It has become clear in the last few years that human cells contain an enzyme, APOBEC3G (hA3G), that induces profound resistance to infection by certain retroviruses. hA3G protein possesses cytidine deaminase activity, and one mechanism responsible for its antiviral effects is deamination of cytosine residues in minus-strand DNA, producing G-to-A mutation in the coding strand of the provirus. HIV-1 encodes a protein, Vif, that blocks the effects of hA3G by binding to it and promoting its degradation in the proteasome. Despite several years of intensive study, the mechanism of hA3G incorporation into virions and the existence of antiviral effects other than deamination are still unresolved questions. While the interaction of human A3G with HIV-1 has been a central object of investigation in many laboratories, it is clear that other members of the APOBEC3 family can also have antiviral effects, that APOBEC3 family members are present in many mammalian species, and that different viruses have distinct patterns of sensitivity to the different APOBEC3 isoforms. Mice contain only one family member, APOBEC3 (mA3). It has been reported that MLVs are resistant to mA3 because they do not incorporate it into assembling virions, whereas other studies indicate that mA3 is incorporated into MLV particles without a significant antiviral effect. In collaboration with Dr. David Derse, we have re-examined the response of MLV and MLV-derived vectors to mA3, along with their sensitivity to hA3G. We find, contrary to the published reports, that MLV and related vectors are sensitive to mA3, although they are considerably more sensitive to hA3G. Other experiments showed that the potency of mA3 against delta-vif HIV-1 is equal to that of hA3G. We have been unable to detect G:A hypermutation induced by mA3 following MLV infections, although high levels of the mutations are observed with MLV inactivated by hA3G. This observation supports the concept that G:A hypermutation is not the only mechanism by which APOBEC proteins interfere with retroviral infections. In contrast, mA3 has been reported to induce G:A hypermutation in delta-vif HIV-1. We are surprised to find that our results are in conflict with published data from other laboratories. The discrepancies must be due to one or more differences in the reagents or experimental designs we have used. We will attempt to identify the relevant differences;the results of this search might well shed light on important questions in the field, including the mechanism by which APOBEC proteins inhibit retroviral infections. Taken together, the data show that MLV is partially resistant to the antiviral activity of mA3. MLV is a simple retrovirus, encoding only the three polyproteins that are assembled to form infectious progeny virions. Thus, it would be of considerable interest to determine the mechanism of its resistance to mA3. We are pursuing this goal by constructing chimeric MLVs and testing their sensitivity to mA3, in an effort to locate the determinants of resistance. We are also generating the reagents to produce recombinant mA3 protein in insect cells;this reagent will be invaluable in analysis of the mechanism of mA2 restriction and of MLV resistance to this restriction. [Corresponds to Rein Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program]

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010773-04
Application #
8157445
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2010
Total Cost
$159,688
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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Stavrou, Spyridon; Nitta, Takayuki; Kotla, Swathi et al. (2013) Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex. Proc Natl Acad Sci U S A 110:9078-83
Sanchez-Martinez, Silvia; Aloia, Amanda L; Harvin, Demetria et al. (2012) Studies on the restriction of murine leukemia viruses by mouse APOBEC3. PLoS One 7:e38190
Rein, Alan; Datta, Siddhartha A K; Jones, Christopher P et al. (2011) Diverse interactions of retroviral Gag proteins with RNAs. Trends Biochem Sci 36:373-80