This study demonstrates that rabbits bear an antiretroviral TRIM gene which clusters in a phylogenetic tree with antiviral TRIM5s from primates and cattle. Moreover, the human TRIM genes most closely related to TRIM5, TRIM genes 6, 34, and 22, cluster independently with their orthologues from mouse, cattle, and rabbit. We therefore propose that the antiviral rabbit protein is a true TRIM5 orthologue, as is the antiretroviral bovine gene previously referred to as bovine Lv1 (48
). Orthology indicates a common ancestor for these TRIM5 genes, and we presume that it had antiretroviral properties that have since been selected by pathogenic retroviral infection, leading to the species-specific antiviral properties that we observe today.
Reduction of rabbit TRIM5 expression using shRNAs rescues infectivity with the appropriate specificity, increasing the infectivities of viruses that are poorly infectious in rabbit cells, namely, HIV-1, HIV-2, FIV, and EIAV, but not the relatively infectious viruses SIVmac and MLV (Fig. ). This specificity strongly suggests that TRIM5 significantly contributes to the poor permissivity of rabbit cells to HIV-1, HIV-2, EIAV, and FIV. The abilities of two independent shRNA sequences to specifically increase infectivity supports this notion. The partial rescue of infectivity suggests that the reduction of rabbit TRIM5 expression in these experiments might be incomplete. Pools of drug-selected SIRC cells expressing shRNAs against rabbit TRIM5 grew poorly consistently, suggesting that strong reduction of this protein is not tolerated (data not shown). Furthermore, the transient assay employed is unlikely to deliver a high dose of shRNA-bearing vector to all the cells in the well, and this also might contribute to the incomplete reduction in rabbit TRIM5 expression. The modest effect of the shRNA could also be explained by the existence of further, as-yet-unidentified antiviral proteins in rabbit cells, and it is also possible that the rabbit cells might lack factors important for the high infectivity seen in feline CRFK cells.
A role for TRIM5 in restriction of retroviruses in rabbit cells is confirmed by expression in feline CRFK cells (Fig. ). Although it is likely that CRFK cells also express feline TRIM molecules, feline cells have been shown to be very permissive to retroviral vector infection (21
). Expression of rabbit TRIM5 significantly reduces the titers of HIV-1, HIV-2, EIAV, and FIV in these cells. Importantly, it does not reduce the infectivity of SIVmac, a virus that is highly infectious in rabbit cells. Strikingly, MLV-N but not MLV-B infectivity is reduced by overexpression of rabbit TRIM5, despite the fact that SIRC cells do not restrict MLV-N in comparison to MLV-B (57
) (Fig. and ). It is possible that the feline CRFK cells express a cofactor that expands the specificity of rabbit TRIM5 to MLV-N, but it is more likely that overexpression of TRIM5 leads to expanded specificity, as has been described for human TRIM5 (66
). We imagine that low-affinity interactions between TRIM5 and, for example, the MLV-N CA do not lead to restriction at low expression levels, but when rabbit TRIM5 is highly expressed, this interaction leads to restriction, as shown in Fig. . Whether such broadening of specificity represents an artifact of overexpression is unclear, particularly given that TRIM5 expression is up-regulated by interferon (3
Analysis of DNA synthesis by rabbit TRIM5-restricted viruses indicates strong reduction in reverse transcription by restricted viruses. As in most other cases of TRIM-mediated restriction, the block to viral DNA synthesis is weaker than the block to infectivity, suggesting that many viruses synthesize viral DNA but remain uninfectious (7
). This observation has been investigated further by the members of the Hope laboratory, who have shown that inhibition of the proteasome rescues DNA synthesis by TRIM5α-restricted virus but not infectivity (2
). This suggests that TRIM5α blocks infection independently of the proteasome and that rapid recruitment to an active proteasome prevents reverse transcription.
The observation that the CA bears the determinant for sensitivity to rabbit TRIM5 is consistent with determinants of sensitivity to other TRIM5 molecules (7
) and implies conservation in the mechanism of viral restriction. The dominant-negative activities of human TRIM5α and -δ against rabbit TRIM5 are also consistent with previous observations (5
). The simplest explanation for these data is that the human TRIM5 proteins multimerize with rabbit TRIM5 and compromise its ability to restrict through titration of the virus binding B30.2 domains. The stronger activity of TRIM5δ may be due to this shorter molecule being more readily able to interact with a heterologous trimer. It may also be that without a B30.2 domain, it cannot contribute to antiviral activity (39
). The strength of the dominant-negative rescue of infectivity is also related to the strength of the restriction, with FIV being both restricted most strongly and rescued most effectively (Fig. and ).
