Figure 6. Redoxal increases cellular A3G protein levels by inhibiting de novo pyrimidine biosynthesis.

A. Redoxal increases A3G protein levels in virus producer cells and newly produced virions while uridine supplementation reverses these effects of redoxal. 293T cells were co-transfected with 100 ng A3G-3xHA and 1000 ng pNL4-3GFPΔEnv HIV-1 plasmids. At 5 h post transfection, the media was replaced with fresh media supplemented with DMSO or 1250 nM redoxal and supplemented with increasing doses of uridine (0, 10, or 50 μM). At 40 h post transfection, supernatants containing virus were collected, producer cells were lysed, and A3G, Vif, p55 Gag, and β-tubulin protein levels in producer cell lysates were detected by Western blotting (upper panels). Lower panels: A3G incorporation into virions produced from cells analyzed in the upper panels. Virions normalized for equivalent RT units were purified through 20% sucrose and A3G and p24 Gag protein levels in virion lysates were detected by Western blotting (lower panels). Results are representative of two independent experiments.. B. Redoxal increases cellular A3G protein expression in virus producer cells while orotate supplementation reverses this effect of redoxal. 293T cells were co-transfected with 100 ng A3G-3xHA and 1000 ng pCDNA3.1 (empty vector) plasmids. At 5 h post transfection, the media was replaced with fresh media supplemented with DMSO or 1250 nM redoxal and supplemented with increasing doses of orotate (0, 0.5, or 2 mM). At 40 h post transfection, cells were lysed. A3G and β-tubulin protein levels in cell lysates were analyzed by Western blotting. Results are representative of two independent experiments. Values shown below blots in A and B represent relative A3G protein levels in treated cells determined by densitometry using ImageJ software and normalization to A3G protein levels in untreated cells (DMSO control). C. and D. Redoxal attenuates infectivity of VSV-G pseudotyped NL4-3GFPΔEnv viruses produced in A3G-expressing 293T cells, while uridine and orotate supplementation reverse this effect of redoxal. TZM-bl reporter cells were infected with viruses corresponding to 4,000 RT units. Infectivity was measured 48 h post infection. Results are representative of two independent experiments each done in triplicate. Shown in C and D are means ± SD of triplicate samples from independent wells (p-values from student’s t-test).