Induction of APOBEC3 in vivo causes increased restriction of retrovirus infection

It is now well established that proteins belonging to the APOBEC3 (A3) family are potent restriction factors of viruses and retrotransposable elements (6, 16, 46). Several mechanisms have been proposed for the antiviral activity of APOBEC3. Most, if not all, family members have cytidine deaminase (CDA) activity. As for human immunodeficiency virus type 1 (HIV-1) lacking the viral infectivity factor (vif) gene, human APOBEC3G (hA3G) protein is incorporated into budding HIV-1 virions and deaminates cytidine to uridine residues during reverse transcription. This causes hypermutation in the nascent retroviral DNA and restricts infection through increased degradation of the DNA and the generation of nonfunctional proviral genomes (16, 26). This mechanism is counteracted by Vif, which binds and degrades hA3G via the ubiquitin/proteasome pathway in virus-producing cells, thereby preventing it from being packaged (27, 47, 49, 55). A3 proteins also inhibit replication by an undefined CDA-independent mechanism(s) (33, 48). A3 proteins packaged into virions have been shown to inhibit the accumulation of HIV-1 reverse transcription products, plus-strand DNA transfer, and provirus integration, at least in tissue culture cells (18, 24, 29). In addition to A3 proteins inhibiting infection when packaged into virions, there is mounting evidence that these cellular proteins can restrict incoming virus particles. For example, hA3G expressed in recipient resting CD4+ T cells and mature dendritic cells (mDCs) functions as a postentry, Vif-resistant restriction for HIV infection (7, 39). Several A3 proteins are also expressed at higher levels in cells that restrict HIV-1 infection, such as monocytes and mDCs (36, 37, 39). Indeed, hA3G levels increase during dendritic cell (DC) maturation ex vivo, and treatment of several different cell types with alpha interferon (IFN-α) leads to higher levels of hA3G expression (5, 37, 39, 43, 50). Unlike humans, whose genome carries seven A3 genes, mice have a single mA3 gene, and although the mouse protein restricts HIV-1 infection and is a functional CDA, the domain organization of this single gene is different from any of the human genes (8, 14, 23). Previously, we showed that mice with targeted deletion of the mA3 gene supported higher levels of infection by the betaretrovirus mouse mammary tumor virus (MMTV) than their wild-type counterparts (35). Moreover, we showed that this restriction was independent of A3 CDA activity. More recently, several groups have demonstrated that mA3 inhibits infection by murine leukemia virus and that this also occurs without cytidine deamination (3, 42). Thus, although both MMTV and murine leukemia virus are infectious in mice, it is likely that endogenous A3 plays a role in limiting infection and thereby pathogenesis. In vivo, MMTV first infects DCs at the site of infection, which then carry the virus to lymph nodes (9). Infected DCs produce virus and, because they are professional antigen-presenting cells, present the MMTV-encoded superantigen (Sag) to T cells expressing Sag-specific T-cell receptor Vs chains (41). This results in the stimulation of Sag-reactive T cells, proliferation of B and T cells, and subsequent amplification of infection. MMTV's interactions with DCs also induce their maturation and migration, causing increased surface expression of costimulatory molecules and the MMTV entry receptor transferrin receptor 1 (TfR1) (4, 9, 28). Although MMTV infection of DCs leads to maturation and increased TfR1 expression on the cell surface, MMTV nonetheless infects immature DCs (iDCs) more efficiently than it infects mDCs (51). In this study, we examined whether this preferential replication pattern was due to differential restriction of MMTV by mA3 in iDCs versus that in mDCs. We demonstrate that virion-packaged and DC-intrinsic mA3 function together to restrict MMTV infection ex vivo and in vivo and that this restriction is not due to cytidine deamination. We also show that DC activation and maturation increase mA3 levels in target cells and that this increase results in reduced viral infection ex vivo and in vivo. These studies point to the importance of examining the role of host antiviral mechanisms using natural pathogens and their hosts.