L’infection par HIV-1 représente un des problèmes de santé publique majeurs de notre société actuelle. Le traitement HAART (Highly Active AntiRetroviral Therapy) inhibe le cycle réplicatif viral mais ne permet pas l’éradication du HIV-1. La principale cause de cet échec thérapeutique est la persistance de réservoirs cellulaires infectés de manière latente par HIV-1, qui, lors de l’arrêt du traitement HAART, sont à l’origine d’un rebond de la charge plasmatique virale. Le défi actuel est donc de découvrir de nouvelles méthodes d’élimination des cellules réservoirs. Une des stratégies envisagées est de forcer l’expression virale dans les cellules infectées de manière latente afin d’entraîner leur destruction suite à leur détection par le système immunitaire ou suite aux effets cytopathiques viraux. Parallèlement, le traitement HAART serait maintenu afin de limiter la propagation des virions néo-synthétisés. Plusieurs éléments sont impliqués dans la répression transcriptionnelle associée à la latence post-intégrationnelle du virus HIV-1 : la nature du site d’intégration ; l’absence de facteurs cellulaires inductibles tels que NF-κB ; la structure chromatinienne du provirus et les modifications post-traductionnelles des histones ; l’absence de niveaux suffisants de la protéine trans-activatrice Tat. De plus, notre laboratoire a précédemment mis en évidence un lien entre deux de ces éléments, en démontrant, dans une lignée modèle de latence post-intégrationnelle, que la cytokine pro-inflammatoire TNFα, un activateur de la voie de signalisation NF-κB, permet une réactivation synergique de l’expression virale combinée à l’inhibiteur d’histone-désacétylases (HDACI) TSA. Cependant, l’utilisation thérapeutique du TNFα et de la TSA est inenvisageable en raison de leurs toxicités. Au cours de notre thèse, nous avons montré que la prostratine, un ester de phorbol non-tumorigène qui active la cascade NF-κB, combinée à des HDACIs déjà utilisés en thérapie humaine, réactive en synergie l’expression virale dans des cellules modèles de latence post-intégrationnelle (U1, J-Lat 8.4 et 15.4). Nous avons également constaté que des cellules dont l’expression virale n’est pas réactivée par un traitement individuel (prostratine ou HDACIs) peuvent être réactivées par la combinaison prostratine+HDACI. Au niveau moléculaire, nous avons montré que le VPA intensifie la cascade NF-κB induite par la prostratine, et permet, combiné à cette dernière, un remodelage plus efficace du nucléosome répresseur nuc-1 et une activation synergique de la transcription. Nous avons testé les combinaisons prostratine+HDACIs sur des cultures ex vivo de PBMCs (Peripheral Blood Mononuclear Cells) provenant de patients HIV-1+ avirémiques et traités par la HAART. Les combinaisons prostratine+HDACIs réactivent l’expression virale de 25/42 cultures de PBMCs testées avec une réactivation synergique dans 17/25 cas. De plus, la combinaison prostratine+SAHA réactive l’expression virale de 7/9 cultures testées de lymphocytes T CD4+ HLA DR-. Cette étude suggère que des traitements combinant deux types différents d’activateurs de l’expression du HIV-1 pourraient être utilisés comme adjuvant de la thérapie HAART afin d’accélérer la purge des réservoirs cellulaires infectés de manière latente.

The unspliced 9kb HIV-1 RNA (encoding Gag and GagPol) can undergo multiple splicing events to produce members of the 4kb (encoding Env, Vif, Vpr, and Vpu) or 2kb (encoding Tat, Rev and Nef), respectively. The incompletely spliced 9 and 4kb viral RNAs are exported by HIV-1 Rev which interacts with the RRE (Rev responsive element) in these RNAs as well as the nuclear export receptor Crm1. Several proteins can modulate Rev function and/or HIV-1 gene expression, including the nuclear phosphorprotein Sam68. We have found that overexpression of Sam68 stimulates HIV-1 structural gene expression and increases the proportion of unspliced, 3’ end processed viral RNA. This activity requires the RNA binding activity of Sam68. Surprisingly, Sam68 overexpression does not increase the proportion of unspliced, cleaved RNA found in the cytoplasm, suggesting that Sam68 alters the viral RNP to increase its translation. The Sam68 related proteins Slm1 and Slm2 also stimulate 3’ end cleavage and expression of unspliced HIV-1 RNAs. Sam68 and Slm2 were expressed in Hela cells, whereas Slm1 was not. Therefore, we reduced Sam68 expression alone or in combination with Slm2 to determine if these proteins were required for HIV-1 RNA processing or expression. Knockdown of Sam68 and/or Slm2 had little to no effect on viral RNA cleavage or structural gene expression from transiently transfected reporters. Furthermore, depletion of Sam68 only slightly reduced Gag expression from a stably expressed proviral reporter. These results suggest that additional redundant proteins may be present that functionally replace Sam68 and Slm2. We defined a region encompassing the N-terminal GSG (GRP33, Sam68, Gld1) and KH RNA binding motif as the minimal region of Sam68 required to stimulate HIV-1 gene expression in 293 and 293T cells. The minimal mutant enhanced unspliced RNA cleavage in 293T, but not in 293 cells suggesting that Sam68 may act at other stage of the viral lifecycle to increase gene expression.