A number of studies have proposed that rabbits might be developed into an animal model for HIV-1/AIDS. Indeed, several studies have suggested that HIV-1 will replicate in rabbits if they are inoculated with human cells infected with HIV-1 (15
). Unfortunately, these promising early results did not culminate in the development of rabbits as an animal model, perhaps because rabbit cells are generally poorly permissive to HIV-1 infection (6
). Another study has shown that expression of an HIV-1 receptor (CD4) and a coreceptor (CCR5) on rabbit SIRC cells renders these cells permissive to HIV-1 infection (50
). We imagine that these data reflect the fact that while SIRC cells express a restriction factor strongly active against HIV-1, they are not completely nonpermissive to HIV-1 infection (Fig. ) (6
). It is therefore possible that a high-dose infection might lead to infected cells that can secrete HIV-1 into the supernatant, confounding the interpretation of this study. Our data do not rule out the development of rabbits for use as an animal model for HIV/AIDS but do suggest that a knockout of the rabbit TRIM5 gene will be an important component of this work. A recent study has suggested that rabbit cells might be nonpermissive to HIV-1 infection due to a recessive block caused by the lack of a host factor important for HIV-1 replication (11
). This notion was based on the observation that heterokaryons between permissive human cells and rabbit SIRC cells were permissive to HIV-1 infection. These data might now be explained by the observation that human TRIM5α and -δ proteins act as dominant negatives to the anti-HIV-1 activity found in SIRC cells (Fig. ).
The study of antiviral proteins is likely to be influenced by the narrow range of viruses employed. However, it is striking that primate, bovine, and rabbit TRIM5 proteins restrict multiple unrelated retroviruses, whereas the closely related human TRIM proteins 6, 22, and 34 do not (68
). This implies that the antiretroviral activities of the TRIM5 orthologues have evolved from a common antiretroviral ancestor, whereas TRIM proteins 6, 22, and 34 have evolved independently and may have alternative functions. Importantly, restriction by the TRIM5 orthologues has common features, such as targeting the viral CA early after viral entry and sensitivity to dominant-negative activities of related TRIM proteins. These observations suggest a conserved antiviral mechanism and support the notion of orthology.
The most important finding in this study is that rabbits bear an active orthologue of TRIM5. This suggests that rabbits have been under selection pressure from pathogenic retroviruses similar to that for primates and cattle. The recent identification of rabbit endogenous lentivirus type K supports this notion (25
). Moreover, the discovery of rabbit endogenous lentivirus type K extends the age of lentiviruses to approximately 7 million years and increases the likelihood that lentiviruses have contributed to the selection of TRIM5 antiviral activity. The phylogenetic tree in Fig. also indicates that murine TRIM genes 5, 12, and 30 are homologues of primate TRIM5. It is difficult to designate the true murine TRIM5 orthologue, which evolved from a common TRIM5 ancestor, and TRIM5 paralogues, which were derived by duplication of the TRIM5 orthologue, particularly when their antiviral activities remain as yet uncharacterized. The names assigned to the murine TRIM genes predate our study, and we cannot be sure whether the murine TRIM5 or TRIM30 gene is the true orthologue of primate TRIM5. However, we note that murine TRIM12 is likely to be derived by duplication of murine TRIM5, as they are almost identical, except for the loss of the B30.2 domain in TRIM12 (data not shown).
Cattle also appear to have multiple TRIM5 genes, here named TRIM5b and -d. Pigs have a single sequence, although they may bear further, as-yet-unidentified TRIM5-like genes. It seems certain that the TRIM genes in the TRIM5 cluster (Fig. ) are homologues of TRIM5, many of which are likely to have antiretroviral properties. It will be interesting to test this further, although a negative result is difficult to interpret as it may be that the viruses tested simply do not reflect the viruses that have driven TRIM5 selection. However, the TRIM5 proteins identified thus far tend to restrict multiple divergent retroviruses. It is certainly clear that the continued study of TRIM5 and related molecules will help us understand complex host-retrovirus relationships, particularly those that concern species-specific replication and zoonosis. We hope that eventually this information will improve animal models and therapeutics for HIV/AIDS.