Chapter 1: Introductionn Staphylococci are gram positive rotund bacteria that grow in clusters; and hence get their name. The genus of Staphylococcus comprises of over 30 species of which S. epidermidis and S.aureus are significant in their interaction with humans and are known to cause diseases. S.aureus invades various soft tissues and causes a vast multitude of diseases spanning from simple boils and abscesses to osteomyelitis and endocarditis, which can become fatal upon the onset of bacteremia and toxic shock. S. aureus has also been established as one of the leading causes of nosocomial infections especially because of their multi-drug resistant traits and their ability to colonize prosthetic devices and catheters. The increasing incidence of the multi-drug resistant strains and the rising prevalence of community acquired S. aureus infections mandates a comprehensive understanding of the pathogen and its biology, its intracellular fate and the defense mechanisms in the host. Towards this end, we have attempted to delineate some aspects of the pathogen’s virulence and the host responses to them. S. aureus normally inhabits the skin and mucosal surfaces as a commensal. Upon the onset of permissive circumstances it turns into an opportunistic pathogen. Immuno-compromised conditions or breach of skin can serve as the portals of entry for the pathogen. Upon entry, the bacteria encounter macrophages as the first line of defense in the host. Macrophages appear at the site of infection and phagocytose the bacteria, subjecting the pathogen to phagolysosomal degradation which facilitates antigen presentation and pathogen clearance. As part of their immune evasion mechanism, various pathogens are known to adopt a multitude of strategies to subvert this fate and survive in the host cells. This dissertation work aims at gaining insight into the staphylococcus-macrophage interaction in the ongoing host-pathogen duel, to gain better understanding about the pathophysiology and etiology of the disease. Chapter 2: Intracellular Trafficking of Staphylococcus aureus in Macrophages Successful targeting of the pathogen necessitates a comprehensive understanding of its biology and physiology in its interactions with the host. With this objective we undertook a study to uncover the intracellular niche of S. aureus in RAW264.7 murine macrophage-like cells. Any invading pathogen once internalized by the macrophage is contained in a phagosome, which undergoes progressive acidification and maturation from the early endosome to late endosome and ultimately fuses with the phagolysosome, where where the invading pathogen is subject to degradation. Through exhaustive electron microscopy of the infected macrophages, we show that S. aureus is present as a single bacterium per vacuole through the entire period of infection. We have further monitored the intracellular trafficking of the bacteria in the macrophage through confocal studies with endosomal markers which serve as indicators of vesicle maturation. Soon after the onset of the infection, the bacteria were found to be present in the early endosome (EEA-1 positive vesicles) which gradually matured into LAMP1 positive, late endosomal vesicles. However, only a small fraction of the bacteria containing vesicles were found to fuse with the lysosomes, suggesting that the bacteria prevented phagolysosomal fusion. We further observed that the bacteria did not prevent the acidification of the vesicles they resided in, but only limited their fusion with the lysosome. Taken together, our studies delineating the intracellular niche of S. aureus in RAW macrophages revealed that the pathogen has successfully evolved immune evasion mechanisms to overcome its phagolysosomal relegation. Chapter 3: Staphylococcus aureus Succumbs to the Hepcidin in Murine Macrophage We have further attempted to study the intracellular fate of the bacteria in macrophages towards gaining greater insight into its biology. Our studies on the intracellular fate of S. aureus in RAW264.7 cells revealed a distinct biphasic fate of the bacteria. The pathogen was found to replicate initially and this proliferative phase was subsequently followed by a gradual fall in its numbers. Interestingly however, the pathogen is never found to be cleared from the system suggesting the presence of a residual infective pool in the macrophages. We thus explored the possible mechanisms which could attribute to this biphasic intracellular fate of the bacteria. Macrophages come armed with a rich repertoire of defense mechanisms to incapacitate the invading pathogens. They have in their arsenal, reactive oxygen (ROS) and nitrogen species (RNS) and many potent anti-microbial peptides, apart from the lysosomal machinery, to degrade the invading pathogen. Upon investigation, we find that the RAW macrophages do not mount a ROS/RNS response when infected with S. aureus. Induction of these responses in the macrophage by alternate means further reveals that the pathogen is recalcitrant to death by these oxidative/nitrosative