Skip to main content
Figure 1 | Retrovirology

Figure 1

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 1

HERC5 inhibits HIV-1 particle production via an E3 ligase-independent mechanism. A, Schematic of HERC5 domains and the various mutant constructs generated (not to scale). B, Western blot analysis of 293T cells transfected with either empty plasmid or a plasmid encoding flag-tagged HERC5, HERC5-C994A or HERC5-∆RLD. Anti-flag was used to detect HERC5 and anti-β-actin was used as a loading control. Numbers above bands represent densitometric quantification relative to wildtype HERC5 after normalization to β-actin. C, U2OS cells were transfected as described in panel B. Forty-eight hours post-transfection, cells were analyzed by confocal immunofluorescence microscopy using anti-flag. Scale bars = 10 μm. D, 293T cells were co-transfected with pR9 and either empty plasmid, pHERC5, pHERC5-C994A, or pHERC5-∆RLD. Forty-eight hours after transfection, infectious virions released into the supernatant were quantified using GHOST(3) indicator cells. The averages +/- SD from at least three independent experiments are shown. E, Forty-eight hours after 293T cells were transfected as described in panel D, virions released into the supernatant and Gag within cell lysates were analyzed by Western blotting using anti-p24CA or anti-β-actin. F, Densitometric analysis of the indicated bands relative to the empty vector control from at least three independently generated experiments similar to that shown in panel E. Values were normalized to β-actin. ****P < 0.0001; **P < 0.01; *P < 0.05; n.s. P > 0.05 (student’s paired t test). G, Western blot analysis of 293T cells co-transfected with pR9 and either empty plasmid, flag-tagged pHERC5 or pHERC5-C994A. Forty-eight hours after transfection, cell lysates were analyzed using anti-p24CA, anti-flag or anti-β-actin. H, Cells were co-transfected with pUbe1L, pUbcH8 and myc-tagged ISG15 and either empty vector, pUbp43, flag-tagged pHERC5 or pHERC5-C994A. Cell lysates were analyzed by Western blotting using anti-flag or anti-β-actin. Data shown is representative of at least three independent experiments.

Back to article page