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 intend to try to select MLV variants with higher levels of resistance, in order to elucidate possible ways that viruses without a Vif-like protein can block the antiviral effects of APOBEC proteins. We will perform this selection by passaging replication-competent MLV in 293T cells expressing the ecotropic MLV receptor. mA3 will be expressed under the control of an inducible promoter so that its levels can be regulated as the experiment proceeds. If mA3 proves too toxic for chronic expression in the cells, the experiment will be performed in a series of transient steps rather than continuous passage. If an mA3-resistant variant of MLV is obtained, the mutation(s) responsible for the resistance will be identified. [Corresponds to Rein Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program