bursts. Of the antimicrobial peptides (AMPs) harbored by macrophages, we find that Hepcidin is up-regulated upon infection with S. aureus. Hepcidin is a peptide which is known to have a key regulatory role in iron homeostasis in addition to its potent antimicrobial functions. Since Hepcidin is known to be induced upon increased iron availability; we pre-treated the host cells with iron and monitored the effect of the same on bacterial fate. As expected, we observed that Hepcidin induction by pre-treatment with iron equips the macrophage to counter the pathogen better and thus leads to hastened and heightened clearance of the bacteria. This induction of hepcidin is significant at the mRNA and protein levels and is also corroborated by increased co-localisation of the bacteria with the anti-microbial peptide. Our studies thus identify hepcidin as a key line of the host defense towards countering the bacterial infection thus explaining the near complete bacterial clearance observed. Chapter 4: Global gene expression studies offering insight into potential immune evasion strategies of S.aureus in countering host offences. The interactions between host and the pathogen are multi-layered with the involvement of numerous players and many signaling cascades. In this light, we have attempted to get a holistic view of the host-pathogen interplay through microarray studies. These global profiling studies were aimed at identifying the important players in bacterial virulence and the macrophage response factors involved in countering the same in the context of S. aureus infection. The array was uniquely designed to incorporate both bacterial and host probes so as to facilitate parallel analysis of the host and pathogen gene expression profiles in the same sample. The expression profiling studies were carried out at three time points which represent the key phases of the bacterial infection viz. internalization, replication and clearance. A comprehensive analysis of the bacterial and host gene expression profiles under these phases provided insights into bacterial virulence and the host’s strategies to counter the same. We observe a large scale metabolic shut down in S. aureus subsequent to its internalization. We find the distinct up-regulation of a small subset of genes, majority of which are as yet uncharacterized. Amongst these were a few well-characterized virulence genes which remained active, representing the bacterial strategies to subvert the host immune response. The large scale down-regulation of gene expression can be possibly explained as the adaptation of the bacteria to the available metabolites and its submission to a quiescent phase of existence in the macrophage. In parallel, the host system exhibits the induction of TNF-α and up regulation of TLR2 and Nod2, which are typically triggered by a gram-positive infection. But simultaneously, we also observed a marked increase in the expression of anti-apoptotic and anti-inflammatory responses. This was re-iterated by a significant down-regulation in some of the pro-inflammatory, pro-apoptotic and antigen presentation involved genes and processes. We further find that the time course of the infection did not largely influence the gene expression kinetics. The macrophages were influenced and committed to a fate conducive for the bacteria fairly early in the infection regime. Thus, our studies of the expression profiles of the pathogen and the host under the different phases of the infection provide us with a comprehensive understanding the strategies of bacterial offense and host defenses thereby offering a window into this fascinating world of host-pathogen interactions. Chapter 5: Conclusion To summarize, we have attempted to study the intracellular fate of the S. aureus pathogen in macrophages. Our studies suggest that the bacterium attempts to evade clearance by the host immune system by actively preventing fusion with the lysosomal vesicles. We also find that despite these defenses, the pathogen appears to succumb to the host immune system as it is targeted by Hepcidin, an anti-microbial peptide. The lack of complete bacterial clearance under these conditions is however suggestive of an underlying strategy by the pathogen, possibly to maintain a chronic infective state in the host system. The microarray studies, in addition, shed light on the other possible immune evasion strategies that S.aureus might be employing to escape the host offences. The results are indicative of the bacteria influencing anti-apoptotic, anti-inflammatory and antigen presentation responses and thereby prolonging its survival in the macrophage. In conclusion, given the fact that the macrophages are itinerant cells with a long life span, the light thrown by our findings of the various immune evasion strategies that S.aureus is adopting; it suggests that the macrophages could serve as potential carriers which could account for the dissemination of the infection to new sites, which has perpetually been a major concern for any Staphylococcal infection.

