or hepatitis B virus after serological tests were developed to screen for their presence in the blood. The infectious agent was finally identified with the cloning of the cDNA of hepatitis C virus (HCV) using random polymerase chain reaction (PCR) screening of nucleic acids extracted from plasma of a large pool of chimpanzee infected with non-A non-B hepatitis. NS5B, a membrane-associated RNA-dependent RNA polymerase essential in the replication of HCV, initiates the synthesis of a complementary negative-strand RNA from the genomic positive-strand RNA so that more positive-strand HCV RNA can then be generated from the newly synthesised negative-strand template. The crystal structure of NS5B presented typical fingers, palm and thumb sub-domains encircling the GDD active site, which is also seen in other RNA-dependent RNA polymerases, and is similar to the structure of reverse transcriptase of HIV-1 and murine Moloney leukaemia virus. The last 21 amino acids in the C-terminus of NS5B anchor the protein to the endoplasmic reticulum (ER)-derived membranous web. NS5B has been shown to interact with the core, NS3/NS4A, NS4B and NS5A proteins, either directly or indirectly. Numerous interactions with cellular proteins have also been reported. These proteins are mainly associated with genome replication, vesicular transport, protein kinase C-related kinase 2, P68 (DDX5), α-actinin, nucleolin, human eukaryotic initiation factor 4AII, and human VAMP-associated protein. Previous studies have confirmed that NS5B binds to full-length DDX5. By constructing deletion mutants of DDX5, we proceeded to characterize this interaction between DDX5 and HCV NS5B. We report here the identification of two exclusive HCV NS5B binding sites in DDX5, one in the N-terminal region of amino acids 1 to 384 and the other in the C-terminal region of amino acids 387 to 614. Proteins spanning different regions of DDX5 were expressed and purified for crystallization trials. The N-terminal region of DDX5 from amino acids 1 to 305 which contains the conserved domain I of the DEAD-box helicase was also cloned and expressed in Escherichia coli. The cloning, expression, purification and crystallization conditions are presented in this work. Subsequently, the crystal structure of DDX5 1-305 was solved and the high resolution three-dimensional structure shows that in front of domain I is the highly variable and disordered N terminal region (NTR) of which amino acids 51-78 is observable, but whose function is unknown. This region forms an extensive loop and supplements the core with an additional α-helix. Co-immunoprecipitation experiments demonstrated that the NTR of DDX5 1-305 auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-60 residues in NTR and NS5B binding site in DDX5 1-305, presumably located within residues 79-305. Furthermore, co-immunoprecipitation experiments revealed that DDX5 can also interact with other HCV proteins, besides NS5B.

Hepatitis C virus (HCV) is a significant cause of chronic liver disease worldwide. The molecular basis of HCV infection and replication has been extensively studied, leading to the identification of vital viral proteins and their interactions with host factors. The HCV genome encodes a single polyprotein cleaved by host and viral proteases into individual proteins, including the core, envelope glycoproteins (E1 and E2), p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. These viral proteins play critical roles in virus assembly, entry, replication, and evasion of host immune responses. The HCV envelope glycoproteins E1 and E2 are responsible for virus attachment and entry into host cells through interactions with various host receptors, including CD81, scavenger receptor class B type I (SR-BI), and tight junction proteins. The viral protein NS3 has multiple functions, including protease and helicase activities, which are critical for viral RNA replication. NS5A is an essential component of the viral replication complex and regulates viral RNA replication, virion assembly, and modulation of host immune responses. NS5B is the RNA-dependent RNA polymerase responsible for viral RNA synthesis. The molecular mechanisms underlying HCVinduced pathogenesis and the development of chronic infection remain poorly understood. However, recent studies have shed light on the interactions between HCV and host factors, including the innate and adaptive immune responses and the roles of viral proteins in modulating these responses. These insights have led to new antiviral therapies, including direct-acting antivirals (DAAs) that target viral proteins in RNA replication.

Traditional medicine offers a wide range of application for in silico study techniques. This drug research and development strategy is embryonic in the West African context, particularly in Burkina Faso, which is increasingly faced with emerging diseases such as dengue fever. Circulation of the 4 serotypes of this virus has been documented in the country. This study aims to evaluate the therapeutic potential of phytocompounds contained in the West African pharmacopoeia against dengue virus NS2B/NS3 protein, using computational methods integrating several software packages and databases. Based on a literature review, we identified 191 molecules from 30 plants known for their antiviral effects. Five met the inclusion criteria for molecular docking: patulin from calotropis procera, resiniferonol from Euphorbia poissonii, Securinol A from Flueggea virosa, Shikimic acid and Methyl gallate from Terminalia macroptera. The best binding scores were observed between resiniferonol and the serotypes 1, 2 and 4 NS2B/NS3 protease, with binding energies of -7.4 Kcal/mol, -6.8 Kcal/mol and -7.3 Kcal/mol respectively; while the NS2B/NS3 protease of serotype 3 had the best affinity for securinol A (-7 Kcal/mol). This study points the way to further research in computer aided drug design field and calls for multidisciplinary collaboration to promote West African medicinal plants against health challenges.

