Differential effects of Vpu start codon mutation on release and infectivity of viruses from different cell types.

<p>Cell lines (293T, HeLa, NP2 and GHOST) were transfected with equal amount of wild-type pNL-AD8 (WT) plasmid and its Vpu defective variant (ΔVpu). At 48 h post-transfection, supernatants were harvested, clarified by centrifugation and filtered through 0.4 µm syringe filters. At the same time cell lysates were also prepared and cleared of cell debris and nuclei by centrifugation. (A) The amount of virus release in the supernatant was determined by measuring viral reverse transcriptase (RT) activity through RT-ELISA. Quantification of RT activity was done in duplicates, the average percent release and standard error was calculated considering WT release as 100%. Virus release was also evaluated by assessing viral Gag p24 protein in viral supernatants by 10% SDS-PAGE followed by Western blotting with HIV-1 Gag anti-p24 antibody (183-H12-5C). (B) Infectivity of ΔVpu viruses with respect to WT was determined in TZM-bl indicator cells. Clarified viral supernatants from transfected cells were serially diluted and added to the TZM-bl cells. At 48 h post-infection, infectivity was determined by measuring Luciferase activity as RLU (Relative Luminescence unit) using Luminometer. The average of RLU and standard error was calculated. Figure represents percent change in infectivity with respect to WT which was considered as 100%. (C) Western blots shown represent cell-associated and cell-free viral proteins. For blot of viral proteins, viral supernatants were concentrated by ultracentrifugation using 20% sucrose cushion and run in 10% SDS-PAGE. Before loading, viral lysates were normalized by RT value and equal RT values were subjected to SDS-PAGE. Viral proteins of both cell-associated and cell-free lysates were analyzed by using anti-p24 (183-H12-5C) and anti-gp41 (Chessie 8) antibodies. The release and infectivity experiments were performed in duplicates and the results shown are representative of three independent experiments.