Figure 7

Amino acids 2–103 of HERC5 are required for inhibiting nuclear export of HIV-1 genomic RNA. A, Western blot analysis of 293T cells co-transfected with pR9 and either empty plasmid or a plasmid encoding HERC5, HERC5-C994A, HERC5-∆RLD and HERC5-RLD∆2-103. Forty-eight hours after transfection, HIV-1 virions released into the supernatant and Gag levels within the cell lysates were analyzed by quantitative Western blotting using anti-p24CA or anti-β-actin as a loading control. B, 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, HERC5-∆RLD or HERC5-RLD∆2-103. Forty-eight hours post-transfection, cells were fixed, stained with anti-flag and DAPI and imaged using fluorescence confocal microscopy. Representative images of the predominant phenotypes are shown. Blue, nucleus; green MS2-GFP; red, flag-tagged HERC5. Scale bars = 10 μm. C, MS2-GFP localization was assessed in each cell and categorized according to localization in the nucleus only or both the nucleus and cytoplasm. The results of the phenotypic quantification are shown and were obtained from at least three independent experiments (n = 235).