Innate immunity requires cellular restriction factors that inhibit the replication of multiple pathogens. One group of restriction factors is the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (Apobec) family of cytosine deaminases, including activation-induced cytosine deaminase (AID). AID function includes both innate and adaptive immune responses due to its role in control of retrotransposons as well as somatic hypermutation of immunoglobulin variable regions and class switch recombination. Apobec family enzymes are known inhibitors of complex human retroviruses, such as the lentivirus human immunodeficiency virus (HIV-1), as well as DNA-containing viruses. HIV-1 encodes an Apobec antagonist, Vif and, in the absence of Vif, the provirus sustains high numbers of G to A hypermutations due to cytosine deamination on the minus strand during reverse transcription. My laboratory has shown that mouse mammary tumor virus (MMTV) is the only known complex mouse retrovirus with organizational and functional features similar to HIV, such as the regulatory protein Rem. Rem is a precursor protein that is cleaved by signal peptidase at the ER membrane to give an N-terminal signal peptide (SP) that has Rev-like function and a C-terminal protein of unknown function. Inoculation of cloned MMTV lacking Rem expression into BALB/c mice resulted in mammary tumors with a decreased incidence and latency compared to wild-type MMTV. Characterization of MMTV proviruses from these mammary tumors showed extensive G to A as well as other transition mutations on both DNA strands, consistent with AID-type mutations, but also some typical of the consensus sequences and strand specificity of murine Apobec3 (mA3) or other deaminases. Transfection experiments revealed proteasomal degradation of AID in the presence of Rem, but not mA3. These results led us to the hypothesis that Rem is an HIV-1 Vif-like antagonist of multiple Apobec cytosine deaminases. This application will test several aspects of this hypothesis.
In Aim 1, we will determine sequences within Rem and AID that are necessary for proteasomal degradation. We will identify the E3 ligase for AID protein interactions in the presence and absence of Rem using screens that detect biotinylation of interacting proteins by BirA or conjugation to an AID-ubiquitin fusion protein. The role of Rem retrotranslocation for AID degradation will be investigated.
In Aim 2, MMTVs or T-cell variants lacking Rem expression will be used to determine the types of transition mutations observed in proviruses after infections of knockout mice lacking one or more Apobec genes. These studies will use a complex mouse retrovirus to explore aspects of innate immunity that are difficult to test in vivo with human viruses. Our experiments will provide improved understanding of Apobec function as well as potential application to the treatment of human infectious diseases and cancers.
Cellular restriction factors affect innate immune responses and the development of adaptive immunity to multiple pathogens. Mouse mammary tumor virus (MMTV) is a complex retrovirus that models organizational and functional features of complex retroviruses, such as human immunodeficiency virus (HIV). This application takes advantage of mouse genetics for in vivo and in vitro studies of an MMTV- encoded Vif-like factor that antagonizes activation-induced cytidine deaminase (AID) and multiple members of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (Apobec) family to increase our understanding of immune responses and its application to the treatment of human disease.
