The retrovirus XMRV (xenotropic murine leukemia virus-related virus) has been detected in human prostate tumors and in blood samples from patients with chronic fatigue syndrome, but these findings have not been replicated. We hypothesized that an understanding of when and how XMRV first arose might help explain the discrepant results. We studied human prostate cancer cell lines CWR22Rv1 and CWR-R1, which produce XMRV virtually identical to the viruses recently found in patient samples, as well as their progenitor human prostate tumor xenograft (CWR22) that had been passaged in mice. We detected XMRV infection in the two cell lines and in the later passage xenografts, but not in the early passages. Importantly, we found that the host mice contained two proviruses, PreXMRV-1 and PreXMRV-2, which share 99.92% identity with XMRV over >3.2-kb stretches of their genomes. We conclude that XMRV was not present in the original CWR22 tumor but was generated by recombination of two proviruses during tumor passaging in mice. The probability that an identical recombinant was generated independently is negligible (~10-12); our results suggest that the association of XMRV with human disease is due to contamination of human samples with virus originating from this recombination event.

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be ∼10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.

We previously proposed that a balance between nucleotide excision and template RNA degradation plays an important role in nucleoside reverse transcriptase inhibitor (NRTI) resistance. To explore the predictions of this concept, we analyzed the role of patient-derived C-terminal domains of HIV-1 reverse transcriptase (RT) in NRTI resistance. We found that when the polymerase domain contained previously described thymidine analog resistance mutations, mutations in the connection domain increased resistance to 3 -azido-3 -deoxythymidine (AZT) from 11-fold to as much as 536-fold over wild-type RT. Mutational analysis showed that amino acid substitutions E312Q, G335C/D, N348I, A360I/V, V365I, and A376S were associated strongly with the observed increase in AZT resistance; several of these mutations also decreased RT template switching, suggesting that they alter the predicted balance between nucleotide excision and template RNA degradation. These results indicate that mutations in the C-terminal domain of RT significantly enhance clinical NRTI resistance and should be considered in genotypic and phenotypic drug resistance studies.drug resistance ͉ excision ͉ recombination ͉ RNase H ͉ thymidine analog mutations N ucleoside reverse transcriptase inhibitors (NRTIs) constitute a major class of clinically effective antiretroviral drugs (1). HIV-1 populations possess high genetic diversity, which allows them to acquire rapidly resistance to NRTIs and other inhibitors, limiting the effectiveness of antiviral drugs in controlling viral replication and combating AIDS (2). Resistance to the NRTIs 3Ј-azido-3Ј-deoxythymidine (AZT), 2,3-didehydro-2,3-dideoxythymidine (d4T), dideoxyinosine 2Ј,3Ј-dideoxyinosine, 2Ј,3Ј-dideoxycytidine, abacavir, and tenofovir is associated with thymidine analog resistance mutations (TAMs) that are located in the polymerase (pol) domain of HIV-1 reverse transcriptase (RT) (1).We recently observed that AZT treatment increases the frequency of RT template switching in single-cycle assays and that mutations in the RNase H (rh) domain of HIV-1 RT confer high-level resistance to AZT and d4T (3). RT template switching occurs through a proposed mechanism called dynamic copy choice (4), which postulates that a balance between the rates of DNA synthesis and RNA degradation is an important determinant of RT template switching: slowing DNA synthesis increases RT template switching, whereas reducing RNA degradation decreases RT template switching (4, 5). Based on these observations and predictions of the dynamic copy choice model, we proposed a previously undescribed mechanism for NRTI resistance, which states that a balance between degradation of HIV-1 RNA by rh and nucleotide excision from a terminated primer is an important determinant of NRTI resistance. Thus, a reduced rate of RNA degradation is proposed to increase the time period available for excision of incorporated NRTIs, leading to an increase in NRTI resistance.To investigate whether this proposed mechanism contributes to NRTI resistance arising duri...