Xenotropic Murine Leukemia Virus-related Virus Research Articles

Lack of evidence for human infection with Xenotropic murine leukemia virus-related virus in the Brazilian Amazon basin.

This study confirmed the absence of natural infection with Xenotropic murine leukemia virus-related virus (XMRV) or XMRV-related disease in human populations of the Brazilian Amazon basin. We demonstrated that 803 individuals of both sexes, who were residents of Belem in the Brazilian State of Pará, were not infected with XMRV. Individuals were divided into 4 subgroups: healthy individuals, individuals infected with human immunodeficiency virus, type 1 (HIV-1), individuals infected with human T-lymphotrophic virus, types 1 or 2 (HTLV-1/2), and individuals with prostate cancer. XMRV infection was investigated by nested PCR to detect the viral gag gene and by quantitative PCR to detect pol. There was no amplification of either gag or pol segments from XRMV in any of the samples examined. This study supports the conclusions of the studies that eventually led to the retraction of the original study reporting the association between XMRV and human diseases.

Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV.

XMRV low level of expression in human cells delays superinfection interference and allows proviral copies to accumulate

Xenotropic Murine leukemia virus-Related Virus (XMRV) directly arose from genetic recombinations between two endogenous murine retroviruses that occurred during human xenografts in laboratory mice. Studies on XMRV could thus bring clues on how a new retrovirus could circumvent barrier species. We observed that XMRV exhibits a weak promoter activity in human cells, similar to the transcription level of a Tat-defective HIV-1. Despite this low fitness, XMRV can efficiently propagate through the huge accumulation of viral copies (≈40 copies per cell) that compensates for the low expression level of individual proviruses. We further demonstrate that there is an inverse relationship between the maximum number of viral copies per infected cell and the level of viral expression, which is explained by viral envelope interference mechanisms. Low viral expression compensation by viral copy accumulation through delayed interference could a priori contribute to the propagation of others viruses following species jumps.

Endogenous Retroviruses and Human Cancer: Is There Anything to the Rumors?

Xenotropic murine leukemia virus-related virus (XMRV) infection was incorrectly associated with prostate cancer and chronic fatigue syndrome (CFS) in recent years. In this forum, we discuss the story of XMRV and how we can apply lessons learned here to inform the debate surrounding cancers associated with humanendogenous retroviruses (HERVs).

Prevention of Contamination by Xenotropic Murine Leukemia Virus-Related Virus: Susceptibility to Alcohol-Based Disinfectants and Environmental Stability

Xenotropic murine leukemia virus-related virus (XMRV) represents a novel γ-retrovirus that is capable of infecting human cells and has been classified as a biosafety level 2 (BSL-2) organism. Hence, XMRV represents a potential risk for personnel in laboratories worldwide. Here, we measured the stability of XMRV and its susceptibility to alcohol-based disinfectants. To this end, we exposed an infectious XMRV reporter virus encoding a secretable luciferase to different temperatures, pH values, and disinfectants and infected XMRV-permissive Raji B cells to measure residual viral infectivity. We found that 1 min treatment of XMRV particles at 60°C is sufficient to reduce infectivity by 99.9%. XMRV infectivity was maximal at a neutral pH but was reduced by 86% at pH 4 and 99.9% at pH 10. The common hand and surface disinfectants ethanol and isopropanol as well as the cell fixation reagent paraformaldehyde abrogated XMRV infectivity entirely, as indicated by a reduction of infectivity exceeding 99.99%. Our findings provide evidence of specific means to inactivate XMRV. Their application will help to prevent unintended XMRV contamination of cell cultures in laboratories and minimize the risk for laboratory personnel and health care workers to become infected with this biosafety level 2 organism.

Murine Leukemia Virus Uses NXF1 for Nuclear Export of Spliced and Unspliced Viral Transcripts

