Induction of APOBEC 3 G Ubiquitination and Degradation by an HIV-1 Vif-Cul 5-SCF Complex

The vif orf is present in all lentiviruses except EIAV and is required for viral replication and pathogenicity in vivo (1– 12). In the absence of Vif, HIV-1 virions that are produced from non-permissive primary T lymphocytes and certain T-cell lines are defective and cannot initiate productive infection. Recently, CEM15/APOBEC3G (hereafter referred to as APOBEC3G), which is present only in non-permissive cells, has been identified as a mediator of anti-HIV-1 activity, and its activity has been shown to be suppressed by Vif (13). APOBEC3G belongs to a family of proteins that have cytidine deaminase activity and, when packaged into HIV-1 virions, induce modification of newly synthesized minus-strand viral DNA from cytosines to uracils (13–18). We sought to characterize cellular proteins that interact with Vif and regulate its function, as a means of understanding the mechanism of HIV-1 evasion of host antiviral defense. To efficiently precipitate Vif from HIV-1-infected cells and subsequently identify the cellular protein(s) that might interact with Vif, we constructed an infectious HIV-1 clone (HXB2VifHA) in which the end of Vif carried an HA tag. This clone replicated as efficiently as the parental clone HXB2 (8) in H9 cells, which are not permissive for a Vif deletion mutant HXB2¨Vif (fig. S1, SOM Text). An anti-HA antibody immunoprecipitated HA-tagged Vif from lysates of HXB2VifHA-infected H9 cells, but not untagged Vif from control HXB2-infected H9 cells (Fig. 1). Three cellular proteins co-precipitated with Vif-HA but were absent from HXB2 control samples (Fig. 1A). These proteins, of ∼85kDa, 18kDa, and 15 kDa (Fig. 1A), were identified by mass spectrometry as Cullin-5 (Cul5) and Elongins B and C, respectively. Their identities were confirmed by reactivity with Cul5-, Elongin B-, or Elongin C-specific antibodies (Fig. 1B). Although Rbx1 was not clearly visible by silver staining, it also co-precipitated with Vif-HA, as revealed by immunoblotting with anti-Rbx1 (Fig. 1B). In the converse experiment, HIV-1 Vif could also be immunoprecipitated with Cul5 (Fig. 1C). Moreover, when an SLQ ĺ AAA mutation was introduced into the highly conserved lentiviral Vif motif SLQXLA, the interaction between HIV-1 Vif and Cul5-Elongin B-Elongin C was significantly decreased (Fig. 1D). Interaction of HIV-1 Vif with Cul5, Elongin B, and Elongin C was also detected in HXB2VifHA–infected Jurkat cells and transfected 293T cells (fig. S2). These results indicate that HIV-1 Vif interacts with Cul5, Elongin B, Elongin C, and Rbx1 to form a complex reminiscent of an Skp1-Cullin-F box (SCF)-like, or …