Animal models have greatly contributed to the understanding of different aspects of human biology, as well as a variety of human-related pathogens and diseases. In order to study them, humanized mice susceptible to pathogens that replicate in human immune cells have been developed (e.g., humanized Rag2-/-γc-/- mice). These animals are engrafted with human hematopoietic stem cells (HSCs), resulting in the de novo development and maturation of the major functional components of the human adaptive immune system and the production of a variety of human cell types. Primary and secondary lymphoid organs in the mouse are populated with human cells, and animals have long term engraftment. These features make humanized mice an excellent in vivo model to study pathogenesis of human-specific viruses in the context of a human antiviral immune response. In addition, humanized mice have been shown to be useful preclinical models for the development and validation of antiviral therapeutics. In the present study, we aimed to successfully re-establish the humanized Rag2-/-γc-/- mouse model using cord blood-derived HSCs in our laboratory. We have shown that these mice sustain long term engraftment and systemic expansion of human cells, including the major targets of Kaposi's sarcoma Herpesvirus (KSHV) and Human immunodeficiency virus type 1 (HIV-1), in peripheral blood and different lymphoid organs. Further, we have begun to evaluate the susceptibility of the humanized Rag2-/-γc-/- mouse model to infection with KSHV. We demonstrate that human lymphocytes differentiated in reconstituted Rag2-/-γc-/- mice are permissive to KSHV infection ex vivo. This finding was corroborated by detection of KSHV mRNA expression in the spleen of a humanized mouse at 6 months post infection. In a different study, we tested the in vivo antiviral efficacy of a novel HIV-1 fusion inhibitor (PIE-12-trimer) in humanized Rag2-/-γc-/- mice. We have determined the half life of PIE-12-trimer in mouse plasma. Furthermore, the administration of PIE-12-trimer to HIV-1 infected humanized Rag2-/-γc-/- mice prevents depletion of CD4+ T cells in blood, thus it may be useful to prevent AIDS in human patients.

<p>Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance since mice expressing the VH and VL of 2F5 have an arrested B-cell development characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that mitigate effective humoral responses. I hypothesize that discreet human antigens are mimicked by the membrane-proximal external region (MPER) of HIV-1 gp41, and that such mimicry is a wide-spread strategy for HIV-1 to evade immune attacks to its vulnerable neutralizing epitopes.</p><p>In the first part of the study, I propose to identify autoantigens mimicked by the 2F5 and 4E10 epitopes. I used immunoprecipitation coupled with mass spectrometry as well as protein arrays to identify the self-antigens recognized by 2F5 and 4E10. The binding of antigens was confirmed using serological assays and targeted mutagenesis was used to map the binding epitope. We identified human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes a conformational epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but retain all SF3B3 4E10 epitopes. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant.</p><p>Our identification of structural motif shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1. In the second part of the project, I propose to study the mechanisms of immune tolerance to B cells expressing the 2F5 antibodies. To determine the B cell repertoire before and after tolerance checkpoints, I used the Nojima-Kitamura single B-cell culture that supports differentiation into IgG-secreting plasma cells, even autoreactive cells that are normally subject to tolerization in vivo. I found that the pre-tolerance compartment (small pre-B) from 2F5 KI mice are cells that express the 2F5 V(D)J rearrangements and bind HIV-1 gp41, KYNU, and cardiolipin. Mature, post-tolerance B cells from 2F5 KI mice, however, are purged of gp41- and KYNU-reactivity, but retain cardiolipin-binding, and sequence analysis revealed extensive light-chain editing. The anergic B cells in the post-tolerance compartment are enriched with self-reactivity to KYNU and maintain binding to HIV-1 gp41. Our results demonstrate that tolerance of the 2F5 epitope is driven by specific reactivity to KYNU, but not general cross-reactivity to cardiolipin. In addition, that the peripheral anergic B cells retain self-reactivity and binding to HIV-1 gp41 suggests a potential target for activation by immunizations.