Intron-containing mRNAs are subject to restricted nuclear export in higher eukaryotes. Retroviral replication requires the nucleocytoplasmic transport of both spliced and unspliced RNA transcripts, and RNA export mechanisms of gammaretroviruses are poorly characterized. Here, we report the involvement of the nuclear export receptor NXF1/TAP in the nuclear export of gammaretroviral RNA transcripts. We identified a conserved cis-acting element in the pol gene of gammaretroviruses, including murine leukemia virus (MLV) and xenotropic murine leukemia virus (XMRV), named the CAE (cytoplasmic accumulation element). The CAE enhanced the cytoplasmic accumulation of viral RNA transcripts and the expression of viral proteins without significantly affecting the stability, splicing, or translation efficiency of the transcripts. Insertion of the CAE sequence also facilitated Rev-independent HIV Gag expression. We found that the CAE sequence interacted with NXF1, whereas disruption of NXF1 ablated CAE function. Thus, the CAE sequence mediates the cytoplasmic accumulation of gammaretroviral transcripts in an NXF1-dependent manner. Disruption of NXF1 expression impaired cytoplasmic accumulations of both spliced and unspliced RNA transcripts of XMRV and MLV, resulting in their nuclear retention or degradation. Thus, our results demonstrate that gammaretroviruses use NXF1 for the cytoplasmic accumulation of both spliced and nonspliced viral RNA transcripts. Murine leukemia virus (MLV) has been studied as one of the classic models of retrovirology. Although unspliced host messenger RNAs are rarely exported from the nucleus, MLV actively exports unspliced viral RNAs to the cytoplasm. Despite extensive studies, how MLV achieves this difficult task has remained a mystery. Here, we have studied the RNA export mechanism of MLV and found that (i) the genome contains a sequence which supports the efficient nuclear export of viral RNAs, (ii) the cellular factor NXF1 is involved in the nuclear export of both spliced and unspliced viral RNAs, and, finally, (iii) depletion of NXF1 results in nuclear retention or degradation of viral RNAs. Our study provides a novel insight into MLV nuclear export.

The Nef-Like Effect of Murine Leukemia Virus Glycosylated Gag on HIV-1 Infectivity Is Mediated by Its Cytoplasmic Domain and Depends on the AP-2 Adaptor Complex

Human immunodeficiency virus type 1 (HIV-1) Nef enhances the infectivity of progeny virions. However, Nef is dispensable for the production of HIV-1 virions of optimal infectivity if the producer cells are superinfected with certain gammaretroviruses. In the case of the ecotropic Moloney murine leukemia virus (M-MLV), the Nef-like effect is mediated by the glycosylated Gag (glycoGag) protein. We now show that the N-terminal intracellular domain of the type II transmembrane protein glycoGag is responsible for its effect on HIV-1 infectivity. In the context of a fully active minimal M-MLV glycoGag construct, truncations of the cytoplasmic domain led to a near total loss of activity. Furthermore, the cytoplasmic domain of M-MLV glycoGag was fully sufficient to transfer the activity to an unrelated type II transmembrane protein. Although the intracellular region of glycoGag is relatively poorly conserved even among ecotropic and xenotropic MLVs, it was also fully sufficient for the rescue of nef-deficient HIV-1 when derived from a xenotropic virus. A mutagenic analysis showed that only a core region of the intracellular domain that exhibits at least some conservation between murine and feline leukemia viruses is crucial for activity. In particular, a conserved YXXL motif in the center of this core region was critical. In addition, expression of the μ2 subunit of the AP-2 adaptor complex in virus producer cells was essential for activity. We conclude that the ability to enhance HIV-1 infectivity is a conserved property of the MLV glycoGag cytoplasmic domain and involves AP-2-mediated endocytosis. The Nef protein of HIV-1 and the entirely unrelated glycosylated Gag (glycoGag) protein of a murine leukemia virus (MLV) similarly enhance the infectiousness of HIV-1 particles by an unknown mechanism. MLV glycoGag is an alternative version of the structural viral Gag protein with an extra upstream region that provides a cytosolic domain and a plasma membrane anchor. We now show for the first time that the cytosolic domain of MLV glycoGag contains all the information needed to enhance HIV-1 infectivity and that this function of the cytosolic domain is conserved despite limited sequence conservation. Within the cytosolic domain, a motif that resembles a cellular sorting signal is critical for activity. Furthermore, the enhancement of HIV-1 infectivity depends on an endocytic cellular protein that is known to interact with such sorting signals. Together, our findings implicate the endocytic machinery in the enhancement of HIV-1 infectivity by MLV glycoGag.

