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.