Abstract
Human Immunodeficiency Virus type 1 (HIV-1) major structure protein Gag is synthesized in the cytoplasm, assembles on the plasma membrane, subsequently buds and releases. HIV-1 viral particles incorporate a number of host proteins to facilitate or inhibit HIV-1 replication. Here we identify a new host protein, coiled-coil domain containing protein 8 (CCDC8), in HIV-1 particles. Incorporation of CCDC8 into virions is dependent on the interaction between CCDC8 and Gag matrix region. Exogenous overexpression of CCDC8 can strongly inhibit HIV-1 production, up to ~30 fold. CCDC8 is a membrane-associated protein. The interaction between exogenously expressed CCDC8 and Gag on the plasma membrane changes the assembly of Gag, and redirects it into intracellular sites, or causes Gag endocytosis. CCDC8, along with cytoskeleton protein obscuring-like1 (Obsl1) and E3 ligase Cul7, induces Gag polyubiquitination and degradation. Thus we identify a new host protein and a new pathway for HIV-1 Gag polyubiquitination and degradation. This pathway presents potential therapeutic strategies against HIV infection.
Highlights
4 μg BH10 + C8Infectivity test after normalizing CAp2 aBH_10 vGag P BH10 p55 Anti-Gag Anti-coiled-coil domain containing protein 8 (CCDC8)p24 C8 endogenous virus-like particles (VLPs) Virus bBH_10 P subtilisin_treated BH10 P Anti-gp[120 ] gp120
Using mass spectrometry (MS), we analyzed proteins found in Human immunodeficiency virus type 1 (HIV-1) VLPs viral Gag, or protease-negative viral Gag/ GagPol-RRE-P, which produces unprocessed Gag and GagPol
We found a drastic reduction in the amount of virus produced upon exposure to CCDC8, compared with a slower rate of reduction in the negative control (Enhanced Green Fluorescent Protein [pTT5-SH5-EGFP] expression) (Fig. 1a,b)
Summary
Exogenous expression of CCDC8 strongly inhibits HIV-1 production. Using mass spectrometry (MS), we analyzed proteins found in HIV-1 VLPs viral Gag (vGag-RRE), or protease-negative viral Gag/ GagPol-RRE-P- (vGag/GagPol-P-), which produces unprocessed Gag and GagPol. We found a drastic reduction in the amount of virus produced upon exposure to CCDC8, compared with a slower rate of reduction in the negative control (Enhanced Green Fluorescent Protein [pTT5-SH5-EGFP] expression) (Fig. 1a,b) This is accompanied by a mild reduction in cellular Gag production, but a much greater reduction in cellular CAp24, suggesting that Gag proteolytic processing could be impaired or Gag degradation could be increased or both. Co-expression of CCDC8 with HIV-1 BH10 P− resulted in a very strong reduction in virus production accompanied by a milder reduction in cellular Gag (Fig. 1c) This data indicate that the inhibition of HIV-1 production by CCDC8 is independent on viral protease activity. Subtilisin does not penetrate the viral membrane, but processes gp[120] and any contaminant proteins present on
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