</p><p>Lastly, we sought to determine whether the host mimicry by 4E10 and 2F5 epitopes is also present in other HIV-1 epitopes, including additional conserved neutralizing epitopes and more importantly, non-neutralizing epitopes. We used protein microarrays to assess autoreactivity of HIV-1 broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (nnAbs) and found that as a class, bnAbs are more polyreactive and autoreactive than nnAbs. The poly- and autoreactive property is therefore not a result of chronic inflammation, but rather uniquely associated with neutralization, consistent with the role of heteroligation for HIV-1 neutralizing activity. In addition, mutation frequencies of bNAbs and nnAbs per se do not correlate with poly- and autoreactivity. Our results demonstrate that HIV-1 bnAbs are significantly more polyreactive and self-reactive than non-neutralizers, which may subject them to immunological tolerance control in vivo. Infrequent poly- or autoreactivity among nnAbs implies that their dominance in humoral responses is due to the absence of negative control by immune tolerance.</p><p>The results of this study indicate that mimicry of host antigens by HIV-1 is an effective mechanism to camouflage vulnerable neutralizing epitopes of HIV-1 and evade host immune responses. As a result, protective HIV-1 bnAbs are rare and often poly- or autoreactive, constituting a major hurdle that must be overcome to effectively elicit protective responses by an HIV-1 vaccine.</p> / Dissertation

Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2009. / Submitted in fulfilment for the degree MSc in BioMedical Science at Stellenbosch University. / ENGLISH ABSTRACT: HIV-1 was first diagnosed within South Africa in 1982. In the 1980’s homosexual transmission dominated the HIV-1 epidemic within the country. In the late 1980’s the second HIV-1 epidemic was recognized amongst heterosexual individuals. Today heterosexual transmission of HIV-1 dominates the epidemic in South Africa. Subtype C HIV-1 is responsible for the overwhelming majority of heterosexual infections. An estimated 95% of all infections in the country are thought to be subtype C related. To date only a few papers have been published on non-subtype C HIV within the country. This study characterized subgenomic and near full-length sequences of non-subtype C HIV-1 viruses from the Cape Town area. The gag p24, pol-integrase, and env gp41 regions of 11 of the 12 samples were characterized by amplification and direct sequencing. Phylogenetic analysis of the sequenced data, with online subtyping tools (REGA and jpHMM) and the drawing of NJ-trees revealed the presence of subtype A1, B, F1 and recombinant viral forms such as AD, AG and AC. One of the isolates was classified as a subtype C and was included for control purposes. Near full-length characterization of four of the samples were attempted, through full genome PCR amplification and sequencing. Analysis of sequenced data with the use of subtyping-, recombination identification, and tree drawing tools revealed a subtype B, and A1 isolate. The other two isolates were identified as possible AC and AD recombinants. The data that was generated will greatly improve our knowledge of non-subtype C isolates circulating within South Africa. Due to the possible impact that the high degree of genetic variation that HIV may have on vaccine design and development and ARV treatment and HIV diagnosis, ongoing research of the epidemiology and spread of HIV within South Africa are needed. / AFRIKAANSE OPSOMMING: MIV was in 1982 vir die eerste keer in Suid Afrika gediagnoseer en was hoofsaaklik deur homoseksuele kontak oorgedra. Aan die begin van die 1990’s is `n tweede MIV epidemie gewaar onder heteroseksuele individue. Heteroseksuele oordrag van die virus domineer tans die MIV epidemie in Suid Afrika en is meestal subtipe C verwant. Subtipe C, MIV-1 is verantwoordelik vir 95 persent van alle infeksies in die land. Tot hede is slegs `n paar publikasies oor die nie-subtipe C epidemie in die land gepubliseer. Die huidige studie was gemik op die karakterisering van subgenomiese en vollengte genome van nie-subtipe C MIV isolate van die Kaapstad omgewing. Die gag p24, pol-integrase en env gp41 subgenomiese fragmente van 12 monsters was gekarakteriseer deur amplifikasie en DNS nukleotied volgorde bepaling. Filogenetiese analise deur middel van subtipering (REGA en jpHMM aanlyn subtiperings programme) asook NJ-filogenetiese bome van die data het die teenwoordigheid van subtipe A1, B, en F1, asook verskeie rekombinante viruse insluitende AG, AD en AC vorme aangedui. Een van die isolate was geklassifiseer as `n subtipe C maar is in die studie ingevoeg vir kontrole doeleindes. Vollengte karakterisering van 4 uit die 12 isolate was ook gedoen deur vollengte genoom amplifikasie en DNS nukleotied volgorde bepaling. Tydens die analisering van die DNS volgorde data, deur middel van aanlyn subtipering, rekombinasie identifikasie (Simplot en RIP), en filogenetiese boom konstruksie programme is twee isolate geidentifiseer as subtipe B en A1 MIV-1 viruse. Die ander twee isolate was as moontlike AC en AD rekombinante geklassifiseer. Die data van nie-subtipe C MIV isolate sal ons kennis van die nie-subtipe C epidemie in Suid Afrika versterk. As gevolg van die impak wat die hoë graad van genetisie variasie van MIV op die ontwikkeling van entstowwe, sowel as die diagnose en behandeling van pasiente kan hê, is verdere navorsing in die epidemiologie van die MI-virus in Suid Afrika nodig.

