Genetic Characterization of Natural Variants of Vpu from HIV-1 Infected Individuals from Northern India and Their Impact on Virus Release and Cell Death

Background: Genetic studies reveal that vpu is one of the most variable regions in HIV-1 genome. Functional studies have been carried out mostly with Vpu derived from laboratory adapted subtype B pNL 4-3 virus. The rationale of this study was to characterize genetic variations that are present in the vpu gene from HIV-1 infected individuals from North-India (Punjab/Haryana) and determine their functional relevance. Methods: Functionally intact vpu gene variants were PCR amplified from genomic DNA of HIV-1 infected individuals. These variants were then subjected to genetic analysis and unique representative variants were cloned under CMV promoter containing expression vector as well as into pNL 4-3 HIV-1 virus for intracellular expression studies. These variants were characterized with respect to their ability to promote virus release as well as cell death. Results: Based on phylogenetic analysis and extensive polymorphisms with respect to consensus Vpu B and C, we were able to arbitrarily assign variants into two major groups (B and C). The group B variants always showed significantly higher virus release activity and exhibited moderate levels of cell death. On the other hand, group C variants displayed lower virus release activity but greater cell death potential. Interestingly, Vpu variants with a natural S61A mutation showed greater intracellular stability. These variants also exhibited significant reduction in their intracellular ubiquitination and caused greater virus release. Another group C variant that possessed a non-functional beta-TrcP binding motif due to two critical serine residues (S52 and S56) being substituted with isoleucine residues, showed reduced virus release activity but modest cytotoxic activity. Conclusions: The natural variations exhibited by our Vpu variants involve extensive polymorphism characterized by substitution and deletions that contribute toward positive selection. We identified two major groups and an extremely rare b-TrcP binding motif mutant that show widely varying biological activities with potential implications for conferring subtype-specific pathogenesis.