Engineering of HIV-1 and HCV Entry Inhibitor Lectins and Understanding the Atomic Level Details of HIV Cellular Entry Proces
Microvirin (MVN) is a known anti-HIV lectin that consists of two structural domains with only one domain involved in binding to glycan epitopes on the surface of the virus. Generally, size and chemical heterogeneity are the two major contributing factors in immunogenicity of a protein. Considering these factors, we designed two types of MVN variants, one consisting of only the carbohydrate-binding domain with size almost half of the parent protein and the second consisting of two domains with identical amino acid sequence. We produced several mutants of each type of variants to optimize folding. Finally, we were able to engineer a variant that potently inhibited HIV and HCV in viral infectivity assays and showed significantly less cytotoxic and immunogenic effects as compared to MVN. We named this protein as LUMS1 that represents a potential drug candidate against HIV and HCV particularly co-infections of these viruses. In the second project, we engineered a soluble form of the CCR5 receptor by incorporating artificial linkers of amino acid sequences connecting the extracellular regions of the receptor in place of hydrophobic trans-membrane regions and optimized its functional conformation by in vitro folding. We also produced 13C15N-labelled V3 domain of HIV-1 glycoprotein by expressing its gene along with a fusion protein. We determined through SPR and NMR chemical shift perturbation experiments that only the cyclic form of V3 could bind to CCR5 with KD values of around 200 μM. Overall, in this study we successfully engineered a lectin that potently inhibits HIV-1 and HCV cellular entry, and with its extremely low cytotoxicity and negligible potential immunogenicity it represents a promising candidate for anti-viral drug development. Moreover, we obtained several key information related to the binding of the V3 domain to its cellular receptor.
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