<p>The cellular microRNA (miRNA) pathway has emerged as an important regulator of host-virus interactions. While miRNAs of viral and cellular origin have been demonstrated to modulate viral gene expression and host immune responses, reports detailing these activities in the context of mammalian retroviruses have been controversial. Using modern, high-throughput small RNA sequencing we provide evidence that the spumaretrovirus bovine foamy virus expresses high levels of viral miRNAs via noncanonical biogenesis mechanisms. In contrast, the lentivirus human immunodeficiency virus type 1 (HIV-1) does not express any viral miRNAs in a number of cellular contexts. Comprehensive analysis of miRNA binding sites in HIV-1 infected cells yielded several viral sequences that can be targeted by cellular miRNAs. However, this analysis indicated that HIV-1 transcripts are largely refractory to binding and inhibition by cellular miRNAs. In addition, we demonstrate that HIV-1 exerts minimal perturbations on cellular miRNA profiles and that viral replication is not affected by the ablation of mature cellular miRNAs. Together, these data demonstrate that the ability of retroviruses to encode miRNAs is not broadly conserved and that lentiviruses, particularly HIV-1, have evolved to avoid targeting by cellular miRNAs.</p> / Dissertation

L'ARN d'interférence (ARNi) est un mécanisme de régulation du gène qui permet un ciblage spécifique d'ARNmessager (ARNm) par reconnaissance de séquence. Les effecteurs de l'ARNi sont de petites molécules d'ARN non codants (siARN, microARN et piARN). Evoluant dans le contexte de l'ARNi, les virus de différentes familles ont adopté des stratégies afin utiliser l'ARNipour leur propre bénéfice. Le principal objectif de ma thèse a été d'étudier la fonction de l' ARNmiRTAR, un miARN viral dérivé de l'extrémité 3 'de l'ARN VIH-1 TAR dans la réplication du VIH-1. Nous avons constaté que miRTAR réguleà la fois l'activité basale et la transactivation induite par Tat du promoteur du VIH-1. L'effet de miRTAR ne nécessite pas de sa liaison à l'ARN TAR. miRTAR agit en augmentant l'activité de NF-kB, un facteur important pour la transcription du promoteur du VIH-1. En effet, la mutation des sites NF-kB, mais pas des sites Sp1 dans le LTR, abroge l'amélioration miRTARmédiée par la transcription. De plus, l'inhibition de l'expression de NF-kBpardes siARNspécifiquesdes les sous-unités p50 et p65, entraîne une perte d'activité dumiRTAR. Bien que nous n'avons pas pu identifier le(s) gène(s) cellulaire(s) ciblé par miRTAR, sa surexpression conduit à l'activation de la voie NF-kB. Mutation de l'extrémité 3 'du VIH-1 dans les résultats TAR réduction spectaculaire de la réplication virale Tat sans affecter médiée par la transcription, en raison de la perte de production de miRTARsauvage. Enfin, la surexpression de miRTARrestaure la réplication de ce virus