The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation. The CUL5Vif/CBFß complex recruits ARIH2 where it acts to transfer ubiquitin directly to the APOBEC3 targets. ARIH2 is essential for CRL5- dependent HIV infectivity in primary CD4+ T cells. Furthermore, we show that ARIH2 cooperates with CRL5 to prime other cellular substrates for polyubiquitination, suggesting this may represent a general mechanism beyond HIV infection and APOBEC3 degradation. Taken together, these data identify ARIH2 as a co-factor in the Vif-hijacked CRL5 complex that contributes to HIV infectivity and demonstrate the operation of the E1-E2-E3/E3-substrate ubiquitination mechanism in a viral infection context.

We generated three stable Jurkat T cell lines, each expressing a tetracycline-inducible affinity-tagged version of CUL5, ELOB, and CBFß. Cells expressing the tagged proteins as well as parental Jurkat T cells were subjected to infection with replication-deficient, Vesicular Stomatitis Virus Glycoprotein (VSVg) pseudotyped HIV-1 NL4-3 virus, a Vif-deficient version of the same virus strain (HIV ∆Vif), or a mock serving as a non-infection condition (mock). Following 24h of infection, anti-FLAG immune complexes were purified and subjected to global, quantitative analysis by MS. Specific interactors of the three bait proteins were determined using SAINT high confidence interactor scoring by comparing affinity purifications from bait-expressing versus parental Jurkat T cells. Proteins with a false discovery rate (FDR) below 0.15 were subsequently validated using a targeted proteomics approach, which allows for accurate, reproducible and consistent quantification across samples and conditions.