Genetic and Functional Characterization of HIV-1 Vpu from HIV-1-Infected North Indian Population

Abstract

Acquired immunodeficiency syndrome is a pandemic disease due to increased variability in causative agent in global distribution; it is attributed to various complications in developing the vaccine, namely, error-prone reverse transcriptase, rapid replication, and high recombination rate. Vpu downmodulates CD4 in infected cells, and it targets the newly synthesized CD4 molecules from the endoplasmic reticulum. The aim of this study was to identify the level of genetic changes in the Vpu gene from HIV-1-infected North Indian individuals and determine the functional relevance with respect to the CD4 downregulation potential of this protein. Genomic DNA was isolated from peripheral blood mononuclear cells, and the Vpu gene was polymerase chain reaction amplified with specific primers followed by cloning, sequencing, and sequence analyses using bioinformatic tools for predicting HIV-1 subtypes, recombination events, conservation of domains, and phosphorylation sites. Among all Vpu variants, three of the variants having serine substitution (serine-52 and serine-56 conversion to isoleucine; S52I and S56I) had lost their functional β-TrcP binding motif. However, the specific determinants for CD4 (V20, W22, S23) and BST-2 (A11, A15, I17, and A19) binding remained highly conserved. The data obtained with Vpu mutants recommend that the serine residue substitutions in cytoplasmic domain distress the CD4 downregulation activity of Vpu. These events are likely to have implications for viral pathogenesis and vaccine formulations.

Keywords: HIV-1; Indian population; Vpu gene; major histocompatibility complex.

FIG. 1.

PCR amplification of Vpu gene from genomic DNA samples of HIV-1-infected individuals. PCR, polymerase chain reaction.

FIG. 2.

Phylogenetic analysis of Vpu consensus sequences and variant tree. The phylogenetic analysis was carried out using MEGA 4.1 software using the neighbor joining method.

FIG. 3.

Multiple sequence alignment of unique primary isolates of HIV-1 Vpu. Functional domains are mentioned above the sequences. Dots indicate a match with consensus C subtype and dashes indicate gaps, unique mutations are denoted by different colors.

FIG. 4.

Subcloning and expression of Vpu variants. (A) Vpu variants were subcloned into the pCMV-Myc vector and were confirmed by restriction digestion. (B) HEK 293T cells were transfected with empty vector/Npu B/Npu C and variants. Immunoblot analysis was done using the anti-Myc antibody. GAPDH was used as a loading control. HEK 293T, human embryonic kidney 293T; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

FIG. 5.

CHX-chase to check kinetic stability of various Vpu variants. (A) Cycloheximide chase assay to check kinetic stability of Vpu variants. (B) After densitometric quantification, the signals obtained were used to calculate Myc-Vpu wild-type or variant ratios that were plotted against time intervals. CHX, cycloheximide.

FIG. 6.

CD4 downregulation potential of Vpu variants. Vpu wild type and mutants were transfected into Tzmbl cells. Forty-eight hours post-transfection, cells were harvested and subjected to flow cytometric analysis.