contenant une mutation au niveau de la séquence TAR. En conclusion, nos résultats démontrent clairement que lemiRTARencodé par le VIH-1 joue un rôle clé dans la réplication du virus. Sur la base de ces résultats, nous proposons le modèle suivant pour la fonction de miRTAR. La transcription du LTR du VIH-1 conduit à la production de courts ARN dénommés TAR contenant une structure en épingle à cheveux. TAR est « processé » pour générer le miARN miRTAR. L'ARN miRTAR est ensuite chargé dans le complexe RISC et régule l'expression de plusieurs gènes cellulaires impliqués dans la régulation négative de NF-kB. miRTARmédiée par l'activation de NF-kB résultats dans la régulation de gènes viraux et par conséquent augmente la production de virus. L'ensemble de nos résultats montrent que le VIH-1 utilise la voie de l'ARNiinterférence pour optimiser l'environnement intracellulaire requis pour une réplication optimale. / RNA interference (RNAi) is a gene regulatory mechanism that offers a sequence specific targeting of mRNA. Evolving in the context of RNAi, viruses of different families adopted strategies to use RNAi for their benefit. The main objective of my thesis was to understand the function of miRTAR, a viral miRNA derived from 3' end of the HIV-1 TAR RNA, in HIV-1 replication. We found that miRTAR regulates both basal and Tat-mediated transactivation of HIV-1 promoter. The effect of miRTAR does not require its binding to TAR RNA. miRTAR acts by inducing NF-κB transcription factor important for the LTR activity. Indeed, mutation of NF-κB sites but not Sp1 sites within the LTR abrogate miRTAR-mediated enhancement of transcription from the LTR. Additionally, Inhibition of NF-κB using specific siRNA directed against p50 and p65 subunits results in loss of miRTAR activity. Although, we were unable to identify the cellular gene(s) targeted by miRTAR, its overexpression lead to the activation of NF-κB pathway. Mutation of the 3' end of HIV-1 TAR results in dramatic reduction of viral replication without affecting Tat-mediated transcription. Importantly, overexpression of miRTAR rescued the replication of miRTAR HIV-1 mutant virus.In conclusion, our results strongly demonstrate that the HIV-1 encoded miRTAR plays a key role in virus replication. On the basis of these findings, we propose the following model for the function of miRTAR. Transcription of the HIV-1 LTR leads to production of short, TAR containing, RNA hairpin sequences. TAR is processed to generate miRNA, miRTAR. miRTAR is then loaded into the RISC complex and regulates the expression of several cellular genes involved in the negative regulation of NF-κB. miRTAR-mediated activation of NF-κB results in up regulation of viral genes and consequently enhances virus production. Taken together, our results demonstrate that HIV-1 uses RNAi pathway to optimize the intracellular environment for optimal replication.

