Viral replication usually requires that innate intracellular lines of defence be overcome, a task usually accomplished by specialized viral gene products. The virion infectivity factor (Vif) protein of human immunodeficiency virus (HIV) is required during the late stages of viral production to counter the antiviral activity of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a protein expressed notably in human T lymphocytes. When produced in the presence of APOBEC3G, vif-defective virus is non-infectious. APOBEC3G is closely related to APOBEC1, the central component of an RNA-editing complex that deaminates a cytosine residue in apoB messenger RNA. APOBEC family members also have potent DNA mutator activity through dC deamination; however, whether the editing potential of APOBEC3G has any relevance to HIV inhibition is unknown. Here, we demonstrate that it does, as APOBEC3G exerts its antiviral effect during reverse transcription to trigger G-to-A hypermutation in the nascent retroviral DNA. We also find that APOBEC3G can act on a broad range of retroviruses in addition to HIV, suggesting that hypermutation by editing is a general innate defence mechanism against this important group of pathogens.

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Host cells impose a broad range of obstacles to the replication of retroviruses. Tetherin (also known as CD317, BST-2 or HM1.24) impedes viral release by retaining newly budded HIV-1 virions on the surface of cells. HIV-1 Vpu efficiently counteracts this restriction. Here, we show that HIV-1 Vpu induces the depletion of tetherin from cells. We demonstrate that this phenomenon correlates with the ability of Vpu to counteract the antiviral activity of both overexpressed and interferon-induced endogenous tetherin. In addition, we show that Vpu co-immunoprecipitates with tetherin and β-TrCP in a tri-molecular complex. This interaction leads to Vpu-mediated proteasomal degradation of tetherin in a β-TrCP2-dependent manner. Accordingly, in conditions where Vpu-β-TrCP2-tetherin interplay was not operative, including cells stably knocked down for β-TrCP2 expression or cells expressing a dominant negative form of β-TrCP, the ability of Vpu to antagonize the antiviral activity of tetherin was severely impaired. Nevertheless, tetherin degradation did not account for the totality of Vpu-mediated counteraction against the antiviral factor, as binding of Vpu to tetherin was sufficient for a partial relief of the restriction. Finally, we show that the mechanism used by Vpu to induce tetherin depletion implicates the cellular ER-associated degradation (ERAD) pathway, which mediates the dislocation of ER membrane proteins into the cytosol for subsequent proteasomal degradation. In conclusion, we show that Vpu interacts with tetherin to direct its β-TrCP2-dependent proteasomal degradation, thereby alleviating the blockade to the release of infectious virions. Identification of tetherin binding to Vpu provides a potential novel target for the development of drugs aimed at inhibiting HIV-1 replication.

APOBEC3G (also known as CEM15) is an innate intracellular antiretroviral factor that is counteracted by the Vif protein of lentiviruses. While APOBEC3G orthologues from several species are active against a broad range of retroviruses, given Vif proteins have a narrow spectrum of activity. For instance, HIV-1 Vif efficiently blocks APOBEC3G from human but not African green monkey (AGM), whereas the reverse is observed with SIV AGM Vif. Here, we demonstrate that a single amino acid at position 128 of human and AGM APOBEC3G governs the virus-specific sensitivity of these proteins to Vif-mediated inhibition. Furthermore, we show that this phenotype correlates with the ability of Vif to bind APOBEC3G and interfere with its incorporation into virions. These results shed light on an important determinant of the tropism of primate lentiviruses.The replication of a virus within its host and its spread to a new species require that innate lines of defense be overcome. APOBEC3G 1 is a cytidine deaminase that confers broad protection against retroviruses and limits the cross-species transmission of these pathogens (1-5). Packaged during viral assembly, APOBEC3G associates with the retroviral reverse transcription complex, where it deaminates cytosine residues to uracil in the growing minus-strand viral DNA (4 -7). These U-rich transcripts are either degraded or yield proviruses that are largely non-functional due to G-to-A hypermutation. APOBEC3G is counteracted by the Vif (virion infectivity factor) protein of lentiviruses, which associates with the enzyme to prevent its virion incorporation and trigger its proteasomal degradation (5, 8 -12). In the absence of Vif, human APOBEC3G is active against a broad spectrum of retroelements, as it can inhibit the replication of lentiviruses such as human and simian immunodeficiency virus (HIV and SIV, respectively) and equine infectious anemia virus (EIAV), of the gammaretrovirus murine leukemia virus (MLV) (4, 7), and of the hepadnavirus hepatitis B virus (13). APOBEC3G orthologues are also effective against several of these viruses. Vifdefective HIV-1, for instance, is blocked by APOBEC3G from human, rhesus macaque, African green monkey, and mouse (5). In contrast, a far greater degree of specificity is noted in the Vif sensitivity of these antiviral factors. As an example, the Vif protein of HIV-1 can only counter human and chimpanzee APOBEC3G, but is ineffective against the rhesus macaque, AGM, and mouse orthologues of the enzyme. Conversely, Vif from SIV AGM is active against AGM but not human APOBEC3G (5). Here, we took advantage of these speciesspecific differences to explore further the mechanism of Vif action. EXPERIMENTAL PROCEDURESExpression Vectors-Wild type and vif-defective HIV-1 proviral clones were described previously (14). To permit its trans-expression, a His-tagged form of HIV-1 Vif was inserted into the pEF1/Myc-His plasmid (Invitrogen), yielding the pEF1-VifHis plasmid. The plasmids expressing the HA-tagged form of huAPOBEC3G (3) and the SIV AGM.TAN Vif (pgVif/...

During the early phase of the retroviral life cycle, only a fraction of internalized virions end up integrating their genome into the chromosome, even though the resulting proviruses are almost systematically expressed. Here, we reveal that incoming retroviral preintegration complexes trigger the exportin-mediated cytoplasmic export of the SWI/SNF component INI1 and of the nuclear body constituent PML. We further show that the HIV genome associates with these proteins before nuclear migration. In the presence of arsenic, PML is sequestered in the nucleus, and the efficiency of HIV-mediated transduction is markedly increased. These results unveil a so far unsuspected cellular response that interferes with the early steps of HIV replication.