Phenotypic characterization of natural Vpu variants with respect to viral replication.

<p>A) HeLa cells were transfected with an equal amount of various proviral DNA constructs and total virus released in culture supernatant was quantitated using HIV indicator Tzmbl cells by β-galactosidase staining. B) Relative number of infected cells was counted for each sample and C) plotted. These results are representative of three independent experiments.</p

Genetic variation in HIV-1 <i>vpu</i> gene from North Indian HIV-1 infected individuals.

<p><b>A)</b>The phylogenetic tree was constructed using the Neighbor Joining (NJ) method with the Kimura two-parameter distance matrix. The first letter of the reference sequence denotes the type or subtype or CRF, the second letter denotes the country from sequence sampled and the third letter denotes the accession number. Filled triangle mark the subtype B variants and filled circles mark the subtype C variants sampled from North India. The accession numbers for Indian samples (NII-PGI-IND-vpu sequences) were marked within the brackets. The main supported clades were marked with asterisk (*) along the branch represents the bootstrap support >70%. The scale bar represents the evolutionary distance of 0.1 nucleotides per position in the sequence. <b>B)</b> Multiple sequence alignment of primary isolates of HIV-1 Vpu collected from HIV-1 infected individuals from North India<b>.</b> The color coding generated by software represents difference in color for amino acid with different physiochemical properties. Identical residues are represented as dots.</p

The clinical data for HIV-1 infected individuals from North India.

<p>The clinical data for HIV-1 infected individuals from North India.</p

The rate of accumulation of non-synonymous and synonymous substitution.

<p>The rate of accumulation of non-synonymous and synonymous substitution.</p

The S61A mutation conferred enhanced intracellular stability.

<p>A) Equal amounts of various pCMV HA Vpu expression constructs were transfected into HEK-293T cells by Lipofectamine 2000 for forty eight hours. Thereafter cell lysate were subjected to immunoblotting to measure relative abundance of various Vpu variants. B) CHX-chase assay was performed to check kinetic stability of various Vpu variants by transfecting HEK-293T cells with various Vpu constructs and thereafter treated with 100 µg/ml of CHX for indicated time period and harvested for immunoblotting. C) HEK-293T cells were co-transfected with His-ubiquitin (His-Ub), and various Vpu constructs. After thirty six hours, cells were treated with MG132 for eight hours followed by lysis in denaturation buffer and then total ubiquitinated proteins were pulled down using Ni-NTA beads and Vpu ubiquitination was checked by immunoblotting using anti-HA antibody. These results are representative of three independent experiments.</p

Phenotypic characterization of natural Vpu variants with respect to induction of cell death upon infection.

<p>A) Infected MOLT-4 T Cells were harvested, stained with Propidium iodide (10 µg/ml) and analyzed by flow cytometry to determine the extent of cell death. The extent of cell death is indicated in upper right corner of each panel. The FACS data were analyzed by WinMDI 2.9 software. B) Equal amounts of lysate from infected cells were subjected to immunoblotting to measure Vpu expression. These results are representative of three independent experiments.</p