Suite à une agression inflammatoire, telle que le choc septique, des brulures graves ou un traumatisme sévère, le système immunitaire répond par une modulation massive du transcriptome dans le sang. On propose d’explorer un autre répertoire que l’expression des gènes et de s’intéresser aux éléments répétés du génome, peu étudiés dans ces contextes, et plus particulièrement aux rétrovirus endogènes humains (HERV). Ils représentent plus de 8% du génome chez l’Homme. Certains sont exprimés dans des situations similaires à l’agression inflammatoire (cancer, maladies auto-immunes) et ont un impact sur la réponse immunitaire.Dans ce travail, nous cherchons à décrire et comprendre la contribution des HERV, au sein de la réponse immunitaire de l’hôte à l’agression inflammatoire. Pour cela, nous avons développé des méthodes et outils spécifiquement dédiés à la description du HERVome, au niveau génomique et transcriptomique. Nous montrons que les HERV sont exprimés dans le sang, modulés chez les patients, et que certains pourraient jouer un rôle sur l’expression de gènes de la réponse immunitaire situés à proximité. Nous évaluons également le polymorphisme de présence des HERV dans le génome de plus de deux mille individus répartis dans les populations humaines. On met en évidence que le polymorphisme HERV est globalement important, qu’il est lié à la population d’appartenance et que certains loci sont absents dans la majorité des génomes étudiés. Finalement, par différentes approches, nous identifions des associations entre gènes de la réponse immunitaire et HERV, suggérant que ces éléments peuvent jouer un rôle important dans la réponse de l’hôte à l’agression inflammatoire / Following inflammatory injury, like a septic shock, severe burn or important trauma, the immune system responds by a massive modulation of its transcriptome in the blood. We propose to explore another repertoire than gene expression and to focus on repeated elements, especially on HERVs. They represent more than 8% of the human genome. HERVs are expressed in similar settings (cancer or auto-immune diseases) and impact immune response. In this project, we describe and aim to better understand the HERV contribution in host immune response, following inflammatory aggression. To bring elements of response, we developed specifically dedicated tools to describe the HERVome, either at genomic or transcriptomic level. We show HERVs are expressed in blood in these settings, modulated in patients and could play a role on nearby gene expression. We also evaluate the polymorphism of presence of HERV loci on more than two thousands individuals, grouped into human populations. We show an important HERV polymorphism, that it is population-specific, and that some loci are absent in the majority of the analyzed genomes.Finally, with different approaches, we identify associations between immune-response genes and HERVs, suggesting these elements can play a role in host immune response following inflammatory aggressions

Mycobacterium tuberculosis, the etiological agent of tuberculosis, has adapted with the host environment and evolved to survive in harsh conditions in the host. The pathogen has successfully evolved strategies not only to evade the host immune system but also to thrive within the host cells. Upon infection, the pathogen is either cleared due to the host immune response, or it survives and causes active tuberculosis (TB) infection. In a number of cases however, the pathogen is neither killed nor does it actively proliferate, but it remains dormant in the host until the environment becomes favorable. This dormant state of pathogen is responsible for latent TB infection (LTBI). WHO reports indicated that as much as a third of the whole world’s population is exposed to the pathogen, of which a significant proportion could be latently infected (WHO report, 2015). These individuals do not show symptoms of active TB infection and hence are difficult to detect. The latent TB infected (LTBI) individuals serve as a reservoir for the pathogen, which can lead to epidemics when the conditions change. Hence, it is necessary to understand the host -pathogen interactions during LTBI, as this might provide clues to developing new strategies to detect and curb a latent infection. Host-pathogen interactions are multifaceted, in which both species attempt to recognize and respond to each other, all of these through specific molecules making distinct interactions with the other species. The outcome of the infection is thus decided by a complex set of host-pathogen interactions. The complexity arises since a large number of molecular components are involved, also multiplicity of interactions among these components and due to several feedback, feed forwards or other regulatory or influential loops within the system. The complexity of biological systems makes modeling and simulation an essential and critical part of systems– level studies. Systems biology studies provide an integrated framework to analyze and understand the function of biological systems. This work addresses some of these issues with an unbiased systems-level analysis so as to identify and understand the important global changes both