The treatment of human immunodeficiency virus (HIV-1) infection faces the challenge of drug resistance. The high mutation rate of HIV-1 allows it to develop resistance against all available drugs. New mechanisms of intervention that do not succumb to failure through resistance are thus being explored. Mutagens that increase the viral mutation rate are a promising class of drugs. They can drive HIV-1 past a critical mutation rate, called the error threshold, and induce a catastrophic loss of genetic information. The treatment duration for a mutagen to drive HIV-1 beyond this error threshold is not yet estimated. We devise a detailed stochastic simulation of HIV-1 infection to estimate this duration. The simulations predict that the required duration is inversely proportional to the difference between the mutation rate induced by a mutagen and the error threshold. This scaling is robust to changes in simulation parameters. Using this scaling, we estimate the required duration of treatment with mutagens to be many years. Unfortunately, all available drugs, including mutagens, fail to clear the infection because HIV-1 establishes a reservoir of latently infected cells harbouring silent HIV-1 integrated genomes. A new \shock and kill" strategy that aims to activate latent cells and render them susceptible to immune killing or viral cytopathicity and thus to eradicate the HIV-1 latent reservoir has been suggested. Several latency reversal agents (LRAs) have been developed. Individual LRAs fail to show any decline in the HIV-1 latent reservoir in clinical trials. Combinations of LRAs have been tested in a few in-vitro and ex-vivo experiments. It has been found that in combination LRAs act synergistically. Finding the drug concentrations that yield the maximum synergy may be helpful in achieving a sterilizing cure. Here, we develop an intracellular model to estimate these drug concentrations. We choose drugs from two different classes of LRAs and show that our model captures quantitatively recent in-vitro experiments of their activity individually and in combination. With this model, we estimate the concentrations of the drugs required to obtain the maximum synergy. Strong CD8+ T cell responses against viruses have been associated with low levels of viremia. Elite controllers of HIV-1, who are known to have low or undetectable viremia, mount a cross-reactive CD8+ T cell response against the pathogen which controls viral mutation-driven escape from immune activity. These cross-reactive responses are against specific epitopes of HIV-1. Our goal was to examine whether such epitopes could be identified systematically so that a cross-reactive immune response could be induced by using these epitopes as immunogens. Immune recognition of an epitope involves two parts: presentation of the epitope, or peptide, by the major histocompatibility complex (MHC) molecules in the host and high a finity binding of the peptide-MHC complex with a T cell receptor (TCR). Immune escape could occur at either of these steps. Here, we examined the first step. We devise the following procedure to identify peptides that sustain HLA binding despite mutations. First, from the full length HIV-1 (HCV) proteome, we identify viral peptides that bind tightly with MHC molecules using the software NetMHCpan2.8. Next, we pick the peptides and their complementary MHC molecules that yield tight binding and mutate the peptides bit by bit to examine whether binding was compromised. We identify several viral peptide-MHC pairs that display tight binding despite all possible single mutations of the peptides both with HIV-1 and HCV. These peptides present candidates which can be tested for their TCR binding and cross-reactive immunogenic potential.

Since, its discovery over three decades ago, HIV has wrecked havoc worldwide. According to the UNAIDS report 2011, at present 34 million people is living with HIV and AIDS vaccine with broadly neutralizing activity still remains elusive. The envelope glycoproteins on the virion surface, is the most accessible component to the host immune system and therefore is targeted for vaccine design. However, the virus has employed various strategies to avoid the host immune response. The extremely high rate of mutations, extensive glycosylation of the envelope glycoprotein, conformational flexibility of the envelope, has made all the efforts aimed to design a broadly neutralizing immunogen futile. In Chapter1, we briefly discuss about the structural and genomic organization of the HIV-1 along with various strategies the virus has employed to evade the immune system. We also present the progress and failures encountered in the past three decades, on the way to design protective HIV vaccine and inhibitors. On the host cell surface, HIV-1 glycoprotein gp120 binds to the cell surface receptor CD4 and leads to the fusion of viral and host cellular membranes. CD4 is present on the surface of T-lymphocytes. It consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1−D4. sCD4 has been used as an entry inhibitor against HIV-1. However, this molecule could not neutralize primary isolates of the virus. Previously, from our lab, we had reported the design and characterization of a construct consisting of the first two domains of CD4 (CD4D12), that binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192−16202]. In Chapter 2, we describe the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L51I/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. All the mutations in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC50s as 4-domain CD4. These stabilized derivatives of human CD4 are useful starting points for the design of other more complex viral entry inhibitors. Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the env surface protein. Native env consists of a trimer of gp120−gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs)-directed neutralizing antibodies over non-neutralizing ones. One group of cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface and the other group claimed that the V1V2 loop region is far from the apex of the trimer. To further investigate the position of the V1V2 region, in the native envelope trimer, in Chapter 3, we describe the design and characterization of cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1V2 loop. h-CMP-V1cyc is one such variant in which residues 153 and 142 are the N- and C-terminal residues, respectively, of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity than wild-type gp120. It binds non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cyc1, bound the V1V2 loop-directed broadly neutralizing antibodies PG9 and PG16 with ~15-fold higher affinity than wild-type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support env models in which the V1V2 loops are proximal to the trimer interface. HIV-1 envelope (env) protein gp120 has approximately 25 glycosylation sites of which ~4 are located in the inner domain, ~7-8 in the V1/V2 and V3 variable loops and the rest in the outer domain (OD) of gp120. These glycans shield env from recognition by the host immune system and are believed to be indispensable for proper folding of gp120 and viral infectivity. However, there is no detailed study that describes whether a particular potential n-linked glycan is indispensable for folding of gp120.Therefore, in Chapter 4, using rationally designed mutations and yeast surface display (YSD), we show that glycosylation is not essential for the correct in vivo folding of OD alone or OD in the context of core gp120. Following randomization of the remaining four glycosylation sites, we isolated a core gp120 mutant, which contained a single inner domain glycan and retained yeast surface expression and broadly neutralizing antibody (bNAb) binding. Thus demonstrates that most gp120 glycans are dispensable for folding in the absence of gp41. However in the context of gp160, we show that all core gp120 glycans are dispensable for folding, recognition of bNAbs and for viral infectivity. We also show that deglycosylated molecules can serve as a starting point to re-introduce epitopes for specific glycan dependent bNAbs. Several of these constructs will also be useful for epitope mapping and env structural characterization. Glycosylation of env is known to inhibit binding to germline precursors of known bNAbs. Hence the present results inform immunogen design, clarify the role of glycosylation in gp120 folding and illustrate general methodology for design of glycan free, folded protein derivatives. On the virion surface env glycoproteins gp120 and gp41 interact via non-covalent interactions and form trimers of heterodimers. Upon binding cell surface receptor CD4 and co-receptor CCR5/CXCR4, gp120 and gp41 undergo a lot of conformational changes, which ultimately lead to the fusion of viral and cellular membranes by formation of six-helix bundle in gp41. High resolution structural information is available for core gp120 and post-fusion six-helix bundle conformation of gp41. However, the structural information about the native gp120:gp41 interface in the native trimer is lacking. In Chapter 5, we describe the design and characterization of various single chain derivatives of gp120 inner doamin and gp41. Among the designed constructs, gp41-id2b is folded but is a mixture of dimer and monomer under native conditions. To facilitate, trimer formation, two trimerization domains (h-CMP and Foldon) were individually fused to the N-terminus of gp41-id2b to generate h-CMP-gp41-id2b and Foldon-gp41-id2b. Although, these molecules were proteolytically more stable than gp41-id2b, they did not form trimer under native conditions. All the single chain derivatives were designed based on the crystal structure of gp120, which was devoid of C1 and C5 domains (PDBID 1G9M). A new set of constructs to mimic the native gp120:gp41 interface will be designed and characterized based on the recently solved crystal structure of gp120 with the C1 and C5 domains (PDBID 3JWD and 3JWO). Helix-helix interactions are fundamental to many biological signals and systems, found in homo- or hetero-multimerization of signaling molecules as well as in the process of virus entry into the host. In HIV, virus-host membrane fusion during infection is mediated by the formation of six helix bundle (6HB) from homotrimers of gp41, from which a number of synthetic peptides have been derived as antagonists of virus entry. Yeast surface two-hybrid (YS2H) system is a platform, which is designed to detect protein-protein interactions occurring through a secretory pathway. In Chapter 6, we describe the use of aYS2H system, to reconstitute 6HB complex on the yeast surface and delineate the residues influencing homo-oligomeric and hetero-oligomeric coiled-coil interactions. Hence, we present YS2H as a platform for facile characterization of hetero-oligomeric interactions and design of antagonistic peptides for inhibition of HIV and many other enveloped viruses relying on membrane fusion for infection, as well as cellular signaling events triggered by hetero-oligomeric coiled coils. However, using this YS2H platform, the native hetero-oligomeric complex of gp120 and gp41 could not be captured. In Appendix 1, we report cloning, expression and purification of PΔGgp120 and ΔGgp120 from methylotrophic yeast Pichia pastoris. PΔGgp120 was purified as a secreted protein. However, in electrophoretic analyses the molecule ran as a heterogeneous smear. Further optimization of the purification protocol and biophysical characterizations of this molecule will be performed in future. In Appendix 2, gp41 variants were expressed on the yeast cell surface as a C-terminally fused protein and its interaction with externally added gp120 was monitored by FACS. The surface expression of the gp41 constructs was poor and they did not show any interaction with gp120.

