Human immunodeficiency virus-1 (HIV-1) Vif is essential for viral evasion of host antiviral factor CEM15/APOBEC3G. We report that Vif interacts with cellular proteins Cul5, elongins B and C, and Rbx1 to form an Skp1-cullin-F-box (SCF)-like complex. The ability of Vif to suppress antiviral activity of APOBEC3G was specifically dependent on Cul5-SCF function, allowing Vif to interact with APOBEC3G and induce its ubiquitination and degradation. A Vif mutant that interacted with APOBEC3G but not with Cul5-SCF was functionally inactive. The Cul5-SCF was also required for Vif function in distantly related simian immunodeficiency virus mac. These results indicate that the conserved Cul5-SCF pathway used by Vif is a potential target for antiviral development.

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G [A3G]) and other APOBEC3 proteins (25) are related to a family of proteins that also includes apolipoprotein B-editing catalytic subunit 1 (APOBEC1), APOBEC2, and activation-induced cytidine deaminase (AID) (23,66). These proteins have cytidine deaminase activities that modify RNA or DNA. A3G was the first APOBEC3 protein to be identified as a potent inhibitor of HIV-1 in the absence of Vif (59). A major outcome of virion packaging of A3G is the induction of C-to-U mutations in the minus-strand viral DNA during reverse transcription (22,32,42,43,63,73,77). Virion-packaged A3G and A3F can also reduce the accumulation of viral DNA (3,21,27,40,45,57,71) and the formation of proviral DNA (40,45).

Cullin-Ring E3 ubiquitin ligases target substrates for ubiquitindependent, proteasome-mediated degradation and regulate critical cellular processes. These cullins assemble with cellular substrate receptor proteins through specific adaptor molecules. F-box-and BC-box-containing receptors use Skp1, ElonginB, and ElonginC as adaptors to recruit Cul1͞Cul7 and Cul2͞Cul5, respectively. At present, the determinants of Cul2 vs. Cul5 specificity for the BC-box-containing receptors are poorly defined. Here, we demonstrate that primate lentiviral Vif (virion infectivity factor) proteins represent previously uncharacterized substrate receptor proteins that contain divergent BC-box motifs. These molecules selectively assemble with a Cul5-E3 ligase to suppress the antiviral activity of autologous cytidine deaminase APOBEC3G. A previously unrecognized Hx 5Cx17-18Cx3-5H motif that is highly conserved among all primate lentiviral Vif proteins was found to be critical for the selective assembly and activity of Vif-Cul5-E3 ligase. Non-primate lentiviral Vif proteins, which lack this HCCH motif, displayed reduced interaction with Cul5. These data suggest that in addition to target protein specificity, substrate receptor proteins play important roles in cullin selection and functional assembly of cullinRing E3 ligases. The discovery of these viral substrate receptor molecules that recruit Cul5 through distinct mechanisms from cellular proteins may facilitate the identification of additional cellular factors that regulate cellular functions through Cul5-E3 ligase. Motifs in Vif that are absent from cellular proteins could also be targets for the development of innovative therapeutics. cullin-E3 ubiquitin ligase ͉ HIV-simian immunodeficiency virus virion infectivity factorT argeted protein degradation is one mechanism by which critical processes such as mitosis and the cell cycle are regulated (1). The specificity of protein degradation is mediated by members of the E3 ubiquitin ligase family, including the cullin-based E3 ligases. Cullins form a scaffold on which other components of the E3 ligase organize to bring the substrate into close proximity with the E2 ubiquitin-conjugating enzyme (2-5). E3 ligases are substratespecific, and cullin-based E3 ligases display striking similarities. In SCF (Skp1-Cul1-F-box) complexes, the adaptor protein Skp1 bridges the interaction between the Cul1 and substrate receptor proteins through specific interaction with an F-box (2-5). These substrate receptor proteins bind substrates through distinct protein-protein interaction domains (e.g

APOBEC3G (A3G) is a single-stranded DNA cytidine deaminase that targets retroviral minus-strand DNA and has potent antiviral activity against diverse retroviruses. However, the mechanisms of A3G antiviral functions are incompletely understood. Here we demonstrate that A3G, A3F, and, to a lesser extent, the noncatalytic A3GC291S block human immunodeficiency virus type 1 (HIV-1) replication by interfering with proviral DNA formation. In HIV-1 virions, A3G interacted with HIV-1 integrase and nucleocapsid, key viral factors for reverse transcription and integration. Unlike A3G, the weak antiviral A3C cytidine deaminase did not interact with either of these factors and did not affect viral reverse transcription or proviral DNA formation. Thus, multiple steps of the HIV-1 replication cycle, most noticeably the formation of proviral DNA, are inhibited by both cytidine deamination-dependent and -independent mechanisms.