Effect of Vpu start codon mutation and Gag L30E mutation on infectivity and Env incorporation of primary viruses of diverse origin.

<p>Viruses were produced by co-transfection of 293T cells with combinations of Env deficient backbone 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 a graph. Infectivity experiments were performed in duplicates and the results shown are representative of three independent experiments. Viral supernatants were 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 was 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<i>⁻</i> primary viruses. (C) Densitometry analysis of band intensities of Vpu⁺ and Vpu<i>⁻</i> 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 Gag mutation (L30E) using Student’s <i>t</i>-test: * means p<0.05 and ** means P<0.005. (E) Western blot analysis of L30E and L30E-ΔVpu (double mutant) primary viruses. (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.