Human immunodeficiency virus type 1 (HIV-1), the pathogen responsible for the onset of acquired immuno-deficiency syndrome (AIDS) in humans has reached pandemic proportions. To this day, no cure is available for infection with this virus and the only treatment option for this chronic infection is the life-long adherence to anti-retroviral therapy. Efforts in the quest to control the worldwide AIDS pandemic include the search for an effective anti-HIV-1 vaccine. Providing hope in this endeavor are a few monoclonal antibodies possessing broad neutralizing characteristics (bnmAbs) that have been isolated from the sera of rare patients that have a delayed progression to AIDS. In this thesis, one of these bnmAbs, 2F5 is extensively characterized at the atomic level to better understand its binding and neutralization mechanism. In total, 27 crystal structures of the 2F5 Fab’ in complex with various peptides representing its linear gp41 membrane proximal external region (MPER) epitope are presented. Furthermore, expression of the 2F5 Fab in a bacterial system allowed to design mutants of the 2F5 Fab and therefore investigate the implication of specific domains of 2F5 in mediating binding and neutralization. Atomic level characterization of this immune complex revealed a somewhat promiscuous recognition of 2F5 for its 664DKW666 epitope as long as the following characteristics were conserved: the aspartate’s negative charge, the hydrophobic alkyl-pi stacking arrangement between the beta-turn lysine and tryptophan, and the positive charge of the former. Moreover, it was demonstrated that 2F5 has an elongated and flexible complementary determining region 3 loop of the heavy chain (CDR H3), which is required for neutralization and is involved in secondary binding interactions other than to its core linear epitope. These contributions will significantly help in guiding the structure-based design of an HIV-1 vaccine looking to elicit 2F5-like antibody responses.

The unspliced 9kb HIV-1 RNA (encoding Gag and GagPol) can undergo multiple splicing events to produce members of the 4kb (encoding Env, Vif, Vpr, and Vpu) or 2kb (encoding Tat, Rev and Nef), respectively. The incompletely spliced 9 and 4kb viral RNAs are exported by HIV-1 Rev which interacts with the RRE (Rev responsive element) in these RNAs as well as the nuclear export receptor Crm1. Several proteins can modulate Rev function and/or HIV-1 gene expression, including the nuclear phosphorprotein Sam68. We have found that overexpression of Sam68 stimulates HIV-1 structural gene expression and increases the proportion of unspliced, 3’ end processed viral RNA. This activity requires the RNA binding activity of Sam68. Surprisingly, Sam68 overexpression does not increase the proportion of unspliced, cleaved RNA found in the cytoplasm, suggesting that Sam68 alters the viral RNP to increase its translation. The Sam68 related proteins Slm1 and Slm2 also stimulate 3’ end cleavage and expression of unspliced HIV-1 RNAs. Sam68 and Slm2 were expressed in Hela cells, whereas Slm1 was not. Therefore, we reduced Sam68 expression alone or in combination with Slm2 to determine if these proteins were required for HIV-1 RNA processing or expression. Knockdown of Sam68 and/or Slm2 had little to no effect on viral RNA cleavage or structural gene expression from transiently transfected reporters. Furthermore, depletion of Sam68 only slightly reduced Gag expression from a stably expressed proviral reporter. These results suggest that additional redundant proteins may be present that functionally replace Sam68 and Slm2. We defined a region encompassing the N-terminal GSG (GRP33, Sam68, Gld1) and KH RNA binding motif as the minimal region of Sam68 required to stimulate HIV-1 gene expression in 293 and 293T cells. The minimal mutant enhanced unspliced RNA cleavage in 293T, but not in 293 cells suggesting that Sam68 may act at other stage of the viral lifecycle to increase gene expression.

