HIV-1 and other retroviruses occasionally undergo a high rate of G-to-A substitutions, a phenomenon named hypermutation. APOBEC3G, a dominant-acting host restriction factor, is packaged into virions in the absence of HIV-1 Vif expression. The cytidine deaminase activity of APOBEC3G deaminates cytidines in HIV-1 minus-strand DNA, leading to G-to-A hypermutation. The HIV-1 Vif protein binds to APOBEC3G and induces its proteasomal degradation, thereby preventing its incorporation into HIV-1 virions.Using mutational analysis, we showed that a single amino acid (D128K) is responsible for the species specificity of APOBEC3G proteins. The D128 residue either directly interacts with Vif or is involved in conformational changes that occur upon Vif binding. To gain insights into the mechanism by which APOBEC3G inhibits HIV-1 replication, we recently developed a sensitive cytidine deamination assay using scintillation proximity beads. Using this assay, we demonstrated that interactions with viral and nonviral RNAs that are packaged are sufficient for APOBEC3G virion incorporation and that interactions with viral proteins are not essential for virion incorporation. We are determining the stoichiometry of APOBEC3G in HIV-1 virions produced in primary CD4+ T cells in the absence of Vif expression. We are carrying out biochemical and genetic studies to elucidate the structure and function of APOBEC3G. We are purifying APOBEC3G to characterize its interactions with DNA, RNA, and other proteins. We are also carrying out mutational studies to characterize the domains of APOBEC3G and Vif that interact, and domains that are critical for its virion incorporation. We are developing high-throughput assays for the Vif-APOBEC3G interaction to identify small molecule inhibitors that block the ability of Vif to overcome APOBEC3G antiviral activity. In collaboration with Dr. Steven Zeichner, we are analyzing the effect of HIV-1 Vpr on transcriptional regulation of APOBEC3G. TRIM5alpha is a host restriction factor that targets the HIV-1 capsid, and inhibits viral replication in restricted cells. The mechanism by which TRIM5alpha inhibits viral replication is not understood. We are collaborating with Matt Stremlau and Dr. Joseph Sodroski to understand the effects of TRIM5alpha on HIV-1 reverse transcription. We are using the novel strand-specific amplification assay (SSA) that we have developed to determine the stage at which reverse transcription is inhibited by TRIM5alpha. Host restriction genes like APOBEC3G and TRIM5alpha suppress HIV-1 replication and act as dominant-acting inhibitors of HIV-1 replication. In contrast to the restriction genes, there are host genes that are used by HIV-1 to facilitate its replication. Because mammalian cells are diploid, it has been difficult to identify the recessive host genes that facilitate HIV-1 replication by mutational inactivation of the gene because it is necessary to inactivate both copies of the gene of interest. We are developing novel genetic screens to identify host genes that facilitate HIV-1 replication.