No Detection of Xenotropic Murine Leukemia Virus-Related Viruses in Prostate Cancer in Sanandaj, West of Iran

Multiple etiologies have been hypothesized for prostate cancer, including genetic defects and infectious agents. A recently reported gamaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) has been reported to be detected in prostate cancer. However, this virus has not been detected in similar groups of patients in other studies. Herein, we sought to detect XMRV in prostate cancers and benign controls in Sanandaj, west of Iran. In a case-control study, genomic DNA was extracted from formalin fixed and paraffin embedded prostate tissues from a total of 163 Iranian patients. We developed a conventional and a nested PCR assay using primers targeting to an env specific sequence of XMRV. PCR assays were carried out on 63 prostate cancers and 100 benign prostate hyperplasias. Beta-actin sequences were successfully detected in the DNA extracts from all prostate tissues, confirming DNA extraction integrity. We did not detect XMRV in samples either from prostate cancers or benign prostate hyperplasias using XMRV specific primers. We conclude that in our population XMRV does not play a role in genesis of prostate cancer.

A novel approach to achieving modular retrovirus clearance for a parvovirus filter

Viral filtration is routinely incorporated into the downstream purification processes for the production of biologics produced in mammalian cell cultures (MCC) to remove potential viral contaminants. In recent years, the use of retentive filters designed for retaining parvovirus (~20 nm) has become an industry standard in a conscious effort to further improve product safety. Since retentive filters remove viruses primarily by the size exclusion mechanism, it is expected that filters designed for parvovirus removal can effectively clear larger viruses such as retroviruses (~100 nm). In an attempt to reduce the number of viral clearance studies, we have taken a novel approach to demonstrate the feasibility of claiming modular retrovirus clearance for Asahi Planova 20N filters. Porcine parvovirus (PPV) and xenotropic murine leukemia virus (XMuLV) were co-spiked into six different feedstreams and then subjected to laboratory scale Planova 20N filtration. Our results indicate that Planova 20N filters consistently retain retroviruses and no retrovirus has ever been detected in the filtrates even when significant PPV breakthrough is observed. Based on the data from multiple in-house viral validation studies and the results from the co-spiking experiments, we have successfully claimed a modular retrovirus clearance of greater than 6 log10 reduction factors (LRF) to support clinical trial applications in both USA and Europe.

Crystal Structures of Beta- and Gammaretrovirus Fusion Proteins Reveal a Role for Electrostatic Stapling in Viral Entry

Membrane fusion is a key step in the life cycle of all envelope viruses, but this process is energetically unfavorable; the transmembrane fusion subunit (TM) of the virion-attached glycoprotein actively catalyzes the membrane merger process. Retroviral glycoproteins are the prototypical system to study pH-independent viral entry. In this study, we determined crystal structures of extramembrane regions of the TMs from Mason-Pfizer monkey virus (MPMV) and xenotropic murine leukemia virus-related virus (XMRV) at 1.7-Å and 2.2-Å resolution, respectively. The structures are comprised of a trimer of hairpins that is characteristic of class I viral fusion proteins and now completes a structural library of retroviral fusion proteins. Our results allowed us to identify a series of intra- and interchain electrostatic interactions in the heptad repeat and chain reversal regions. Mutagenesis reveals that charge-neutralizing salt bridge mutations significantly destabilize the postfusion six-helix bundle and abrogate retroviral infection, demonstrating that electrostatic stapling of the fusion subunit is essential for viral entry. Our data indicate that salt bridges are a major stabilizing force on the MPMV and XMRV retroviral TMs and likely provide the key energetics for viral and host membrane fusion.

Utilization of Replication-Competent XMRV Reporter-Viruses Reveals Severe Viral Restriction in Primary Human Cells

The gammaretrovirus termed xenotropic murine leukemia virus-related virus (XMRV) was described to be isolated from prostate cancer tissue biopsies and from blood of patients suffering from chronic fatigue syndrome. However, many studies failed to detect XMRV and to verify these disease associations. Data suggesting the contamination of specimens in particular by PCR-based methods and recent reports demonstrating XMRV generation via recombination of two murine leukemia virus precursors raised serious doubts about XMRV being a genuine human pathogen. To elucidate cell tropism of XMRV, we generated replication competent XMRV reporter viruses encoding a green fluorescent protein or a secretable luciferase as tools to analyze virus infection of human cell lines or primary human cells. Transfection of proviral DNAs into LNCaP prostate cancer cells resulted in readily detectably reporter gene expression and production of progeny virus. Inoculation of known XMRV susceptible target cells revealed that these virions were infectious and expressed the reporter gene, allowing for a fast and highly sensitive quantification of XMRV infection. Both reporter viruses were capable of establishing a spreading infection in LNCaP and Raji B cells and could be easily passaged. However, after inoculation of primary human blood cells such as CD4 T cells, macrophages or dendritic cells, infection rates were very low, and a spreading infection was never established. In line with these results we found that supernatants derived from these XMRV infected primary cell types did not contain infectious virus. Thus, although XMRV efficiently replicated in some human cell lines, all tested primary cells were largely refractory to XMRV infection and did not support viral spread. Our results provide further evidence that XMRV is not a human pathogen.