<p>The humoral immune system plays a critical role in the clearance of numerous pathogens. In the setting of HIV-1 infection, the virus infects, integrates its genome into the host's cells, replicates, and establishes a reservoir of virus-infected cells. The initial antibody response to HIV-1 infection is targeted to non-neutralizing epitopes on HIV-1 Env gp41, and when a neutralizing response does develop months after transmission, it is specific for the autologous founder virus and the virus escapes rapidly. After continuous waves of antibody mediated neutralization and viral escape, a small subset of infected individuals eventually develop broad and potent heterologous neutralizing antibodies years after infection. In this dissertation, I have studied the ontogeny of mucosal and systemic antibody responses to HIV-1 infection by means of three distinct aims: 1. Determine the origin of the initial antibody response to HIV-1 infection. 2. Characterize the role of restricted VH and VL gene segment usage in shaping the antibody response to HIV-1 infection. 3. Determine the role of persistence of B cell clonal lineages in shaping the mutation frequencies of HIV-1 reactive antibodies. </p><p>After the introduction (Chapter 1) and methods (Chapter 2), Chapter 3 of this dissertation describes a study of the antibody response of terminal ileum B cells to HIV-1 envelope (Env) in early and chronic HIV-1 infection and provides evidence for the role of environmental antigens in shaping the repertoire of B cells that respond to HIV-1 infection. Previous work by Liao et al. demonstrated that the initial plasma cell response in the blood to acute HIV-1 infection is to gp41 and is derived from a polyreactive memory B cell pool. Many of these antibodies cross-reacted with commensal bacteria, Therefore, in Chapter 3, the relationship of intestinal B cell reactivity with commensal bacteria to HIV-1 infection-induced antibody response was probed using single B cell sorting, reverse transcription and nested polymerase chain reaction (RT- PCR) methods, and recombinant antibody technology. The dominant B cell response in the terminal ileum was to HIV-1 envelope (Env) gp41, and 82% of gp41- reactive antibodies cross-reacted with commensal bacteria whole cell lysates. Pyrosequencing of blood B cells revealed HIV-1 antibody clonal lineages shared between ileum and blood. Mutated IgG antibodies cross-reactive with both Env gp41 and commensal bacteria could also be isolated from the terminal ileum of HIV-1 uninfected individuals. Thus, the antibody response to HIV-1 can be shaped by intestinal B cells stimulated by commensal bacteria prior to HIV-1 infection to develop a pre-infection pool of memory B cells cross-reactive with HIV-1 gp41.</p><p>Chapter 4 details the study of restricted VH and VL gene segment usage for gp41 and gp120 antibody induction following acute HIV-1 infection; mutations in gp41 lead to virus enhanced neutralization sensitivity. The B cell repertoire of antibodies induced in a HIV-1 infected African individual, CAP206, who developed broadly neutralizing antibodies (bnAbs) directed to the HIV-1 envelope gp41 membrane proximal external region (MPER), is characterized. Understanding the selection of virus mutants by neutralizing antibodies is critical to understanding the role of antibodies in control of HIV-1 replication and prevention from HIV-1 infection. Previously, an MPER neutralizing antibody, CAP206-CH12, with the binding footprint identical to that of MPER broadly neutralizing antibody 4E10, that like 4E10 utilized the VH1-69 and VK3-20 variable gene segments was isolated from this individual (Morris et al., 2011). Using single B cell sorting, RT- PCR methods, and recombinant antibody technology, Chapter 4 describes the isolation of a VH1-69, Vk3-20 glycan-dependent clonal lineage from CAP206, targeted to gp120, that has the property of neutralizing a neutralization sensitive CAP206 transmitted/founder (T/F) and heterologous viruses with mutations at amino acids 680 or 681 in the MPER 4E10/CH12 binding site. These data demonstrate sites within the MPER bnAb epitope (aa 680-681) in which mutations can be selected that lead to viruses with enhanced sensitivity to autologous and heterologous neutralizing antibodies. </p><p>In Chapter 5, I have completed a comparison of evolution of B cell clonal lineages in two HIV-1 infected individuals who have a predominant VH1-69 response to HIV-1 infection--one who produces broadly neutralizing MPER-reactive mAbs and one who