Analyses of Vpx revertant viruses emerging in macaques K42 and JA4X.

<p>ConA-activated rhesus PBMCs were infected at a MOI = 0.01 with (A) the WT SIVmac239, SIVmac239 X-Q76A Vpx mutant, or the virus recovered at week 40 PI from infected macaque K42 or (B) the WT SIVmac316, SIVmac316 X-Q76AVpx mutant, or the virus recovered at week 40 PI from infected macaque JA4X. Virus replication was monitored by measuring the particle-associated ³²P RT activity released into culture supernatants. Results are shown as mean +/- s.e.m. The parametric unpaired t test was performed using PRISM software. The significant p values (* p<0.05, ** p<0.01) for putative revertant viruses compared to the starting X-Q76A Vpx mutant refer to the indicated time points during the infection. (C) ConA- activated enriched rhesus CD4⁺ T lymphocytes were infected with the indicated viruses at MOI = 0.2. Whole cell lysates were prepared on day 3 PI and levels of endogenous SAMHD1 present in virus or mock infected cell extracts from 3 x 10⁵ cells/lane were determined by immunoblotting as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004928#ppat.1004928.g004" target="_blank">Fig 4C</a>. The numbers below each lane indicate relative densitometric intensities of SAMHD1 bands relative to that of GAPDH and normalized to that present in Mock infected cells. (D) Rhesus PBMC were infected with the WT SIVmac316 or the molecularly cloned SIVmac316 derivatives containing the X-Q76A, Q76S, or the Q76A/I32T Vpx substitutions, using virus stocks prepared in transfected 293T cells. Cell cultures were infected with equivalent amounts of virus inocula, based on particle-associated RT activity (approximately 5 × 10^{6 32}P cpm). Progeny virion production is shown as mean +/—s.e.m. The parametric unpaired t test was performed using PRISM software. The significant p values (* p<0.05) for SIVmac316 X-Q76S or SIVmac316 X-Q76A/I32T versus the starting SIVnac316 X-Q76A Vpx mutant are indicated. Representative results from at least two experiments are shown.