Molecular and Genetic Characterization of Natural Variants of HIV-1 Nef Gene from North India and its Functional Implication in Down-Regulation of MHC-I and CD-4

Background: HIV-1 Nef is an important accessory protein with multiple effector functions. Genetic studies of the HIV-1 Nef gene show extensive genetic diversity and the functional studies have been carried out mostly with Nef derived from regions dominated by subtype B (North America & Europe). Objective: This study was carried out to characterize genetic variations of the Nef gene from HIV-1 infected individuals from North India and to find out their functional implications. Methods: The unique representative variants were sub-cloned in a eukaryotic expression vector and further characterized with respect to their ability to downregulate cell surface expression of CD4 and MHC-1 molecules. Results: The phylogenetic analysis of Nef variants revealed sequence similarity with either consensus subtype B or B/C recombinants. Boot scan analysis of some of our variants showed homology to B/C recombinant and some to wild type Nef B. Extensive variations were observed in most of the variants. The dN/dS ratio revealed 80% purifying selection and 20% diversifying selection implying the importance of mutations in Nef variants. Intracellular stability of Nef variants differed greatly when compared with wild type Nef B and C. There were some variants that possessed mutations in the functional domains of Nef and responsible for its differential CD4 and MHC-1 downregulation activity. Conclusion: We observed enhanced biological activities in some of the variants, perhaps arising from amino acid substitutions in their functional domains. The CD4 and MHC-1 down-regulation activity of Nef is likely to confer immense survival advantage allowing the most rare genotype in a population to become the most abundant after a single selection event.