APOBEC3 (A3) proteins are members of an innate immune response that provide a defense against HIV-1 and other pathogens. In the absence of the HIV-1 protein Vif (Vif1), the A3 proteins are incorporated into virions in the virus producer cells and inhibit viral replication by deaminating cytidines in the minus-strand of viral DNA during reverse transcription in the target cells, resulting in extensive G-to-A hypermutation of the viral genome. In addition to inactivating most of the viral genomes through lethal hypermutation, we and others have shown that A3G and A3F also inhibit viral DNA synthesis and integration. To overcome these host defenses, Vif1 binds to the A3 proteins and targets them for proteasomal degradation, preventing their incorporation into virions. Defining the interactions of Vif1 with A3G and A3F at the molecular level could provide two potential targets for the development of antiviral drugs to suppress A3G and A3F degradation. ____Our goal is to understand the structure and function of Vif and A3 proteins. We will gain insights into the structures of Vif:A3 complexes through mutational and comparative analyses and generate reagents for structural studies. We recently determined that Vif1 and HIV-2 Vif (Vif2) interact with A3 proteins by using completely different determinants. We determined the relative restriction potential of A3G and A3F in primary CD4+ T cells by using Vif1 mutants that specifically failed to induce degradation of A3G or A3F, and showed that A3G exerts a greater restriction effect on HIV-1 than the combined activity of A3F and A3D. In collaboration with Dr. Kei Sato (Institute for Virus Research, Kyoto University), we also determined the replication potential in humanized mice of Vif1 mutants that are deficient in inducing degradation of A3G or A3F and found that both A3 proteins potently inhibited HIV-1 replication. A3G is localized to cytoplasmic RNA processing bodies (P bodies); we previously found that Mov10, a putative RNA helicase, is also localized to P bodies and inhibits HIV-1 replication. We therefore explored the significance of A3G and Mov10 localization to P bodies to their antiviral activity and concluded that virion incorporation and antiviral activities of A3G and Mov10 do not require localization to P bodies. In collaboration with Dr. Jeffrey Lifson, we found that replication of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus, is severely restricted in pigtailed macaques; in agreement with our in vitro studies, XMRV restriction was associated with extensive G-to-A hypermutation, suggesting powerful restriction by A3 proteins. _____To identify host factors that facilitate Vif1-mediated degradation of A3G, we performed a genome-wide siRNA screen in collaboration with Dr. Scott Martin (NIH Chemical Genomics Center). These studies revealed that UBA52, a fusion protein of ubiquitin and ribosomal protein L40, is a major source of ubiquitin for Vif1-mediated degradation of A3G and that a UBA52 mutant is a dominant inhibitor of Vif1-induced A3G degradation. The UBA52 mutant did not inhibit Vif2-induced degradation of A3G, indicating that Vif2 uses a different mechanism. We will define the mechanism that Vif2 uses to induce A3G degradation, and in collaboration with Dr. Eric Barklis (Oregon Health & Science University), we will use E. coli biotin ligase and mass spectrometry to identify and characterize Vif2- and Vif1-interacting proteins to gain insights into their different mechanisms. We will also characterize Vif proteins of HIV-1 groups M, N, O, and P to compare their mechanisms and interactions with human A3 proteins. The ultimate goal of these comparative studies is to generate stable Vif:A3 complexes and to determine their structures in collaboration with Dr. Yong Xiong (Yale University). _____We are studying the mechanisms by which A3 proteins inhibit viral replication and how they potentially affect viral genetic variation and evolution. Although A3G, A3F, A3D, and A3H have been shown to inhibit HIV-1 replication, it is not known whether they can copackage into the same virions and comutate the HIV-1 genomes. We found that A3G and other A3 proteins can copackage and comutate the same viral genomes and do not synergize or antagonize each other's antiviral activities. We sought to determine the potential contribution of A3-induced G-to-A hypermutation to viral genetic variation. We examined the potential for hypermutation to affect recombination frequency and induce sublethal mutagenesis in vivo, and for recombination to assort G-to-A hypermutations. Our results indicate that the contribution of A3-induced hypermutation to viral genetic variation is far less than that of error-prone viral replication. _____We have developed lentiviral vectors that can efficiently deliver Vif1-resistant A3 genes to T cells. To elucidate the evolutionary potential of Vif1 to evolve and overcome host restriction proteins, we will express Vif1-resistant A3 proteins in T-cell lines and determine the potential for Vif1 to evolve and acquire the ability to degrade these restriction factors in the presence and absence of wild-type A3 proteins. These studies will provide insights into the evolutionary interplay between HIV-1 and host restriction factors. _____[Corresponds to Pathak Project 1 in the July 2016 site visit report of the HIV Dynamics and Replication Program]

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Maiti, Atanu; Myint, Wazo; Kanai, Tapan et al. (2018) Crystal structure of the catalytic domain of HIV-1 restriction factor APOBEC3G in complex with ssDNA. Nat Commun 9:2460
Desimmie, Belete A; Smith, Jessica L; Matsuo, Hiroshi et al. (2017) Identification of a tripartite interaction between the N-terminus of HIV-1 Vif and CBF? that is critical for Vif function. Retrovirology 14:19
Chen, Jianbo; Rahman, Sheikh Abdul; Nikolaitchik, Olga A et al. (2016) HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein. Proc Natl Acad Sci U S A 113:E201-8
Delviks-Frankenberry, Krista A; Nikolaitchik, Olga A; Burdick, Ryan C et al. (2016) Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation. PLoS Pathog 12:e1005646
Sardo, Luca; Hatch, Steven C; Chen, Jianbo et al. (2015) Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly. J Virol 89:10832-40
Chikaev, Anton N; Bakulina, Anastasiya Yu; Burdick, Ryan C et al. (2015) Selection of peptide mimics of HIV-1 epitope recognized by neutralizing antibody VRC01. PLoS One 10:e0120847
Sato, Kei; Takeuchi, Junko S; Misawa, Naoko et al. (2014) APOBEC3D and APOBEC3F potently promote HIV-1 diversification and evolution in humanized mouse model. PLoS Pathog 10:e1004453
Smith, Jessica L; Izumi, Taisuke; Borbet, Timothy C et al. (2014) HIV-1 and HIV-2 Vif interact with human APOBEC3 proteins using completely different determinants. J Virol 88:9893-908
Desimmie, Belete A; Delviks-Frankenberrry, Krista A; Burdick, Ryan C et al. (2014) Multiple APOBEC3 restriction factors for HIV-1 and one Vif to rule them all. J Mol Biol 426:1220-45
Burdick, Ryan C; Hu, Wei-Shau; Pathak, Vinay K (2013) Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes. Proc Natl Acad Sci U S A 110:E4780-9

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