Although expression of the viral Vif protein is critical for HIV-1 replication in primary cells, the mechanism of action of Vif has, until recently, been largely unclear. However, recent data from the Malim laboratory suggest that Vif functions by blocking the ability of a host factor called CEM15 or APOBEC3G to compromise the infectivity of progeny virions. APOBEC3G is not expressed in certain cell lines that are permissive for Vif- HIV-1 replication and expression of APOBEC3G in trans is sufficient to render these cells non-permissive for Vif- HIV-1. Although the mechanism of action of APOBEC3G, or how Vif prevents this action, remains unknown, APOBEC3G belongs to a family of cytidine deaminases that includes a member known to edit mRNA. We therefore hypothesize that APOBEC3G may block HIV-1 replication by editing HIV-1 mRNA and inducing a mutational error catastrophe. Alternately, APOBEC3G may inhibit HIV-1 infectivity after being packaged into virions and directly interfering with an early step in the viral infection cycle. The focus of this grant is on determining how APOBEC3G blocks HIV-1 replication, how Vifprevents this block and finally to examine whether APOBEC3G contributes to HIV-1 species tropism. We have already demonstrated a specific interaction between HIV-1 Vif and hAPOBEC3G in vivo and will further characterize this interaction in vitro, including testing whether it is direct. We will test whether APOBEC3G is a cytidine deaminase and whether this activity is required for its effect on HIV-1 replication. We will also sequence HIV-1 genomic RNAs produced in the presence of APOBEC3G, and absence of Vif, to detect editing. We will test whether non-human, including simian, forms of APOBEC3G inhibit HIV-1 replication and are responsive to HIV-1 Vif. Based on evidence showing that murine APOBEC3G blocks Vif- HIV-1 infectivity yet is not responsive to Vif, we intend to construct chimeric enzymes derived from Vif-responsive human and Vif-non-responsive animal APOBEC3G variants to map residues critical for Vif function. We will also attempt to use genetic and biochemical techniques to identify the viral nucleic acid or protein sequences that cause HIV-1 to be specifically targeted by APOBEC3G. Together, this wide-ranging series of experiments should lead to considerable insight into how HIV-1 Vif functions and potentially suggest approaches to inhibit Vif function.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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AIDS Molecular and Cellular Biology Study Section (AMCB)
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Sharma, Opendra K
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Duke University
Schools of Medicine
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Gooch, Barry D; Cullen, Bryan R (2008) Functional domain organization of human APOBEC3G. Virology 379:118-24
Bogerd, Hal P; Cullen, Bryan R (2008) Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation. RNA 14:1228-36
Bogerd, Hal P; Wiegand, Heather L; Doehle, Brian P et al. (2007) The intrinsic antiretroviral factor APOBEC3B contains two enzymatically active cytidine deaminase domains. Virology 364:486-93
Bogerd, Hal P; Wiegand, Heather L; Doehle, Brian P et al. (2006) APOBEC3A and APOBEC3B are potent inhibitors of LTR-retrotransposon function in human cells. Nucleic Acids Res 34:89-95
Cullen, Bryan R (2006) Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors. J Virol 80:1067-76
Russell, Rebecca A; Wiegand, Heather L; Moore, Michael D et al. (2005) Foamy virus Bet proteins function as novel inhibitors of the APOBEC3 family of innate antiretroviral defense factors. J Virol 79:8724-31
Doehle, Brian P; Schafer, Alexandra; Wiegand, Heather L et al. (2005) Differential sensitivity of murine leukemia virus to APOBEC3-mediated inhibition is governed by virion exclusion. J Virol 79:8201-7
Dutko, James A; Schafer, Alexandra; Kenny, Alison E et al. (2005) Inhibition of a yeast LTR retrotransposon by human APOBEC3 cytidine deaminases. Curr Biol 15:661-6
Doehle, Brian P; Schafer, Alexandra; Cullen, Bryan R (2005) Human APOBEC3B is a potent inhibitor of HIV-1 infectivity and is resistant to HIV-1 Vif. Virology 339:281-8
Wiegand, Heather L; Doehle, Brian P; Bogerd, Hal P et al. (2004) A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins. EMBO J 23:2451-8

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