Novel insights from structural analysis of lentiviral and gammaretroviral reverse transcriptases in complex with RNA/DNA hybrids

Structures of HIV-1 reverse transcriptase (RT) have been reported in several forms, but only one contains an RNA/DNA hybrid, the conformation of which has been controversial. We have been successful in obtaining three structures of HIV-1 RT complexed with a non-nucleoside RT inhibitor (NNRTI) and an RNA/DNA hybrid [1]. In the presence of an NNRTI, our RNA/DNA structure differs from all prior nucleic acid bound to RT including the previously-reported RNA/DNA hybrid derived froom the polypurine tract. The enzyme structure observed in our cocrystals also differs from all previous RT-DNA complexes. As a result, the hybrid has ready access to the ribonuclease H (RNase H) active site. These observations collectively reinforce previous proposals that an RT-nucleic acid complex may be required to adopt independent structural states competent for DNA synthesis and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites map to the unique RT-hybrid interface that undergoes conformational changes between two catalytic states. Structural features of the nucleoprotein complex, including drug resistance mutations, have been verified by site-directed mutagenesis, and will be presented. Although the single-subunit RT of Moloney murine leukemia virus (Mo-MLV) has been extensively characterized biochemically, structural information is lacking that describes the substrate binding mechanism for this RT species. We also present data on the first crystal structure of a complex between an RNA/DNA hybrid and the 72 kDa single-subunit RT from the related xenotropic murine leukemia virus-related virus (XMRV) [2]. A comparison of this structure with its HIV-1 counterpart shows that substrate binding around the DNA polymerase active site is conserved but differs between the two enzymes in their thumb and connection subdomains. Small-angle X-ray scattering (SAXS) was used to model full-length XMRV RT, demonstrating its flexible RNase H domain becomes ordered in the presence of substrate, a key difference between monomeric and dimeric RTs.

Generation of Multiple Replication-Competent Retroviruses through Recombination between PreXMRV-1 and PreXMRV-2

We previously identified two novel endogenous murine leukemia virus proviruses, PreXMRV-1 and PreXMRV-2, and showed that they most likely recombined during xenograft passaging of a human prostate tumor in mice to generate xenotropic murine leukemia virus-related virus (XMRV). To determine the recombination potential of PreXMRV-1 and PreXMRV-2, we examined the generation of replication-competent retroviruses (RCRs) over time in a cell culture system. We observed that either virus alone was noninfectious and the RNA transcripts of the viruses were undetectable in the blood and spleen of nude mice that carry them. To determine their potential to generate RCRs through recombination, we transfected PreXMRV-1 and PreXMRV-2 into 293T cells and used the virus produced to infect fresh cells; the presence of reverse transcriptase activity at 10 days postinfection indicated the presence of RCRs. Population sequencing of proviral DNA indicated that all RCRs contained the gag and 5' half of pol from PreXMRV-2 and the long terminal repeat, 3' half of pol (including integrase), and env from PreXMRV-1. All crossovers were within sequences of at least 9 identical nucleotides, and crossovers within each of two selected recombination zones of 415 nucleotides (nt) in the 5' untranslated region and 982 nt in pol were required to generate RCRs. A recombinant with the same genotype as XMRV was not detected, and our analysis indicates that the probability of generating an identical RCR is vanishingly small. In addition, the studies indicate that the process of RCR formation is primarily driven by selection for viable cis and trans elements from the parental proviruses.