Human immunodeficiency virus type 1 (HIV-1), the pathogen responsible for the onset of acquired immuno-deficiency syndrome (AIDS) in humans has reached pandemic proportions. To this day, no cure is available for infection with this virus and the only treatment option for this chronic infection is the life-long adherence to anti-retroviral therapy. Efforts in the quest to control the worldwide AIDS pandemic include the search for an effective anti-HIV-1 vaccine. Providing hope in this endeavor are a few monoclonal antibodies possessing broad neutralizing characteristics (bnmAbs) that have been isolated from the sera of rare patients that have a delayed progression to AIDS. In this thesis, one of these bnmAbs, 2F5 is extensively characterized at the atomic level to better understand its binding and neutralization mechanism. In total, 27 crystal structures of the 2F5 Fab’ in complex with various peptides representing its linear gp41 membrane proximal external region (MPER) epitope are presented. Furthermore, expression of the 2F5 Fab in a bacterial system allowed to design mutants of the 2F5 Fab and therefore investigate the implication of specific domains of 2F5 in mediating binding and neutralization. Atomic level characterization of this immune complex revealed a somewhat promiscuous recognition of 2F5 for its 664DKW666 epitope as long as the following characteristics were conserved: the aspartate’s negative charge, the hydrophobic alkyl-pi stacking arrangement between the beta-turn lysine and tryptophan, and the positive charge of the former. Moreover, it was demonstrated that 2F5 has an elongated and flexible complementary determining region 3 loop of the heavy chain (CDR H3), which is required for neutralization and is involved in secondary binding interactions other than to its core linear epitope. These contributions will significantly help in guiding the structure-based design of an HIV-1 vaccine looking to elicit 2F5-like antibody responses.

The molecular mechanisms involved in the regulation of protease (PR) activity and human immunodeficiency virus type 1 (HIV-1) viral maturation are incompletely elucidated. To better understand the importance of the cleavage event between HIV-1 PR and reverse transcriptase (RT), we have selectively mutagenized specific residues at the junction between these genes to produce a PR-RT fusion protein in the context of a full-length proviral construct. Mutant viruses derived from COS-7 cells transfected with this construct were analyzed in regard to each of viral replication, maturation, and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existed as a PR:RT fusion protein in each of cellular and viral lysates. Interestingly, intracellular PR that existed within the PR-RT fusion protein not only remained functionally active, bid also processed HIV-1 precursor proteins with slightly increased efficiency as shown in time-course experiments in transfected COS-7 cells. In contrast, the RT component of the fusion protein was active at wild-type levels in in vitro and endogenous RT assays. Electron microscopy revealed that mutant viruses containing the cleavage site mutation between PR and RT possessed wild-type morphology. These viruses also displayed wild-type sensitivities to inhibitors of each of HIV-1 PR and RT activities. However, viruses containing the PR-RT fusion protein were 20-times less infectious than Wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed as a consequence of an additional mutation that interfered with frameshifting. Thus, unlike cleavage site mutations at the N-terminus of PR, a cleavage site mutation between PR and RT did not prevent proteolytic processing and abolish infectivity; rather, viruses containing PRAT fusion proteins were viable, in agreement with the notion that C-terminal liberation of PR is not as critical for

During HIV-1 assembly, the Gag structural protein specifically encapsidates two copies of viral genomic RNA in the form of a dimer. An RNA stem-loop structure (SL1) in the 5' untranslated region, known as the dimerization initiation site (DIS), is important for dimerization and packaging of HIV-1 genomic RNA; however, the mechanisms involved are not fully understood. The major goal of this PhD study was to further understand HIV-1 RNA dimerization, and to study the role of the Gag protein in the dimerization and packaging processes. Despite the known involvement of the DIS in RNA dimerization, DIS-mutated viruses still contain significant levels of dimerized RNA, and electron microscopy studies suggest that the RNA molecules are linked at the extreme 5' end. We show here that RNA sequences on both sides of the DIS are also required for HIVA genome dimerization, suggesting that multiple RNA elements are involved. We have also examined the contribution of specific amino acids within Gag to the dimerization and packaging processes. Previous work showed that partial deletion of the DIS impacted on viral replication capacity, but could largely be corrected by compensatory point mutations within Gag. To further elucidate the mechanism(s) of these compensatory mutations, we generated DIS mutants lacking the entire SL1, or only the SL1 loop sequences, and combined these deletions with various combinations of compensatory mutations. Analysis of virion-derived RNA showed that the relevant mutant viruses contained increased levels of spliced viral RNA compared to wild type, indicating that a defect in genome packaging specificity was present. However, this defect was corrected by our compensatory mutations, and a T121 substitution in p2 was shown to be solely responsible for this activity. These results suggest that the p2 spacer peptide plays a critical role in the specific packaging of viral genomic RNA. In summary, these findings provide new insig