does not. Autologous neutralization in the plasma takes ~12 weeks to develop (Gray et al., 2007; Tomaras et al., 2008b). Only a small subset of HIV-1 infected individuals develops high plasma levels of broad and potent heterologous neutralization, and when it does occur, it typically takes 3-4 years to develop (Euler et al., 2010; Gray et al., 2007; 2011; Tomaras et al., 2011). The HIV-1 bnAbs that have been isolated to date have a number of unusual characteristics including, autoreactivity and high levels of somatic hypermutations, which are typically tightly regulated by immune control mechanisms (Haynes et al., 2005; 2012b; Kwong and Mascola, 2012; Scheid et al., 2009a). The VH mutation frequencies of bnAbs average ~15% but have been shown to be as high as 32% (reviewed in Mascola and Haynes, 2013; Kwong and Mascola, 2012). The high frequency of somatic hypermutations suggests that the B cell clonal lineages that eventually produce bnAbs undergo high-levels of affinity maturation, implying prolonged germinal center (GC) reactions and high levels of T cell help. To study the duration of HIV-1- reactive B cell clonal persistence, HIV-1 reactive and non HIV-1- reactive B cell clonal lineages were isolated from an HIV-1 infected individual that produces bnAbs, CAP206, and an HIV-1 infected individual who does not produce bnAbs, 004-0. Single B cell sorting, RT-PCR and recombinant antibody technology was used to isolate and produce monoclonal antibodies from multiple time points from each individual. B cell sequences clonally related to mAbs isolated by single cell PCR were identified within pyrosequences of longitudinal samples of these two individuals. Both individuals produced long-lived B cell clones that persisted from 0-232 weeks in CAP206, and 0-238 weeks in 004-0. The average length of persistence of clones containing members isolated from two separate time points was 91.5 weeks both individuals. Examples of the continued evolution of clonal lineages were observed in both the bnAb and non-bnAb individual. These data indicated that the ability to generate persistent and evolving B cell clonal lineages occurs in both bnAb and non-bnAb individuals, suggesting that some alternative host or viral factor is critical for the generation of highly mutated broadly neutralizing antibodies. </p><p> Together the studies described in Chapter 3-5 show that multiple factors influence the antibody response to HIV-1 infection. The initial antibody response to HIV-1 Env gp41 can be shaped by a B cell response to intestinal commensal bacteria prior to HIV-1 infection. VH and VL gene segment restriction can impact the B cell response to multiple HIV-1 antigens, and virus escape mutations in the MPER can confer enhanced neutralization sensitivity to autologous and heterologous antibodies. Finally, the ability to generate long-lived HIV-1 clonal lineages in and of itself does not confer on the host the ability to produce bnAbs.</p> / Dissertation

Philosophiae Doctor - PhD / The HIV-1 envelope (Env) glycoprotein is the primary target of the humoral immune response and a critical vaccine candidate. However, Env is densely glycosylated and thereby substantially protected from neutralisation. Despite the importance of the HIV- 1 Env glycans, limited computational analyses have been employed to analyse these glycans. Here, the Env glycans of two HIV-1 wild-type subtype C isolates are examined, in detail, using computational approaches. These particular strains were used since in vitro data showed that the removal of a single glycan had a substantially different impact on the neutralisation sensitivity of the two strains. Molecular dynamics simulations, and the subsequent analyses, were carried out on the computationally determined, fully glycosylated, Env structures of these two wild-type strains and their N301A mutant counterparts. Detailed comparison of the molecular dynamics simulations demonstrated that unique glycan dynamics and conformations emerged and that, despite shared HXB2 reference sequence positions, the glycans adopted distinct conformations specific to each wild-type model. Furthermore, different changes in conformations were observed for each wild-type model compared to its N301A mutant counterpart and, interestingly, these N301A mutant model-specific glycan conformations were directly associated with the protein residues ultimately found to be exposed, which may explain the varied resistance to neutralising antibodies observed, in vitro, for the two N301A mutant strains.

