Apobec-Mediated Retroviral Hypermutation In Vivo is Dependent on Mouse Strain

Retroviruses cause lifelong infections resulting from their ability to thwart innate immunity. The Apobec family of cytidine deaminases are part of the innate immune response that recognizes and mutates foreign nucleic acids, including those from multiple viruses. Multiple retroviral antagonists of Apobecs have been identified, including mouse mammary tumor virus (MMTV)-encoded Rem protein. Previous experiments have shown that Rem-null MMTV or closely related TBLV proviruses from BALB/c tumors accumulate G-to-A and C-to-T mutations typical of Apobecs compared to wild-type proviruses expressing Rem. The difference in mutations between Rem-expressing and non-expressing MMTV strains largely disappeared in mice lacking the Apobec family member, activation-induced cytidine deaminase (AID). These results suggested that Rem is an AID antagonist. In this study, we attempted to study AID-mediated mutations of TBLV proviruses lacking Rem expression obtained from tumors in C57BL/6 (B6) wild-type and AID-knockout backgrounds. Surprisingly, no differences in G-to-A mutations were observed in TBLV proviruses regardless of Rem expression, yet such mutations were significantly reduced in proviruses obtained from mA3/AID-double knockout mice relative to those from wild-type B6 or AID-knockout mice. Many cellular mRNAs involving the innate immune response, but not Apobecs, were elevated in the absence relative to the presence of Rem expression on the B6 AID-knockout background. These results revealed that Apobec-mediated mutagenesis is dependent on mouse strain and suggested a second means of Rem-dependent immune evasion. Overall design: Each of two mouse mammary tumor virus (MMTV) strains (TBLV-WT = Rem-expressing or TBLV-SD = lacks Rem expression) was used to generate tumors in C57BL/6 (B6) mice or AID-knockout B6 mice. Three tumors from each mouse strain and virus were extracted for total DNA, and used for PCR to amplify the envelope-LTR region of the TBLV genome. Libraries of viral PCR fragments were prepared prior to MiSeq sequencing and comparison to the cloned TBLV-WT sequence (total of 12 samples). Nanopore sequencing was employed for high-throughput sequence analysis of TBLV proviruses from tumors induced by low-dose TBLV infection. For Nanopore sequencing, we used DNA extracted separately from three tumors for each mouse strain/virus strain combination. Mouse strains used for virus infection were wild-type B6, AID-knockout, and mA3-AID double knockout (6 tumors/strain X 3 strains = 18 tumors for Nanopore sequencing). DNA samples from each of the 18 samples was used for PCR to amplify the envelope-LTR region of the TBLV genome. Libraries of viral PCR fragment were prepared prior to Nanopore sequencing and comparison to the cloned TBLV wild-type (Rem+) sequence.