Dendritic cells (DCs) are specialized members of the innate immune system that are responsible for the initiation of primary adaptive immune responses whose purpose is to resolve infection and inflammation. During most viral infections, mature dendritic cells present critical viral antigens to naïve T-cells within secondary lymphoid organs, resulting in the generation of an antigen-specific adaptive immune response and clearance of the virus. During infection with HIV-1 however, the virus is not cleared and a chronic systemic infection develops characterized by immune dysfunction, CD4+ T-cell depletion, systemic inflammation, and opportunistic infections. A growing body of evidence indicates that HIV-1 subversion of DCs contributes to both HIV-1 pathologies and viral dissemination. A number of similar effects by accessory HIV-1 peptides on DC physiology have also been reported. In vitro studies demonstrate that HIV-1 inhibits DC maturation and function. Ex vivo studies on the other hand describe partially mature, dysfunctional DCs collecting in secondary lymphoid organs. In vitro studies examining the effects of HIV-1-Tat and HIV-1-Vpr have described opposing effects on DC maturation. Therefore we undertook experiments to comprehensively describe the effects of HIV-1 and the Tat and Vpr accessory peptides on DC maturation and function. To understand the contributions of individual viral proteins to DC dysfunction we infected DCs with a dual tropic HIV-1 and examined phenotypic and functional changes after maturation with inflammatory cytokines. Following this we examined the influence of exogenous and endogenous HIV-1-Tat and HIV-1-Vpr on MDDC maturation and function using recombinant proteins and deletion mutant lab adapted HIV-1 strains. Live dual tropic HIV-1 was found to selectively inhibit aspects of phenotypic maturation as well as antigen capture and presentation functions. MDDC MAPK responsiveness to bacterial LPS remained intact however. Exogenous accessory HIV-1 Tat and Vpr did not affect MDDC phenotype but inhibited dextran endocytosis and viral peptide presentation. HIV-1-gp120 increased iMDDC maturation while blunting cytokine induced decreases in MDDC antigen capture abilities. The deletion of HIV-1-Tat did not affect MDDC phenotype, but was found to affect antigen capture decreases by R5 tropic HIV-1BaL. Deletion of HIV-1-Vpr likewise did not affect MDDC phenotype, however it was found to be influential in HIV-1 induced decreases in MDDC antigen presentation to autologous T-cells. These accumulated results indicate that HIV-1 subverts DC maturation and function through whole virus effects and individual accessory peptide influences. Understanding the mechanisms of DC dysfunction in HIV infection may provide some insight into infection prevention strategies and therapies leading to adaptive immune system activation and viral clearance.

The unusually long Cytoplasmic tail (CT) of Human Immunodeficiency Virus Type-1 (HIV-1) glycoprotein-41 (gp41) is highly conserved and engineered large truncations often render the virus non-infectious in a cell-type dependent manner. While large CT truncations occur infrequently in natural isolates, little is known about the mechanisms involved in infectious virions harbouring a large CT truncation. This thesis characterises RFgp34, a replication competent laboratory HIV-1 isolate with an acquired 100 amino acid CT truncation, and how it diverged from wildtype RF. The CT truncation and two possible compensatory mutations in Matrix (E40K and F44I) were introduced into the HIV-1 isolate NL4-3. These mutants were tested for infectivity, syncytia formation and glycoprotein incorporation into virions, alternative co-receptor usage and sensitivity to the fusion inhibitor T-20. Compared with RFwt, RFgp34-infected cultures displayed delayed viral replication kinetics in all cell types. Similar sized (MT-4 cells, PBMC) or larger and more numerous syncytia (Hut78 cells) were detected in RFgp34-infected cultures. Similar (Hut78 cells) or decreased (MT-4 cells, PBMC) amounts of glycoprotein was incorporated into RFgp34 virions, compared with RFwt virions. The increased syncytia in RFgp34-infected Hut78 cultures and the reduced glycoprotein incorporation into RFgp34 virions from MT-4 cells and PBMCs may explain the delayed RFgp34 replication kinetics. The Matrix E40K and F44I mutations were not able to directly compensate for the CT truncation to restore infectivity in Hut78 and MT-4 cells, as secondary mutations or the reversion of the CT truncation to a full-length CT were observed. In PBMCs the Matrix mutations alone were able to partially restore infectivity, suggesting specific mutations may compensate for the CT truncation in different cell types. None of the viruses utilised alternative HIV-1 co-receptors, nor were more resistant to T-20 than wildtype HIV-1 suggesting that the CT does not directly play a role in these viral functions. This thesis suggests that the sequence of mutations acquired by RFgp34 to compensate for the CT truncation and restore infectivity in multiple cell types may have occurred in a specific order and the evolution of RFgp34 to out-compete RFwt occurred over many passages.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.