Bien que le traitement antirétroviral (ART) supprime efficacement la multiplication du VIH-1 chez les patients infectés, l’ART ne guérit pas l'infection. En effet, si l'ART est arrêté, nous observons un rebond viral. Celui–ci est principalement dû à l'activation stochastique de cellules latentes qui contiennent le génome viral intégré mais ne produisent pas de virus et ne sont donc pas ciblées par l'ART ou le système immunitaire. Ces cellules latentes sont peu nombreuses (1-10 par million de cellules T-CD4+ quiescentes) mais elles apparaissent rapidement après la primo infection et constituent donc un obstacle majeur à l'éradication virale. La stratégie la plus prometteuse pour supprimer ces cellules, dite "Shock and Kill", est de les réactiver pour qu'elles soient ensuite ciblées par l'ART et/ou lysées par les cellules T cytotoxiques. Un certain nombre d’agents de sortie de latence (LRAs) ont été mis au point pour réactiver ces cellules. Ils ciblent les protéines cellulaires telles que les Histone-désacétylases (HDAC) ou la protéine kinase C. La plupart d'entre eux présentent donc des effets non spécifiques et parfois une toxicité. Tat est la protéine du VIH-1 qui permet la transcription virale et favorise la traduction des gènes viraux. Tat est la protéine clé pour la levée de latence et l'initiation de la production des protéines virales par la cellule latente. Sur la base des structures RMN de Tat disponibles, nous avons identifié par dynamique moléculaire les conformations les plus stables de Tat. Cela nous a permis d'identifier par criblage in silico des ligands potentiels de Tat. Dix molécules ont été sélectionnées. Une molécule appelée D10 se fixe spécifiquement à la protéine Tat et augmente son activité de transactivation d'environ 4 fois. De plus, D10 présente une activité LRA sur les lignées cellulaires latentes JLat-9.2 et OM-10.1. L’activité LRA de D10 sur ces lignées représente 50 à 70% de celle du SAHA (vorinostat), un inhibiteur des HDAC candidat LRA en cours d’essais cliniques (Phase 2). Sur les cellules latentes de patients VIH traités, D10 à 50 nM a une activité LRA très efficace, 80% supérieure à celle de la bryostatine-1 qui agit sur la PKC et est considéré comme le LRA le plus prometteur actuellement. Le mécanisme d’action de D10, à semble être la stabilisation du complexe de transcription Tat-TAR. Cet effet est observé à 30 nM D10. En utilisant une approche chémoinformatique nous avons sélectionné 11 analogues de D10, dit N1-N11. Certains de ces analogues (N5, N8) montrent un effet plus fort que D10 sur l’augmentation de la transactivation de Tat ainsi que pour l’effet LRA sur les lignées cellulaires latentes. Ce résultat nous a permis d'ébaucher une relation structure chimique / activité LRA de ces molécules. Nous avons donc identifié de nouveaux agents de sortie de latence du VIH-1 ciblant Tat, plus spécifiques que les LRAs ciblant les protéines cellulaires. Ce sont les premiers activateurs de Tat identifiés. / Despite its efficiency to prevent viral multiplication, antiretroviral therapy (ART) is unable to cure patients with HIV-1. Indeed if ART is stopped, a viral rebound is observed. This increase in blood viral load is due to the activation of HIV-1 reservoirs, among which latently-infected memory CD4+ T cells. These cells are rare (1 per million of quiescent T cells) and appear very quickly following infection. To purge this long-lived reservoir the "Shock and Kill" approach was developed. This strategy relies on the use of latency reversing agents (LRAs) to induce reservoir activation. All LRAs developed until now target cellular proteins such as Histone deacetylases or protein kinase C. These LRAs are not specific for viral transcription and displayed modest effects ex vivo. Here we present a new LRA family that binds to and activates HIV-1 Tat which is the key regulator for viral transcription and latency reversal. These compounds are not cytotoxic and specifically activate Tat transcriptional activity. They were less efficient than available LRAs on HIV-1 latent cell lines. Nevertheless, when tested on latent T-cells from HIV-1 patients, the lead compound D10 was ~ 80% more efficient than bryostatin-1, one of the best LRA available to date. This effect was observed at 50 nM, which corresponds to the D10 concentration required for this compound to stabilize the Tat-TAR transcription complex. These molecules are the first Tat activators available.

