A W252A RT mutation inhibits HIV-1 reverse transcription and replication.

<p>(<b>A</b>) Proviral plasmids transiently expressing wild type or W252A RT mutant HIV-1 in HEK293T cells were visualized by western blot using a rabbit anit-HIV-1 antibody. Western blot samples were normalized for transfection efficiency using a co-transfected <i>Gaussia</i> luciferase reporter plasmid. (<b>B</b>) The amount of virus present in culture supernatant was measured using a CA ELISA. Results are normalized for transfection efficiency as determined by secreted <i>Gaussia</i> luciferase. (<b>C</b>) Western blot of concentrated HIV-1 virus particles from culture supernatants expressing wild type or W252A RT HIV-1, detecting with anti-HIV-1 (left, human serum, NIH AIDS reagent) or anti-RT antibodies (right). (<b>D</b>) The RT activities of wild type and W252A RT mutated viruses were measured using a standard RT colorimetric assay and normalized to CA levels in each sample. (<b>E</b>) Jurkat cells were infected with wild type or W252A RT mutant virus using equivalent amount of CA (500 ng CA/10⁷ cells) or (<b>F</b>) equivalent amount of RT (5 ng of RT/10⁷ cells). Virus replication was monitored by using an ELISA to measure CA in the culture supernatant at 0, 3, 7 and 14 days post-infection. The mean values and standard deviations from an experiment performed in triplicate are shown. The experiment was repeated twice with similar results.