Viruses causes severe infections and associated and linked with outbreaks of some major global diseases such as HIV/AIDS, COVID-19, influenza virus, dengue fever, common cold, and hepatitis. The infective process of viruses varies depending on the viral species; however, they follow certain steps in the infective process. The typical stages in their life cycle include (Fig 1): attachment and entry, viral uncoating, replication and transcription of viral genome, protein synthesis, assembly, and release of progeny virus. Antiviral drugs are used in combating viral infections and the development of these drugs has been major achievement in the global effort to mitigate infectious diseases. Antiviral drugs are available for a number of viruses including severe acute respiratory coronavirus 2 (SARS-CoV-2), hepatitis-A and-B virus, influenza virus, Papillomavirus. Human Immunodeficiency Virus (HIV), respiratory synctial virus, human cytomegalovirus (HCMV). Although most of these agents are not curative, they can efficiently control viral replications. They can be either small or large molecules, synthetic or natural. Based on their mode of action (MOA), antiviral agents are divided into two classes: 1, inhibitors of the viruses or 2. Inhibitors of the target host cells. Viral –targeting antivirals (VTAs) act by either directly or indirectly inhibiting the biological functions of viral protein which results in the inhibition of the ideal viral replication machinery. Host-targeting antivirals (HATs) on the other hand target the host protein that are associated with the viral replication cycle, regulating the function of the immune system or other cellular processes in the host (3) The Food & Drug Administration Board has approved more than hundred antiviral agents which are based on either monotherapy and combined therapies (https://www.fda.gov). The approved drugs have different MOAs based on their functions or structures. These includes structural analogues, entry inhibitors, integrase inhibitors, essential enzyme inhibitors such as nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors, protease inhibitors, inhibitors that are specific to certain viruses such as influenza virus and HCV NS5A protein and NS5B polymerase inhibitors, immunomodulators, interferons, antimitotic inhibitors, and oligonucleotides

INTRODUÇÃO: A detecção de HCV na triagem, em doadores de sangue da Fundação Hemopa, é realizada simultaneamente por um teste sorológico e um teste molecular. No caso de discordância entre estes, é realizado um teste confirmatório. Geenius HCV Supplemental Assay é utilizado como teste confirmatório suplementar para análise da presença de anticorpos específicos para HCV, utilizando os antígenos específicos NS3, NS4, NS5 e capsídeo. OBJETIVO: Determinar a freqüência de resultado de imunocromatografia Geenius HCV Confirmatory Assay (BioRad) em amostras Eclesys Anti-HCV reagentes (positivos e inconclusivos) e teste de ácido nucléico (NAT) para HCV indetectáveis. MATERIAIS E MÉTODOS: Foram avaliadas 55 amostras entre o período de Setembro de 2021 a Março de 2022. Para este estudo, todas as amostras selecionadas apresentaram sorologia Anti HCV reagente (valor de leitura da amostra/ valor de “cut off” (S/CO) >0,8) e NAT indetectável. Foram utilizados, para detecção de anticorpos anti-HCV, o ensaio Eclesys Anti-HCV com metodologia de eletroquimioluminescência, e para detecção de material genético de HCV, o Kit NAT HIV/HCV Bio-Manguinhos com metodologia de PCR em tempo real. A realização do teste Geenius HCV Supplemental Assay se dá de acordo com a utilização da proteína A de ligação ao anticorpo, conjugada com partículas coloridas de celulose e os antígenos específicos ligados à membrana da fita. Como resultado, pode haver a captura dos anticorpos Anti-HCV, produzindo uma reação colorimétrica na área teste do cassete e na área de controle da reação. RESULTADOS: Conforme o ensaio sorológico Eclesys Anti-HCV, de 55 amostras, 32,8% (17/55) apresentaram resultado de sorologia inconclusivo (valor de “cut off” (S/CO) 0,8 a 1,2) e 67,2% (37/55) apresentaram resultado de sorologia positivo (valor de “cut off” (S/CO) > 1,2) . Entre as amostras que tiveram resultado de sorologia inconclusiva, 100% (17/17) apresentaram resultados de imunocromatografia negativos. Entre as amostras que tiveram sorologia positiva, 97,2% (36/37) apresentaram resultados de imunocromatografia negativos e 2,8% (1/37) apresentaram resultados de imunocromatografia indeterminados. Não houveram resultados positivos.CONCLUSÃO: A imunocromatografia mostrou-se ferramenta eficiente para confirmar a não exposição ao HCV em 98% (54/55) dos doadores de sangue com resultados discordantes do teste de triagem sorológico-molecular para o HCV, permitindo que os doadores recebessem a devida orientação. PALAVRAS-CHAVE: HCV, IMUNOCROMATOGRAFIA, TESTE