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Figure 2 | Retrovirology

Figure 2

From: Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag

Figure 2

HERC5 restricts HIV-1 Gag particle production by a mechanism dependent on its E3 ligase activity. A, U2OS cells were co-transfected with pGag, pUbe1L, pUbcH8, pMyc-ISG15 and either empty plasmid, pHERC5 or pHERC5-C994A. Gag particles released into the supernatant and intracellular Gag protein expression were analyzed after 24 hours by quantitative Western blotting using anti-p24CA and anti-β-actin as a loading control. The same blot was stripped and tested for ISG15 conjugates using anti-myc (bottom blot). B, Cells were co-transfected with empty plasmid or pHERC5 and pUbe1L, pUbcH8, pHis-ISG15 and pGag. Cells were lysed 72 hours later and histidine-tagged proteins were purified using nickel agarose. Purified proteins were resolved using SDS-PAGE and subjected to Western blotting using anti-p24CA. C, Cells were co-transfected with empty vector or pHERC5, pUbe1L, pUbcH8, pHis-ISG15 and pGag with or without pUbp43. Gag particles released into the supernatant and intracellular Gag protein expression were analyzed after 72 hours by quantitative Western blotting using anti-p24CA or anti-β-actin. D, In a similar transfection as in C, histidine-tagged proteins were purified from cell extracts using nickel agarose and subjected to Western blotting using anti-p24CA. The two lanes were digitally separated from the same image. Numerical values on the blots display the densitometric quantification of the specified bands. All Western blots shown are representative of at least two independent experiments.

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