The Influence of the Xenotropic Murine Leukaemia Virus-Related Virus (XMRV) Research Findings on the Thoughts and Feelings of People Living with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis: An Interpretative Phenomenological Analysis

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a condition of unknown aetiology characterised by severe physical and mental fatigue, musculoskeletal pain, and cognitive disturbances. Due to the lack of a clear biological marker, those with CFS/ME frequently encounter stigma and delegitimation of illness, causing them psychological distress. Individuals with CFS/ME tend to reject the psychogenic model associated with CFS/ME in favour of a viral or immunological explanation: a perception which appears to aid their understanding of their illness. A recent, widely publicised study by Lombardi et al. (2009) claimed to have identified a link between xenotropic murine leukaemia virus-related virus (XMRV) and CFS/ME. In this context, an investigation of the influence of these research findings on the thoughts and feelings of people living with CFS/ME was conducted. Nine semi-structured telephone interviews were conducted with support group members in the United Kingdom. Interpretative Phenomenological Analysis (IPA; Smith 1996) was used to uncover three superordinate themes: XMRV and the legitimation of CFS/ME, a cautious response to the research findings, and criticism of the reaction to the XMRV research of fellow individuals with CFS/ME. The findings are discussed in relation to the existing literature surrounding illness representations, with a critique of the present study.

The rise and fall of XMRV

Due to the relatively recent emergence of the human T-lymphotropic and the human immunodeficiency viruses, enthusiasm for the identification of novel viruses, especially retroviruses, with pathogenic potential in humans, remains high. Novel technologies are now available with the ability to search for unknown viruses, such as gene arrays and new generation sequencing of tissue and other samples. In 2006, chip technology identified a novel retrovirus in human prostate cancer (PCa) tissue samples. Due to close homology to a mouse retrovirus, the virus was named xenotropic murine leukaemia virus-related virus (XMRV). Ever since the initial disease association with PCa, XMRV has stirred a lot of attention and concern worldwide for the medical community, public health officials and in particular global transfusion services. Public response, in this new era of electronic communication and advocacy was rapid, wide and unprecedented. In this review, we outline the course of biomedical research efforts that were put forward internationally in the process of determining the risk to the human population, the response of the blood banking community and review the current state of knowledge of xenotropic murine retroviruses. Although XMRV is no longer regarded as an infection of humans, a lesson was learnt in modern virology that holds deeper implications for biomedical research, particularly stem cell generation and transplantation practices.

Revisiting a role for a mammary tumor retrovirus in human breast cancer

There remains great controversy as to whether mouse mammary tumor virus (MMTV), the etiological agent of mammary cancer in mice, or a closely related human retrovirus, plays a role in the development of breast cancer in humans. On one hand, retroviruses such as human T-cell lymphotropic virus and human immunodeficiency virus (HIV) are known causative agents of cancer (in the case of HIV, albeit, indirectly), but attempts to associate other retroviruses with human cancers have been difficult. A recent, high profile, example has been the postulated involvement of another mouse virus, xenotropic murine leukemia virus-related virus, in human prostate cancer, which is now thought to be due to contamination. Here, we review some of the more recent evidence for and against the involvement of MMTV in human breast cancer and suggest future studies that may allow a definitive answer to this conundrum.

Xenotropic MLV envelope proteins induce tumor cells to secrete factors that promote the formation of immature blood vessels

BackgroundXenotropic Murine leukemia virus-Related Virus (XMRV) is a γ-retrovirus initially reported to be present within familial human prostate tumors and the blood of patients with chronic fatigue syndrome. Subsequent studies however were unable to replicate these findings, and there is now compelling evidence that the virus evolved through rare retroviral recombination events in human tumor cell lines established through murine xenograft experiments. There is also no direct evidence that XMRV infection has any functional effects that contribute to tumor pathogenesis.ResultsHerein we describe an additional xenotropic MLV, “B4rv”, found in a cell line derived from xenograft experiments with the human prostate cancer LNCaP cell line. When injected subcutaneously in nude mice, LNCaP cells infected with XMRV or B4rv formed larger tumors that were highly hemorrhagic and displayed poor pericyte/smooth muscle cell (SMC) investment, markers of increased metastatic potential. Conditioned media derived from XMRV- or B4rv-infected LNCaPs, but not an amphotropic MLV control virus infected LNCaPs, profoundly decreased expression of marker genes in cultured SMC, consistent with inhibition of SMC differentiation/maturation. Similar effects were seen with a chimeric virus of the amphotropic MLV control virus containing the XMRV env gene, but not with an XMRV chimeric virus containing the amphotropic MLV env gene. UV-inactivated XMRV and pseudovirions that were pseudotyped with XMRV envelope protein also produce conditioned media that down-regulated SMC marker gene expression in vitro.ConclusionsTogether these results indicate that xenotropic MLV envelope proteins are sufficient to induce the production of factors by tumor cells that suppress vascular SMC differentiation, providing evidence for a novel mechanism by which xenotropic MLVs might alter tumor pathogenesis by disrupting tumor vascular maturation. Although it is highly unlikely that either XMRV or B4Rv themselves infect humans and are pathogenic, the results suggest that xenograft approaches commonly used in the study of human cancer promote the evolution of novel retroviruses with pathogenic properties.

