APOBEC3G Incorporation Research Articles

Specific packaging of APOBEC3G into HIV-1 virions is mediated by the nucleocapsid domain of the gag polyprotein precursor

In cells infected by HIV-1 mutants lacking a functional Vif protein, APOBEC3G is specifically packaged into progeny virions and then interferes with the process of virus infection. Here, we show that incorporation of APOBEC3G into HIV-1 virions is mediated by the specific interaction of APOBEC3G with the carboxy-terminal nucleocapsid/p6 domain of the Gag polyprotein precursor. As a result, HIV-1 virus-like particles that lack the nucleocapsid domain fail to package APOBEC3G. Surprisingly, RNA was also found to be essential for formation of the nucleocapsid–APOBEC3G complex in vitro, thus raising the possibility that RNA may form a bridge between these two proteins.

The Interaction between HIV-1 Gag and APOBEC3G

APOBEC3G, a member of an RNA/DNA cytidine deaminase superfamily, has been identified as a cellular inhibitor of HIV-1 infectivity, possibly through the dC to dU deamination of the first minus strand cDNA synthesized during reverse transcription. Virions incorporate APOBEC3G during viral assembly in non-permissive cells, and this incorporation is inhibited by the viral protein Vif. The mechanism of APOBEC3G incorporation into HIV-1 is examined in this report. In the absence of Vif, cytoplasmic APOBEC3G becomes membrane-bound in cells expressing HIV-1 Gag, and its incorporation into Gag viral-like particles (VLPs) is proportional to the amount of APOBEC3G expressed in the cell. The expression of Vif, or mutant Gag unable to bind to membrane, prevents the APOBEC3G association with membrane. HIV-1 Gag alone among viral proteins is sufficient for packaging of APOBEC3G into Gag VLPs, and this incorporation requires the presence of Gag nucleocapsid. The presence of amino acids 104-156 in APOBEC3G, located in the linker region between two zinc coordination motifs, is also required for its incorporation into Gag VLPs. Evidence against an RNA bridge facilitating the Gag/APOBEC3G interaction includes data indicating that 1) the incorporation of APOBEC3G occurs independently of viral genomic RNA, 2) a Gag/APOBEC3G complex is immunoprecipitated from cell lysate after RNase treatment, and 3) the zinc coordination motif, rather than the regions flanking this motif, have been implicated in RNA binding in another family member, APOBEC1.

Vif Overcomes the Innate Antiviral Activity of APOBEC3G by Promoting Its Degradation in the Ubiquitin-Proteasome Pathway

Viruses must overcome diverse intracellular defense mechanisms to establish infection. The Vif (virion infectivity factor) protein of human immunodeficiency virus 1 (HIV-1) acts by overcoming the antiviral activity of APOBEC3G (CEM15), a cytidine deaminase that induces G to A hypermutation in newly synthesized viral DNA. In the absence of Vif, APOBEC3G incorporation into virions renders HIV-1 non-infectious. We report here that Vif counteracts the antiviral activity of APOBEC3G by targeting it for destruction by the ubiquitin-proteasome pathway. Vif forms a complex with APOBEC3G and enhances APOBEC3G ubiquitination, resulting in reduced steady-state APOBEC3G levels and a decrease in protein half-life. Furthermore, Vif-dependent degradation of APOBEC3G is blocked by proteasome inhibitors or ubiquitin mutant K48R. A mutation of highly conserved cysteines or the deletion of a conserved SLQ(Y/F)LA motif in Vif results in mutants that fail to induce APOBEC3G degradation and produce non-infectious HIV-1; however, mutations of conserved phosphorylation sites in Vif that impair viral replication do not affect APOBEC3G degradation, suggesting that Vif is important for other functions in addition to inducing proteasomal degradation of APOBEC3G. Vif is monoubiquitinated in the absence of APOBEC3G but is polyubiquitinated and rapidly degraded when APOBEC3G is coexpressed, suggesting that coexpression accelerates the degradation of both proteins. These results suggest that Vif functions by targeting APOBEC3G for degradation via the ubiquitin-proteasome pathway and implicate the proteasome as a site of dynamic interplay between microbial and cellular defenses.

HIV-1 Vif Blocks the Antiviral Activity of APOBEC3G by Impairing Both Its Translation and Intracellular Stability

The human immunodeficiency virus type 1 (HIV-1) relies on Vif (viral infectivity factor) to overcome the potent antiviral function of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, also known as CEM15). Using an APOBEC3G-specific antiserum, we now show that Vif prevents virion incorporation of endogenous APOBEC3G by effectively depleting the intracellular levels of this enzyme in HIV-1-infected T cells. Vif achieves this depletion by both impairing the translation of APOBEC3G mRNA and accelerating the posttranslational degradation of the APOBEC3G protein by the 26S proteasome. Vif physically interacts with APOBEC3G, and expression of Vif alone in the absence of other HIV-1 proteins is sufficient to cause depletion of APOBEC3G. These findings highlight how the bimodal translational and posttranslational inhibitory effects of Vif on APOBEC3G combine to markedly suppress the expression of this potent antiviral enzyme in virally infected cells, thereby effectively curtailing the incorporation of APOBEC3G into newly formed HIV-1 virions.