Background:  APOBEC3G induces hypermutations in newly synthesized viral DNA and its activity is suppressed by HIV-1 Vif. HIV-1 Vif recruits Cul5-ElonginB-ElonginC E3 ubiquitin ligase, which targets APOBEC3G for polyubiquitination and degradation. It is not clear whether HIV-1 Vif is an integral part of the Cul5-ElonginB-ElonginC E3 ubiquitin ligase or merely a linker protein that connects the target protein to the E3 ligase complex. 

Methods:  Protein sequences of HIV-1 Vif and cellular SOCS-box-containing proteins were aligned, showing that Vif contains a putative SOCS-box motif. We tested the effect of various Vif mutants on viral infectivity determined by MAGI assay, and their protein-to-protein interactions with ElonginC or E3 ligase complex by immunoprecipitation and immunoblot analysis. Direct protein interactions were examined between recombinant ElonginC and Vif proteins in vitro. Mutants of ElonginC and Cul5 were also examined for interaction with HIV-1Vif, and other Cullin family members were tested for recruitment by HIV-1 Vif.

Results:  A novel SOCS-box motif in HIV-1 Vif was identified to mediate its interaction with ElonginC. HIV-1 Vif can functionally tolerate a change of alanine 149 to cysteine within the SOCS-box motif. The interface of ElonginC critical for interaction with SOCS-box of VHL is also required for HIV-1 Vif interaction. N-terminal region of Cul5 interacting with ElonginC is critical for Cul5-ElonginB-ElonginC-Vif Complex formation. HIV-1 Vif selectively recruits Cul5 versus Cul2 to form an E3 ubiquitin ligase.

Conclusions:  HIV-1 Vif is a SOCS-box-containing protein that acts as an adaptor protein bridging target protein(s) to E3 ligase complex. The SOCS-box motif of HIV-1 Vif interacting with Elongin C was necessary but not sufficient for interaction with Cul5-ElonginB-ElonginC complex. Our studies suggest that selective assembly with Cul5 vs Cul2 E3 may require protein interfaces in addition to the SOCS-box-ElonginC interaction.

Keywords: HIV-1 Vif; APOBEC3G; Ubiquitin E3 Ligase