Abstract
BackgroundGenetic 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.MethodsFunctionally 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.ResultsBased 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 β-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.ConclusionsThe 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 β-TrcP binding motif mutant that show widely varying biological activities with potential implications for conferring subtype-specific pathogenesis.
Highlights
The pathogenesis of HIV infection is remarkably different from other primate lentiviruses in many ways
To explore the genetic and functional implications of natural variations displayed by vpu alleles in HIV-1 infected patients (Clinical parameters summarized in Table 1), we polymerase chain reaction (PCR) amplified complete open reading frames (ORFs) corresponding to the vpu locus of HIV-1 genome
The vpu variants were PCR amplified with consensus Vpu B and C-specific primers using DNA isolated from peripheral blood mononuclear cells (PBMCs) of infected individuals as reported by us earlier [17]
Summary
The pathogenesis of HIV infection is remarkably different from other primate lentiviruses in many ways. It manifestates immunodeficiency, the pathological hallmark of HIV infection caused by severe depletion of infected as well as uninfected lymphocyte populations [1,2]. Quasispecies are continuously generated in infected individuals as they are subjected to various selection pressures such as drugs, immune response, genetic factors as well as anti-retroviral restriction factors. Yusim et al (2002) studied relative variability of HIV-1 proteins and there analysis showed that Vpu displayed highest entropy of variation in infected individual [8]. 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
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