A3 proteins are potent retrovirus restriction factors which have been shown to inhibit infection by a number of viruses, most notably HIV-1. hA3G and human APOBEC3F (hA3F) inhibition of HIV-1 occurs through two mechanisms. In the absence of Vif, these hA3 proteins are packaged into viral particles and inhibit infection via CDA of the reverse-transcribed DNA and other more poorly defined means. In addition, cell-intrinsic hA3G and, to a lesser extent, hA3F can inhibit infection in DCs and resting T cells. However, little is known about which of these mechanisms is critical to the antiviral properties of A3 proteins in vivo.
Here, we showed that increasing A3 levels in vivo results in greater A3-mediated restriction of virus infection. We showed previously that MMTV infection of mA3−/−
mice was dramatically increased compared to that of their wild-type littermates (35
). Since MMTV depends on DCs to initiate infection in vivo (9
) and little was known about mA3 expression in murine DCs, we first determined that mA3 expression levels increased upon treatment with LPS. LPS induces DC maturation and cytokine production, notably of type I IFNs, which are inducers of hA3G transcription, as well as of other retroviral restriction factors such as TRIM5α and tetherin (1
). The increase in mouse A3 levels upon LPS-induced DC activation and maturation or IFN-α treatment was similar to that observed by others for hA3G (5
) and supports previous studies demonstrating that mDCs are less infected by MMTV than iDCs (51
). Interestingly, most if not all of the antiviral effect induced by LPS was the result of mA3 restriction, since in mA3−/−
DCs, LPS treatment had little or no effect on ex vivo (Fig. ) or in vivo infection (Fig. ). Thus, at least in murine DCs, mA3 and not other cytokine-induced factors such as tetherin seems to be the major anti-MMTV restriction factor induced upon their maturation.
We also examined the relative importance of virion-packaged and cell-intrinsic mA3 in restriction of MMTV infection using three different experimental systems. First, we demonstrated that mA3-transfected 293T cells, which do not express endogenous A3 proteins, restricted MMTV infection compared to their untransfected counterparts. More importantly, BMDCs from mA3+/+ mice expressing physiologically relevant levels of mA3 showed much lower levels of infection than those from mA3−/− mice. Finally and most importantly, we observed that infection of DCs was higher in mA3−/− mice than in mA3+/+ mice, demonstrating that cell-intrinsic expression of mA3 restricts virus infection in vivo. In all three cases, this restriction occurred whether the incoming particle contained or was deficient in packaged mA3. Interestingly, in all the experimental systems, cell-intrinsic mA3 inhibited MMTV infection to a greater extent than did packaged mA3 with either one-hit or replication-competent viruses.
Strikingly, packaged mA3 appeared to synergize with cell-intrinsic mA3 to decrease virus infection in most experiments. For example, in vivo infection of mA3+/+
mice was decreased by more than fourfold when virions contained mA3; in contrast, mA3 in virions decreased infection only ~1.4-fold in mA3−/−
mice (Fig. ). This suggests that cell-intrinsic mA3 accesses the reverse transcription complex in incoming virions. Interestingly, packaged mA3 more potently restricted MMTV and HIV-1 infection in mA3+
cells than in mA3−
cells in vitro, ex vivo, and in vivo; this direct comparison of the relative inhibitory effects of packaged versus cell-intrinsic A3 has not been previously described. CDAs like hA3G and mA3 are believed to function as dimers or tetramers, and one recent study by Wedekind and colleagues (52
) suggested that hA3G self-associates into higher-order structures (13
). Thus, mA3 that is already present in MMTV cores may “attract” cell-intrinsic mA3 through self-association, and if dimers, tetramers, or higher-order mA3 structures are more effective inhibitors of, for example, reverse transcription, this would explain the enhanced activity of virion-packaged mA3 in mA3+
cells. How cell-intrinsic mA3 accesses viral cores in infected cells and inhibits reverse transcription is currently under investigation.
We did not observe editing of proviral DNA when mA3 was packaged into virions or when intrinsically expressed in DCs (Table ) (35
). Instead, we found that intrinsic mA3 exerts its effects at an early step of virus infection, since even strong-stop, ERT levels were reduced (Fig. ). This supports previous observations that both T cell- and DC-intrinsic expression of hA3G and hA3F inhibited HIV-1 infection but resulted in only low-level hypermutation of LRT (7
). A number of in vitro studies have suggested that A3 proteins in particles can inhibit binding of the
primer to the viral RNA or accumulation of HIV-1 reverse transcription products (2
). Whether mA3 inhibition of MMTV infection, either in the intrinsic or virion-packaged form, is preventing reverse transcriptase processivity or functions at an earlier step is currently under investigation.
As is believed to be the case for HIV-1, DCs are critical early targets for MMTV infection (9
). Expression of A3 in DCs may play a significant role in restriction of infection because they are the first cells to be infected by retroviruses like HIV and MMTV in vivo. Increased levels of cell-intrinsic A3 would render the host cell nonpermissive to initial infection and also increase the level of virion-packaged A3 protein. Thus, treatments that increase A3 expression would have a dual effect on suppression of virus infection in DCs. Whether this would also be the case for other lymphoid targets of MMTV infection such as B and T cells in mice is currently under investigation. In addition, we can test whether long-term administration of agents that increase A3 expression, such as IFN-α, will suppress viral loads in chronically infected mice.
While mice have a single A3 gene in contrast to humans, who have seven genes, these model studies suggest that increasing A3 expression in vivo has potential as an antiretroviral therapy, particularly because hA3G and hA3F levels are induced by IFN-α and other factors in many of the cell types that are targets for HIV-1 infection (5
). Because A3 proteins are CDAs, it has been speculated that their misexpression could lead to increased genomic instability or even cancer (15
), although there is, at present, no direct evidence that this occurs in mammalian cells. The mouse thus also provides a model system for testing whether increasing A3 levels as a means of antiretroviral therapy does indeed lead to genomic changes in vivo.