in the host and in the pathogen during LTBI. The broad objectives of the work was to identify the key processes that vary in the host during latent infection, the set of metabolic reactions in the host which can be modulated to control the reactivation of infection, global adaptation in Mycobacterium tuberculosis (Mtb) and then to utilize this knowledge to identify strategies for tackling latent infection. A review of literature of the current understanding of latency from the pathogen and the host perspective is described in chapter 1. From this, it is clear that most available studies have focused on the role of individual molecules and individual biological processes such as granuloma formation, toll-like receptor signaling, T cell responses as well as cytokine signaling, in either initiating or maintaining a latent infection, but there is no report till date about whether and how these processes are connected with each other. While transcriptome based studies have identified lists of differentially expressed genes in LTBI as compared to healthy controls, no further understanding is currently available for many of them, regarding the processes they may be involved in and what interactions they make, which may be important for understanding LTBI. The first part of the work is a systematic meta-analysis of genome-scale protein interaction networks rendered condition-specific with transcriptome data of patients with LTBI, which has provided a global unbiased picture of the transcriptional and metabolic variations in the host and in the pathogen during the latent infection. To start with, publicly available gene expression data related to LTBI, active TB and healthy controls were considered. In all, 183 datasets summing up to 105 LTBI, 41 active TB and 37 healthy control samples were analyzed. (Chapter 2). Standard analysis of the transcriptome profiles of these datasets indicated that there was zero overlap among them and that not a single gene was seen in common among all datasets for the same condition. An extensive human protein-protein interaction network was constructed using information available from multiple resources that comprehensively contained structural or physical interactions and genetic interactions or functional influences. Nodes in this network represented individual proteins and edges represented interactions between pairs of nodes. The identity of each node and the nature of interaction of each edge along with the type of evidence that was used as the basis for drawing the edge, was collated for the network. The gene expression data was integrated into the human protein-protein interaction (PPI) network for each condition, which essentially had weighted nodes and directed edges, specific to that condition, from which specific comparative networks were derived. The highest ranked perturbations in LTBI were identified through a network mining protocol previously established in the laboratory. This involved computing all versus all shortest paths on the comparative network, scoring the paths based on connectedness and various centrality measures of the nodes and the edges and finally ranking the paths based on the cumulative path scores. Intriguingly, the top-ranked set of perturbations were found to form a connected sub-network by themselves, referred to as a top perturbed sub-network (top-net), indicating that they were functionally linked or perhaps even orchestrated in some sense. Th17 signaling appears to be dominant. About 40 genes were identified in the unique set of LTBI condition as compared to the active TB condition, and these genes showed enrichment for processes such as apoptosis, cell cycle as well as natural killer cell mediated toxicity. Construction and analysis of a miRNA network indicated that 32 of these have strong associations with miRNA explaining the role of the latter in controlling LTBI. 3 other genes from the top-net are already established drug targets for different diseases with known drugs associated with them, which are BCL2, HSP90AA1 and NR3C1. These 3 proteins can be explored further as drug targets in LTBI whose manipulation using existing drugs may result in inhibiting the underlying biological process and thereby result in disturbing the state of latency. As a second objective, global variations in the host transcriptome were identified during ascorbic acid induced dormancy (Chapter 3). Ascorbic acid or Vitamin C is a nutrient supplement required in the diet. This organic compound has a known antioxidant property, as it is known to scavenge the free radicals. In a recent study, Taneja et al, demonstrated that Vitamin C could induce dormancy in Mtb. On similar lines, experiments were done in THP-1 cells infected with Mtb to determine the host responses during ascorbic acid (AA) induced dormancy. The raw gene expression data was provided by our collaborator Prof. Jaya Tyagi that included 0 hour, 4 days and 6 days time points with infection and vitamin C versus infection alone or vitamin C alone as controls. The transcriptome data was normalized and integrated into the human PPI network as described for the meta-analyses. It was experimentally determined that ascorbic acid induces dormancy