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est l’agent étiologique du SIDA, un rétrovirus complexe encodant les protéines accessoires : Nef, Vif, Vpr et Vpu. La fonction principale de ces protéines est de moduler l’environnement cellulaire afin de promouvoir la réplication virale. Les travaux présentés dans cette thèse portent sur la protéine virale Vpr, une protéine bien connue pour son activité d’arrêt du cycle cellulaire en phase G2/M dans les cellules en division et pour l’avantage réplicatif qu’elle confère au virus durant l’infection de cellules myéloïdes. Les évènements sous-jacents à ces deux activités restent pour l’heure mal compris. Le but des travaux regroupés dans cet ouvrage est d’identifier de nouveaux facteurs cellulaires pouvant éventuellement expliquer les activités de Vpr précédemment décrites. Pour ce faire, nous avons utilisé une approche d’identification des partenaires de proximité par biotinylation, appelée BioID. L’avantage du BioID est de permettre un marquage in cellulo des protéines à proximité de la protéine d’intérêt. La mise en place et la caractérisation de cette approche font l’objet de la première section de cette thèse. En utilisant cette approche, nous avons défini un réseau de 352 partenaires cellulaires de la protéine Vpr. Parmi ces partenaires de Vpr, plusieurs sont organisés sous forme de complexes ou réseaux protéiques incluant notamment le complexe promoteur de l’anaphase/cyclosome (APC/C) et les centrosomes. Étant donné que le complexe APC/C est l’un des principaux régulateurs du cycle cellulaire, nous avons décidé d’analyser sa relation avec Vpr. Nous avons découvert que Vpr formait un complexe non seulement avec APC1, une sous-unité essentielle du complexe APC/C, mais aussi avec les coactivateurs (CDH1 et CDC20) de ce complexe. Nous avons par la suite démontré que Vpr induisait la dégradation d’APC1 et que celle-ci pouvait être prévenue par une double-mutation N28S-G41N de Vpr. Cette dégradation d’APC1 ne semblerait pas être reliée aux activités précédemment décrites de Vpr. Ces travaux font l’objet de la seconde section de cette thèse. Enfin, dans une troisième section, des travaux effectués en collaboration et analysant la relation entre les centrosomes et Vpr sont présentés. Cette thèse identifie 200 nouveaux partenaires de Vpr, ouvrant la porte à l’exploration de nouvelles cibles et activités de Vpr. Elle décrit également une nouvelle cible de Vpr : le complexe APC/C. Globalement nos résultats contribuent à une meilleure compréhension de la façon dont le VIH-1 manipule l’environnement cellulaire de l’hôte à travers la protéine virale Vpr. / Human immunodeficiency virus (HIV-1) is the AIDS causal agent. This complex retrovirus encodes several accessory proteins; namely Nef, Vif, Vpr and Vpu; whose functions are to manipulate the cellular host environment in order to favor HIV-1 viral replication. This thesis focused on Vpr whose main activities are to induce a cell cycle arrest in the G2/M phase in dividing cells and to provide a replicative advantage to HIV-1 during infection of myeloid cells such as macrophages. The cellular mechanisms underlying these two activities are up to now misunderstood. The main goal of the work presented in this thesis is to identify new cellular factors that could potentially explain the previously described Vpr activities. To do so, we used the proximity labelling approach called BioID. The main strength of BioID is to tag in cellulo partners of the protein of interest. The development as well as optimization of the BioID approach is presented in the thesis first section. Using BioID, we defined a network containing 352 cellular partners in close proximity with the viral protein Vpr. Amongst these cellular partners, several were organized into protein complexes or networks such as the anaphase promoting complex/cyclosome (APC/C) or the centrosome. Given that APC/C is a cell cycle master-regulator, we analyzed the interplay governing Vpr and APC/C interactions. We first demonstrated that Vpr could form a complex containing the scaffolding subunit APC1. APC/C coactivators, namely CDH1 and CDC20, could also be found in association with Vpr. We next showed that Vpr was inducing APC1 degradation and that Vpr residues N28 and G41 were essential to this activity. Surprisingly, the APC1-Vpr interplay does not relate to previously described Vpr activities. This work is presented in the second section of this thesis. Lastly, in the third section, a work done in collaboration analyzed the interplay between Vpr and the centrosomes. In this thesis we identified 200 new potential partners of Vpr, opening the doors to discover novel Vpr targets and activities. This thesis also defined APC/C as new Vpr target. Taken together our results allow a better understanding on how HIV-1 modulates the cellular environment by using the viral accessory protein Vpr.