FIG 2.

Semen exosome-mediated reduction in HIV RNA expression is operative in host cytoplasmic and nuclear subcellular compartments. Vehicle PBS or semen exosomes (100 μg/ml) were incubated with HIV-1 NL4.3 (8 RTU/ml) at 37°C for 1 h before infection of SUPT1 cells. (A and B) Total cellular RNA and RNA from cells fractionated into cytoplasmic and nuclear compartments. Fractionation was evaluated by Western blot analysis of cytoplasmic GAPDH and nuclear PCNA. Shown is one representative experiment. (C) Total, nuclear, and cytoplasmic RNAs were analyzed by qRT-PCR for viral Gag-Pol mRNA expression. GAPDH and pre-GAPDH were used for normalization of total/cytoplasmic and nuclear RNA, respectively. Vehicle was set as a reference for RNA analysis from each cellular compartment. (D) Cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Vehicle was used for normalization and set to 100%. (E) Cellular cytoplasmic RNA and progeny viral RNA were analyzed by qRT-PCR for Gag-Pol expression. GAPDH was used for normalization. Vehicle was set as a reference. Encapsidation efficiency was mathematically determined as the ratio of progeny and cytoplasmic Gag-Pol expression. Vehicle was used for normalization and set to 100%. *, P < 0.05; ns, not significant. The error bars indicate standard errors of the mean of the results of triplicate experiments.