MSc (Med) Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand / In recent years there has been much progress in understanding and defining the key protein structure-function relationships that mediate Human Immunodeficiency Virus (HIV-1) entry into host CD4+ cells. This process involves fusion of the virus and host cell membranes, following engagement of corresponding viral (gp120) and target (CD4) receptor proteins. Binding of gp120 to CD4 triggers extensive conformational changes in gp120, exposing binding sites for the co-receptor proteins (CCR5 or CXCR4), and facilitating insertion of gp41, the viral fusion protein, into the target cell membrane. Following insertion of gp41, oligomerisation of fusogenic domains on gp41 is thought to drive the juxtaposition of the virus and host cell and fusion of their membranes. Recent reports suggest that detergent-resistant membrane domains, known as lipid rafts, play a crucial role in orientating the receptor molecules during this step of HIV-1 infection. Lipid rafts are typically rich in cholesterol, sphingolipids and GPI-anchored proteins, and are biophysically distinct from the glycerophosolipid bilayer, which constitutes the bulk of mammalian cell membranes. The role of lipid rafts in virus entry, however, is still controversial, and further experimentation is needed to define their importance in this regard. To provide insight into the role of lipid rafts during HIV-1 entry, we evaluated the natural distribution of the host receptor proteins in HIV-1 target cells (U87.CD4.CCR5/CXCR4). CD4 was detected in membrane samples fractionated by sucrose density gradient centrifugation using immunoblotting techniques, while CCR5 and CXCR4 were detected on whole cells by fluorescence microscopy. We then used a primary CCR5-utilising subtype C HIV-1 isolate (FV5) to characterise dynamic changes in the distribution of these receptors and gp41 during viral entry in real-time. Viral fusion assays were set up by inoculating v target cells with FV5 at 23 ºC, a temperature that allows HIV-1 attachment, but is nonpermissive for advancement of the fusion reaction. This prefusogenic form of the virus-host receptor complex is defined as the temperature-arrested state (TAS). We found that, under normal, uninfected conditions, CD4, CCR5 and CXCR4 are distributed throughout both raft and non-raft microdomains on the U87 cell surface, and there is little evidence for any significant redistribution of these receptors into lipid rafts during the HIV-mediated fusion reaction. Interestingly, we observed a change in the structure of CD4 during the fusion process, which could describe a functionally important event in HIV-1 entry, or be related to compromises in the integrity of the virally-infected membranes. Moreover, we discovered that gp41 is capable of membrane insertion and oligomerisation at TAS, in contrast to previous reports that suggest the fusion peptide is not capable of breaching the membrane at this temperature. Our results provide valuable novel insights into the HIV-1 subtype C entry process, and the involvement of lipid rafts in this stage of the viral lifecycle, that may be relevant to novel therapy and immunogen design.

MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand / Three separate recent publications used genome-wide RNA interference (RNAi) to screen for novel host factors that are required for HIV-1 infection and replication. This was achieved using small interfering RNAs (siRNAs) to silence the expression of ~21 000 human genes and determining the effect of each gene’s loss of function on HIV-1 replication. Collectively, several hundred genes have now been implicated as novel HIV-1 host factors (termed HIV-1 Dependency Factors, HDFs). However, differences in study design resulted in little overlap and limited interpretive value from the three published datasets. To identify novel HDFs that are potential targets for anti-HIV therapy, five putative HDFs (SPTBN1, TMED2, KIAA1012, PRDM14 and SP110) were chosen for validation. RNAi effecters (both siRNAs and expressed short hairpin RNAs) were used to silence the selected genes. Gene suppression was measured by quantitative RT-PCR assay and two candidate genes were studied further (SPTBN1 and SP110) based on efficient mRNA inhibition (over 90%). As efforts to deliver the RNAi effecters to a T-cell line were unsuccessful, the effect of this knockdown on HIV-1 replication (both early- and late-stage) was assessed in cultured TZM-bl cells, a HeLa-derived cell line that expresses HIV-1 entry receptors and an integrated luciferase reporter for HIV-1 transcriptional activity (also used in the first genome-wide RNAi screen). An initial viral challenge assay with Subtype C-enveloped pseudovirus showed a 60% decrease in TZM-bl luciferase reporter activity in cells with suppressed SPTBN1 function, while knockdown of SP110 showed no effect on reporter activity. The final experiment, using fully-replicating Subtype B virus, showed a 75% decrease in late-stage viral replication when SPTBN1 expression was suppressed. In addition, SP110 suppression was confirmed to have no effect on TZM-bl reporter activity during any stage of HIV-1 replication. In conclusion, SPTBN1, but not SP110, is required for late-stage HIV-1 replication, though these results need to be confirmed in CD4+ T-cells. The absence of several important viral accessory factors from vi the virus used in the genome-wide screen may explain these findings and emphasises the need for using physiologically representative viral and cellular models to study the viral/cellular interactome.