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 TZMbl 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 antigp41 (Chessie 8) antibodies. The release and infectivity experiments were performed in duplicates and the results shown are representative of three independent experiments.

https://doi.org/10.1371/journal.pone.0301303.g001

Peripheral blood mononuclear cells (PBMC) were prepared from blood of healthy donors. The experiment was carried out in duplicate and in cells from two different donors and the results presented are averages of duplicate experiments. (A) PBMC obtained from two healthy seronegative donors were infected with equal focus-forming units (FFU) of WT and mutant (ΔVpu, L30E, L30E-ΔVpu) viruses pseudotyped with VSV-G. The amount of virus release in the supernatant was monitored and assessed up to 12 days by RT-ELISA. Virus release from infected PBMC was plotted as RT activity versus number of days. (B) Viral supernatants harvested from infected PBMC were tested for infectivity in TZM-bl indicator cells. Equal amount of viral supernatant (50 μL) was added on TZM-bl cells in duplicates. At 48 h post-infection, TZM-bl cells were fixed and stained with X-Gal substrate for β-galactosidase activity to give blue FFU. Figure shown represents infectivity of viruses harvested at three different days (0, 6^th and 12^th day). (C) Blue cells were counted as infectious units and calculated as FFU per milliliter and plotted as graph.

https://doi.org/10.1371/journal.pone.0301303.g002

Viruses were produced by co-transfection of 293T cells with combinations of Env deleted backbones plasmids containing Gag (L30E) or Vpu mutation and Env plasmids of patient origin. At 48 h post-transfection, supernatants were harvested and infectivity of viruses was determined in TZM-bl cells by measuring RLU. Average of RLU and standard error was calculated and plotted as graph. Infectivity experiments were performed in duplicates. Viral supernatants of independent experiments were pooled and concentrated by ultracentrifugation using 20% sucrose cushion, normalized for RT values and equal quantities were subjected to 10% SDS-PAGE gel, followed by Western blot with anti-p24 (183-H12-5C) and anti-gp41 (Chessie 8) antibody. Western blots shown represent gp41 Env on released virion particles. Viral p24 and gp41 band intensities were determined with Image J software (NIH) and ratio of gp41 to p24 were plotted in a graph to determine Env incorporation. (A) Infectivity of primary viruses in presence (+) and absence (-) of Vpu. (B) Western blot analysis of Vpu⁺ and Vpu^- primary viruses. The vertical black lines in the blot indicates spliced bands. (C) Densitometry analysis of band intensities of Vpu⁺ and Vpu^- viral p24 and gp41 proteins. Ratio of gp41/p24 represents the amount of Env (gp41) incorporation on virions. (D) Infectivity of primary viruses carrying Gag L30E mutation in presence and absence of Vpu. Significance level of improved infectivity of double mutant viruses (L30E-ΔVpu) was determined with respect to viruses possessing only Gag mutation (L30E) using Student’s t-test: * means p< 0.05 and ** means P< 0.005. (E) Western blot analysis of L30E and L30E-ΔVpu (double mutant) primary viruses. The vertical black lines in the blot indicates spliced bands. (F) Densitometry analysis of L30E and L30E-ΔVpu viral proteins, p24 and gp41. Ratio of gp41/p24 represents the amount of Env (gp41) incorporation on virions.

https://doi.org/10.1371/journal.pone.0301303.g003