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Figure 5 | Retrovirology

Figure 5

From: Interferon-induced HERC5 is evolving under positive selection and inhibits HIV-1 particle production by a novel mechanism targeting Rev/RRE-dependent RNA nuclear export

Figure 5

HERC5 interacts with Ran and reduces RanGTP levels and/or its binding with RanBP1. A, 293T cells were co-transfected with either empty vector or a plasmid encoding flag-tagged HERC5. Forty-eight hours after transfection, cells were lysed under non-denaturing conditions and subjected to immunoprecipitation using anti-Ran or anti-flag. Precipitated proteins were separated by SDS-PAGE and subjected to Western blotting using anti-HERC5 or anti-Ran. Numbers below the lower blot represent the densitometric quantification of the non-specific band in the empty vector control and Ran in the HERC5-expressing cells. B, U2OS cells were transiently-transfected with empty vector or a plasmid encoding HERC5. Forty-eight hours after transfection, total cell lysate was mixed with RanBP1-bound agarose beads to selectively isolate and pull-down RanGTP. As a control, cell lysate was incubated with non-hydrolyzable GTPγS prior to incubation with the agarose beads. The eluate, including input lysate (3%), were separated by SDS-PAGE gel and subjected to quantitative Western blotting using anti-Ran. Note, bands shown in the input samples represents total Ran protein and does not distinguish RanGTP or RanGDP. The blot shown is representative of three independent experiments. Numbers represent the densitometric quantification of RanGTP after normalization to Ran levels. C, U2OS cells were co-transfected with plasmids encoding HIV-1 Rev and either flag-tagged HERC5 or HERC5-C994A. Forty-eight hours post-transfection, cells were fixed, stained with anti-flag and/or anti-Rev and DAPI and imaged using fluorescence confocal microscopy. Rev localization was assessed in each cell and categorized as either: near the nuclear membrane, in both the nucleus and cytoplasm, in the cytoplasm only, or nucleolar. Results shown are from at least three independent experiments (n = 500). D, Representative images of the predominant phenotypes. Blue, nucleus; green, flag-tagged HERC5; red, Rev. Scale bars = 10 μm.

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