Figure 3

HERC5 inhibits nuclear export of HIV-1 genomic RNA. A, 293T cells were co-transfected with pR9 and either empty vector or pHERC5. Forty hours post-transfection, cells were treated with the proteasomal inhibitor MG132 (20 μM) or the lysosomal inhibitor amantidine (1.5 mM) for 16 hours. Virus released into the supernatant or total cell lysates were subjected to quantitative Western blot analysis using anti-p24CA and anti-β actin as a loading control. B, Cells were transfected as in (A). Forty-eight hours after transfection, total RNA was extracted and reverse transcribed into cDNA from whole cell lysates or from the cytoplasmic fraction only. Quantitative PCR was performed on each fraction using primers specific to unspliced HIV-1 genomic RNA (e.g. Gag), fully spliced RNA (e.g. Rev), total HIV-1 RNA (e.g. LTR), β-actin (loading control) or GFP (transfection control). The proportion of unspliced or fully-spliced HIV-1 RNA in the cytoplasmic fraction compared to total amount of HIV-1 RNA (nuclear plus cytoplasmic) was determined for control cells and cells expressing HERC5. Fold-change in copy number relative to control cells is shown. Data shown represents the average (+/- SEM) from six independent experiments. ***P = 0.0003; not significant (n.s.) P > 0.05 (student’s paired t test). C and D, HeLa cells were co-transfected with plasmids encoding MS2-GFP alone, MS2-GFP and NL4-3-SL, or MS2-GFP + NL4-3-SL and either flag-tagged HERC5, HERC5-C994A or HERC5-∆RLD. Forty-eight hours post-transfection, cells were fixed, stained with anti-flag and DAPI and imaged using fluorescence confocal microscopy. MS2-GFP localization was assessed in each cell and categorized according to localization in the nucleus only or both the nucleus and cytoplasm (C). Results shown are from at least three independent experiments (n = 331). Representative images of the predominant phenotypes are shown (D). Blue, nucleus; green, MS2-GFP; red, flag-tagged HERC5. Scale bars = 10 μm.