Early studies reported the presence of XMRV in stromal cells from prostate cancer patient samples and also in CFS clinical samples. Some of the subsequent studies confirmed these findings whereas several others failed to identify XMRV in prostate cancer or in CFS patients, even when same samples were used (71
). It was recently reported that human sample contamination with mouse DNA can occur frequently (17
). Moreover, two coauthors from this study have recently demonstrated that XMRV is the product of recombination events between two MLV proviruses, suggesting that XMRV may not be relevant to human disease (18
). Nonetheless, XMRV is still an important human retrovirus and comparisons with HIV can provide valuable insights into the fundamental mechanisms of DNA polymerization, RT inhibition and drug resistance. (75
There is high degree of sequence similarity between the XMRV and MoMLV RTs (95% amino acid identity), and much less so with HIV-1 RT. Based on gel filtration experiments we conclude that unlike HIV-1 RT, but similar to MoMLV RT, XMRV RT exists in solution primarily as a monomer. We also included comparisons with HIV-1 RT in this study as it has been extensively studied and provides an excellent frame of reference.
We report here that there are significant differences in the DNA polymerization efficiency of the three enzymes. Although the polymerase active sites of the XMRV and MoMLV enzymes are almost identical, there is a considerable decrease in the efficiency of nucleotide incorporation by XMRV RT. Most differences in sequence are at the RNase H domain and are likely to affect polymerization by changing the positioning of DNA at the polymerase active site.
We have recently solved the crystal structure of the XMRV RNase H at high resolution (1.5Å) (pdb 3P1G) (Kirby, K.A. et al
., submitted for publication). We observed major differences in affinity for nucleic acid that we determined with gel-mobility shift assays and with pre-steady state kinetics. SPR experiments dissected in more detail the specific defect of XMRV RT in binding DNA. Surprisingly, XMRV RT can associate very rapidly with DNA, even more so than HIV-1 RT ( and ). However, it dissociates from DNA much faster than the HIV enzyme, resulting in an overall reduced binding affinity. A possible reason for the fast association and dissociation rates of XMRV RT may be the apparent monomeric state, which might offer facile access to the nucleic acid binding cleft, although with less contacts and lower affinity than HIV-1 RT, which is a heterodimer (76
). This high rate of XMRV RT dissociation from DNA likely contributes to the decreased processivity observed in our study, and may have consequences in the recombination rates of this virus.
Previous sequences of XMRV from prostate cancer tumors showed low variability, suggesting that the virus may have a high fidelity of replication (1
). Our study demonstrated that HIV-1 RT and MoMLV RT incorporated mismatched nucleotides and extended past the mismatches more efficiently than XMRV RT. Pre-steady state kinetics established that the higher overall fidelity of XMRV RT over MoMLV RT is due to a lower affinity for mismatched nucleotides. When compared to HIV-1 RT, however, XMRV RT has differs in both the nucleotide binding and incorporation steps. Nonetheless, XMRV did not have higher fidelity than a related amphotropic MLV virus or HIV-1 in a cell-based assay. It is possible that the high dNTP concentration in dividing cells can suppress mismatching events. We have previously shown (39
) that as nucleotide concentrations vary in different cell lines, this can affect viral susceptibility to NRTIs, and possibly in this case also incorporation of mismatched nucleotides. Additional cell-based studies using multiple cell lines and a large panel of viruses should provide a better understanding of the relation between in vivo
and in vitro
Early studies have reported susceptibility of XMRV to some antiretrovirals that have been used in the treatment of HIV infection (53–56
). In those studies the compounds were tested at the virus level. To better understand the interactions of inhibitors at their RT target level we tested here the ability of these and several more compounds to block the polymerase activity of XMRV RT. We found that two TDRTIs, EFdA-TP and ENdA-TP were very potent RT inhibitors (IC50
µM and 0.14
µM, respectively). Unlike other NRTIs, these compounds have a 3′ OH group and are known to efficiently inhibit HIV replication by blocking translocation (32
). Preliminary experiments demonstrated that they also block XMRV RT by the same mechanism (data not shown).
In HIV, moderate resistance to EFdA is conferred by the emergence of the M184V mutation at the conserved X position of the conserved YXDD motif of the polymerase active site. Interestingly, XMRV and MoMLV RTs already have a valine (V223) at this position. This difference is likely to contribute to the better potency of EFdA against HIV-1 RT than XMRV RT or MoMLV RT (57
). It may also contribute to the decreased ability of XMRV RT to unblock chain-terminated primers, as was also reported for M184V HIV-1 RT (79
) and to the enhanced fidelity reported here for XMRV RT, which is also reminiscent of the previously reported high fidelity of M184V HIV-1 RT (80
). Nonetheless, despite the presence of a Val in the YMDD motif of XMRV RT we found EFdA to inhibit very efficiently replication-competent or pseudotyped XMRV, with submicromolar EC50
s (40 and 110
Previously, highly potent aptamers were selected to inhibit MoMLV RT (60
). We demonstrate here that the three aptamers we tested have varying potency against XMRV RT. Aptamer m.1.1FL was the most potent inhibitor of XMRV RT and MoMLV RT in in vitro
2 and 4
nM, respectively). The fact that XMRV and MoMLV RTs are inhibited by the same aptamers at comparable efficiencies suggests that the RT residues that are different in the two enzymes are not critical to the binding of the aptamer. In contrast, heterodimeric HIV-1 RT has a very different binding cleft and is not inhibited by these aptamers.
Tenofovir is an essential component of HIV therapies and is also a potent inhibitor of XMRV RT. HIV resistance to tenofovir is conferred by a single codon mutation (K65R). HIV-1 RT residue 65 is known to interact with the incoming dNTP or the activated tenofovir analog (tenofovir diphosphate) (82
). K65R causes resistance to tenofovir by lowering the kpol
for the incorporation of the inhibitor into the nascent DNA. We prepared XMRV RT with the equivalent mutation, K103R, and determined that it has decreased susceptibility to tenofovir. Hence, it is possible for XMRV to develop tenofovir resistance through the same mechanism as HIV-1 RT. HIV resistance to AZT can occur by either decreased binding/incorporation or increased excision of the chain-terminating NRTI (33
). HIV-1 RTs containing the M41L, D67N, K70R, T215Y/F, K219E/Q mutations show enhanced removal of AZT. Our experiments show that unlike wild-type HIV-1 RT, XMRV RT is not able to excise NRTI-terminated primers. Similarly, it was previously shown that MoMLV RT is not capable of unblocking chain-terminated primers (33
In HIV, decreased binding of AZT is conferred initially in the presence of the primary Q151M mutation, followed by secondary mutations F77L, A62V, V75I and F116Y (27
). XMRV RT already differs from wild-type HIV-1 RT in the first three of these residues (P104, Q113 and L115 versus A62, V75 and F77) (). We demonstrated that introducing the primary Q→M mutation at the equivalent XMRV RT site (Q190M) resulted in an enzyme with decreased susceptibility to AZT. Hence, it appears that these residues can confer AZT resistance to XMRV by reduced incorporation of nucleotide analogs, as is the case in HIV-2 (41
). At this point we do not know if introduction of as yet unknown mutations could endow XMRV RT with the ability to unblock chain-terminated nucleic acids. The details of the molecular mechanism of XMRV resistance to tenofovir and AZT are under investigation.
In conclusion, our study provides detailed biochemical analysis of the mechanisms of polymerization, inhibition, fidelity, processivity and drug resistance of XMRV RT and how it compares with the closely related enzyme MoMLV RT and the more distantly related HIV-1 RT. The findings enhance our understanding of the basic mechanisms of reverse transcription.