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Other members of the A3 family are believed to affect other exogenous viruses as well as endogenous retrovirus/retroelement movement within the genome. In particular, human A3A is a potent inhibitor of IAP and MusD and other retrotransposons such as LINE-1 and this inhibition is CDA-independent, at least in cultured cells [18]–[20]. A3A also inhibits adeno-associated virus replication, a nuclear-replicating parvovirus, via CDA-independent means [20]. In monocytes, A3A restricts HIV-1 infection and the decrease in A3A levels that occurs during monocyte-to-macrophage development is concomitant with increased susceptibility to HIV-1 infection [21]. A3A is not packaged into HIV virions and is thought to restrict infection by targeting incoming virus [22]–[24]. In contrast, A3A is packaged in human T-lymphotropic virus type-I virions and restricts infection, at least in transfected cells [25]. A3A preferentially deaminates cytidines that are in a TC motif [26].

To determine the level of transgene expression, we first isolated RNA from different tissues, including peripheral blood mononuclear cells (PBMCs), and performed reverse-transcribed real-time quantitative PCR (RT-qPCR). RNA from human H9 cultured cells and human and C57BL/6 mouse PBMCs served as controls. For each transgene, there was one high- (A3Ghigh, A3Ahigh) and one low- (A3Glow, A3Alow) expressing strain, defined by their relative expression in lymphoid tissues. The A3Ghigh strain expressed higher levels of the transgene than the endogenous mouse gene in spleen and thymus, but similar A3G levels in mouse and human PBMCs, while the A3Glow strain expressed approximately 10-fold lower levels in these tissues (Figure 1A). In contrast, the A3Ahigh strain expressed similar or lower levels than mouse A3; there was also about 2-fold lower expression of A3A in mouse PBMCs than in human PBMCs (Figure 1B). The A3Alow strain had very low but detectable levels of expression in several tissues. Since the β-actin regulatory region was used, transgene expression was seen in many tissues and in several at levels higher than endogenous mouse A3 (e.g. heart, brain and liver) (Figure 1A and 1B). We also performed western blots on different tissues from the 4 different mouse strains, using antiserum that detects both A3A and A3G. The relative protein expression levels were similar to that seen at the RNA level (Figure S1A and S1B).

We next determined if the in vivo-produced A3A and A3G proteins were functionally active. Extracts were prepared from primary splenocyte cultures and equal amounts (total protein concentration/volume) were incubated with FAM-labeled substrates containing the A3A- or A3G-preferred target sequence (S50-TTC and S50-CCC, respectively). As controls, we also performed these assays with extracts prepared from 293T cell lines transfected with A3A or A3G. Activity could be readily detected in transgenic mice expressing high levels of A3A or A3G. Further, in accord with the known specificity of the cytidine deaminases, extracts from the A3Ahigh mice deaminated the TTC- more efficiently than CCC-containing substrates, while those from A3Ghigh mice more efficiently deaminated the CCC substrate (Figure 2). For both A3Alow and A3G low, trace amounts of activity were detectable with the preferred substrates, while no activity was detectable with either endogenous mA3 or from mA3 knockout splenocytes. No deaminase activity was detected with WT mouse extracts, perhaps because the mouse protein has lower overall activity or expression. These data show that the transgenic mice expressed catalytically active human deaminases in these heterologous cells.

Humans have 7 APOBEC3 genes and determining how each specifically functions to inhibit retroviruses like HIV is complicated, because all 7 can be produced in a given cell type or tissue.

To overcome this limitation, we made transgenic mice that express two of the human proteins, APOBEC3A and APOBEC3G in mice that do not express their own APOBEC3. These mice were able to effectively block infection by several mouse retroviruses

We were unable to perform similar assays with in vivo produced MMTV, because the only cell-free virus in mice is found in milk and mammary tumors and we have not yet established breeding colonies of virus-infected human A3 transgenic mice.

A3G and A3A inhibit retrovirus infection by different means.

Two A3A and A3G mouse strains each were generated, expressing levels of these proteins within the range or at levels lower than that seen in human cells. This likely has relevance to what occurs in individual humans, where non-coding region polymorphisms in A3 genes alter expression levels and may influence progression to HIV-induced disease

An additional limitation of previous studies done on human A3 proteins is the reliance on transfecting constructs expressing A3 proteins, which may not reflect the endogenous levels of a protein expression found in vivo

Moreover, we demonstrate that this binding is sufficient to concentrate a recombinant toxin to selectively kill HIV-1-infected cells. Our results suggest that the gp41 C-peptide region is a viable target for development of antiviral therapeutics, neutralizing antibodies, and cytotoxic agents directed against infected cells.

Such a molecule, administered together with agents that induce the expression of dormant integrated provirus (e.g., cytokines or activators of protein kinase C), might help to reduce or possibly eliminate latent reservoirs of HIV-1

Although variation in the entire HLA protein is involved in the differential response to HIV across HLA allotypes, the major genetic effects are condensed to the positions highlighted in this study, indicating a structural basis for the HLA association with disease progression that is probably mediated by the conformation of the peptide within the class I binding groove.

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