in 4 days post infection. The top-ranked paths of perturbation were analyzed and compared for three different conditions: (i) uninfected condition, (ii) AA treated and infected condition, and (iii) AA, isoniazid and infected condition. The dormant pathogen is known to be drug-tolerant and thus as a marker for the state of dormancy, the lack of effect of isoniazid is also monitored in the infected host cells. The analysis revealed that there were some broad similarities as compared to LTBI from patient samples but AA induced dormancy in cell lines stood out a separate group indicating that there were significant differences such as involving Interferon Induced Transmembrane Proteins (IFITMs), vacuolar ATPase as well as GDF15, which belongs to TGF-beta signaling pathway. The highest ranked perturbed paths contained genes involved in innate immune responses of which ISG15, IFITMs, HLAs and ATPases emerge as the most altered in the dormant condition. CCR7 emerges as a key discriminator, which is subdued in the latent samples but highly induced in infection conditions. Pathway-based analysis of different conditions showed that oxidative stress, glutathione metabolism, proteasome degradation as well as type II interferon signaling are significantly up-regulated in AA induced dormancy. The dormant bacteria reside in the host cells and are known to modulate the host metabolism for their own benefit. So, the third objective was to understand the metabolic variations in the host during LTBI (Chapter 4). A genome-scale metabolic (GSM) model of alveolar macrophage was used in this study. The metabolic model contains information of the reactions, metabolites and the genes encoding enzymes that catalyze a particular reaction. Flux balance analysis (FBA), a constraint-based metabolic modeling method, is used for analyzing the alterations in the metabolism under different infection conditions. In order to mimic the physiological condition, gene expression data was used for constraining the bounds of the reactions in the model. Two different expression studies were used for analysis: GSE25534 (from Chapter 2) and ascorbic acid induced dormancy (Chapter 3). The analysis was carried out for latent TB versus healthy control and latent TB versus active TB to identify the most altered metabolic processes in LTBI. Differences in fluxes between the two conditions were calculated. A new classification scheme was devised to categorize the reactions on the basis of flux differences. In this chapter, higher fluxes in LTBI condition were identified for reactions involved in transport of small metabolites as well as amino acids. Solute carrier proteins responsible for the transport of the metabolites were identified and their biological significance is discussed. Reduced glutathione (GSH), arachidonic acid, prostaglandins, pantothenate were identified as important metabolites in LTBI condition and their physiological role has been described. Sub-system analysis for different conditions shows differential regulation for arachidonic acid metabolism, fatty acid metabolism, folate metabolism, pyruvate metabolism, glutathione metabolism, ROS detoxification, triacylglycerol synthesis and transport as well as tryptophan metabolism. From the study, transporter proteins and reactions altered during LTBI were identified, which again provide clues for understanding the molecular basis of establishing a latent infection. Mycoabcterium tuberculosis is known to undergo dormancy during stress conditions. In this chapter, the main objective was to identify the global variations in the dormant Mtb (Chapter 5). To carry out the analysis, the Mtb PPI network was constructed using information from available resources. Gene expression data of two different dormancy models, Wayne growth model and multiple-stress model, were used for the study. To identify the key players involved in reversal of dormancy, the transcriptome data of reaeration condition was also used. In this study, the Max-flow algorithm was implemented to identify the feasible paths or flows in different condition. The flows with higher scores indicate that more information is traversed by the path, and hence is important for the study. From the analysis of Wayne growth model (hypoxia model), important transcriptional regulators such as SigB, SigE, SigH, regulators in the two-component system such as MprA, MtrA, PhoP, RegX3 and TrcR were identified in stress condition. Multiple-stress model studied the growth of bacteria in low oxygen concentration, high carbon dioxide levels, low pH and nutrient starvation. The gene expression data was integrated in the Mtb PPI network and implementation of Max-flow algorithm showed that MprA, part of the MprA-MprB two-component system, is involved in the regulation of persistent condition. WhiB1 also features in the paths of dormant condition and its role in persistence can be explored. In reaeration model, WhiB1 and WhiB4 are present in the top flows of this condition indicating that the redox state is perturbed in the pathogen and the interactions of these proteins are important to understand the reversal of dormant condition. From the study, Rv2034, Rv2035, HigA, Rv1989, Rv1990 and Rv0837 