Using high throughput screening to define virus clearance by chromatography resins

High throughput screening (HTS) of chromatography resins can accelerate downstream process development by rapidly providing information on product and impurity partitioning over a wide range of experimental conditions. In addition to the removal of typical product and process-related impurities, chromatography steps are also used to remove potential adventitious viral contaminants and non-infectious retrovirus-like particles expressed by rodent cell lines used for production. This article evaluates the feasibility of using HTS in a 96-well batch-binding format to study removal of the model retrovirus xenotropic murine leukemia virus (xMuLV) from product streams. Two resins were examined: the anion exchange resin Q Sepharose Fast Flow™ (QSFF) and Capto adhere™, a mixed mode resin. QSFF batch-binding HTS data was generated using two mAbs at various pHs, NaCl concentrations, and levels of impurities. Comparison of HTS data to that generated using the column format showed good agreement with respect to virus retentation at different pHs, NaCl concentrations and impurity levels. Results indicate that NaCl concentration and impurity level, but not pH, are key parameters that can impact xMuLV binding to both resins. Binding of xMuLV to Capto adhere appeared to tolerate higher levels of NaCl and impurity than QSFF, and showed some product-specific impact on binding that was not observed with QSFF. Overall, the results demonstrate that the 96-well batch-binding HTS technique can be an effective tool for rapidly defining conditions for robust virus clearance on chromatographic resins.

Complete Genome Sequences of New Xenotropic Murine Leukemia Viruses from the Senescence-Accelerated Mouse (SAM): Molecular and Phylogenetic Analyses

Approximately 10% of the mouse genome is constituted by endogenous retroviruses (ERVs), and a number of mouse ERVs remain active. Many copies of endogenous murine leukemia viruses (MuLVs) are detected in the genomes of inbred mouse strains. Some of these MuLVs are transcriptionally active or produce infectious virus particles. Previously, we identified partial env sequences of new xenotropic MuLVs (X-MuLVs) from a senescence-accelerated mouse (SAM) strain. In the present study, we investigated and characterized the complete sequences of the X-MuLVs. The complete genomes and open reading frames (ORFs) of two X-MuLVs, designated xmlv15 and xmlv18 (accession nos. HQ154630 and HQ154631, respectively), were molecularly cloned from the genome of the SAM mice. We confirmed that the xmlv15 and xmlv18 sequences are distinct from all known MuLV genomes and are most similar to DG-75 MuLV. Moreover, we found that common strains of laboratory mice carry our newly identified xmlvs. Additionally, the expression levels of xmlv15-related sequences were much higher in C57BL and ICR mice than in the SAM strains without any stimulators. Our findings suggest that a specific group of endogenous MuLVs is constitutively expressed in the brain and that they may participate in normal functions and/or pathogenic conditions.

Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid.

A key step in proliferation of retroviruses is the conversion of their RNA genome to double-stranded DNA, a process catalysed by multifunctional reverse transcriptases (RTs). Dimeric and monomeric RTs have been described, the latter exemplified by the enzyme of Moloney murine leukaemia virus. However, structural information is lacking that describes the substrate binding mechanism for a monomeric RT. We report here the first crystal structure of a complex between an RNA/DNA hybrid substrate and polymerase-connection fragment of the single-subunit RT from xenotropic murine leukaemia virus-related virus, a close relative of Moloney murine leukaemia virus. A comparison with p66/p51 human immunodeficiency virus-1 RT shows that substrate binding around the polymerase active site is conserved but differs in the thumb and connection subdomains. Small-angle X-ray scattering was used to model full-length xenotropic murine leukaemia virus-related virus RT, demonstrating that its mobile RNase H domain becomes ordered in the presence of a substrate—a key difference between monomeric and dimeric RTs.