proteins belonging to toxin-antitoxin systems were also identified in the dormant bacteria, indicating their role in adaptation during stress condition. The role of Rv2034 has been studied in persistence, but the function of other proteins can be analyzed to provide new testable hypotheses about the role of these proteins in dormancy. Thus, the flows or paths perturbed during dormancy were identified in this study. To get a better understanding of the metabolic network active in mycobacteria under different conditions, experiments were performed in Mycobacterium smegmatis MC2 155. The non-pathogenic strain of genus Mycobacteria, Mycobacterium smegmatis, is used as a surrogate to carry out molecular biology studies of Mtb. Mycobacterium smegmatis MC2 155 (Msm) is the commonly used laboratory strain for experimental purpose. In order to obtain a clear understanding of how comparable are the metabolic networks between the virulent M. tuberculosis H37Rv and the model system Msm, the latter model is first studied systematically. In Chapter 6, first the functional annotation of the Msm genome was carried out and the genes were categorized into different Tuberculist classes based on homology with the Mtb genome. A high-throughput growth characterization was carried out to characterize the strain systematically in terms of different carbon, nitrogen or other sources that promoted growth and thus served as nutrients and those that did not, together yielding a genome-phenome correlation in Msm. Gene expression was measured and used for explaining the observed phenotypic behavior of the organism. Together with the genome sequence, the transcriptome and phenome analysis, a set of about 257 different metabolic pathways were identified to be feasible in wild-type Msm. About 284 different carbon, nitrogen source and nutrient supplements were tested in this experiment and 167 of them supported growth of Msm. This indicates that the compounds enter the cells and are metabolized efficiently, thus yielding similar phenotypes. The expressed genes and metabolites supporting growth were mapped to the metabolic network of Msm, thus helping in the identification of feasible metabolic routes in Msm. A comparative study between Msm and Mtb revealed that these organisms share similarity in the nutrient sources that are utilized for growth. The study provides experimental proof to identify the feasible metabolic routes in Msm, and this can be used for understanding the metabolic capability in the two organisms under different conditions providing a basis to understand adaptations during dormancy. In the last part of the work presented in this thesis, the metabolic shift in the pathogen was studied using a genome-scale metabolic model of Mtb (Chapter 7). The model contains information of the reactions, metabolites and genes involved in the reactions. Flux balance analysis (FBA) was carried out by integrating normalized gene expression data (Wayne model and multiple-stress model transcriptome considered in Chapter 5) to identify the set of reactions, which have a higher flux in the dormant condition as compared to the control replicating condition. Glutamate metabolism along with propionyl CoA metabolism emerge as major up-regulated processes in dormant Mtb. Next, with an objective of identifying essential genes in dormant Mtb, a systematic in silico single gene knock-out analysis was carried out where each gene and it's associated reaction was knocked out of the model, one at a time and the ability of the model to reach its objective function assessed. About 168 common genes in Wayne model and multiple-stress model were identified as important in Mtb after the knockout analysis. Essentiality is in essence a systems property and requires to be probed through multiple angles. Towards this, essential genes were identified in Mtb using a multi-level multi-scale systems biology approach. About 283 genes were identified as essential on the basis of combined analysis of transcriptome data, FBA, network analysis and phyletic retention studies in Mtb. 168 genes identified as important in dormant Mtb were compared with 283 essential genes and about 91 genes were found to be essential. Finally, among the set of essential genes, those that satisfy other criteria for a drug target were analyzed using the list of high-confidence drug targets of Mtb available in the laboratory along with their associated drug or drug-like molecules. 38 out of the 168 important genes in Mtb were found to have one or more drugs associated with them from the DrugBank database. Colchicin-Rv1655, Raloxifene-Rv1653, Bexarotene-Rv3804, Rosiglitazone-Rv3804 are top-scoring drug-target pairs that can be explored for killing dormant bacilli. The study has thus been useful in identifying important proteins, reactions and drug targets in dormant Mtb. In summary, the thesis presents a comprehensive systems-level understanding of various aspects of host responses and pathogen adaptation during latent TB infection. Key host and pathogen factors involved in LTBI are identified that serve as useful pointers for deriving strategies for tackling a latent infection.

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.