Infection of female primary lower genital tract epithelial cells after natural pseudotyping of HIV-1: possible implications for sexual transmission of HIV-1.

The global AIDS pandemic continues to expand and in some regions of the world, such as southern Africa, the prevalence of HIV-1 infection exceeds 20%. The devastating spread of the virus in young women in these countries appears disproportional to overall risk of infection. Regions with high prevalence of HIV-1 are often also highly endemic for other pathogenic viruses including HSV, CMV and HTLV. We propose that acquisition by HIV-1 of the envelope glycoproteins of other viruses, in a process we call "natural pseudotyping," expands the cellular tropism of HIV-1, enabling it to infect female genital epithelial cells directly and thereby dramatically increasing risk of infection during sexual intercourse. In this proof-of-concept study, we demonstrate that when HIV-1 co-infects T cells along with the gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV), progeny HIV-1 particles are produced capable of infecting primary vaginal, ectocervical and endocervical epithelial cells. These cell types are normally resistant to HIV-1 infection. Infection of primary genital cells was neutralized by antisera against the XMRV glycoprotein, confirming that infection was mediated by the XMRV glycoprotein acquired through pseudotyping of HIV. Inhibition by AZT showed that active replication of HIV-1 occurred in these cells and ruled out non-specific endocytic uptake of the virus. These results demonstrate that natural pseudotyping can expand the tropism of HIV-1 to include genital epithelial cells and have potential implications for sexual transmission of the virus.

The use of preliminary scientific evidence in public health: a case study of XMRV.

Kumanan Wilson and colleagues explain how the rapid response to XMRV as a novel pathogen has highlighted some challenges pertaining to policy-making and editorial responsibilities. The impact on policy and the propagation of the initial scientific information may not cease if the evidence is disproven and retracted from the peer-reviewed literature, which creates a challenge for regulators and scientific journals. Please see later in the article for the Editors' Summary.

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.

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.

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.

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 avian XPR1 gammaretrovirus receptor is under positive selection and is disabled in bird species in contact with virus-infected wild mice.

Xenotropic mouse leukemia viruses (X-MLVs) are broadly infectious for mammals except most of the classical strains of laboratory mice. These gammaretroviruses rely on the XPR1 receptor for entry, and the unique resistance of laboratory mice is due to two mutations in different putative XPR1 extracellular loops. Cells from avian species differ in susceptibility to X-MLVs, and 2 replacement mutations in the virus-resistant chicken XPR1 (K496Q and Q579E) distinguish it from the more permissive duck and quail receptors. These substitutions align with the two mutations that disable the laboratory mouse XPR1. Mutagenesis of the chicken and duck genes confirms that residues at both sites are critical for virus entry. Among 32 avian species, the 2 disabling XPR1 mutations are found together only in the chicken, an omnivorous, ground-dwelling fowl that was domesticated in India and/or Southeast Asia, which is also where X-MLV-infected house mice evolved. The receptor-disabling mutations are also present separately in 5 additional fowl and raptor species, all of which are native to areas of Asia populated by the virus-infected subspecies Mus musculus castaneus. Phylogenetic analysis showed that the avian XPR1 gene is under positive selection at sites implicated in receptor function, suggesting a defensive role for XPR1 in the avian lineage. Contact between bird species and virus-infected mice may thus have favored selection of mouse virus-resistant receptor orthologs in the birds, and our data suggest that similar receptor-disabling mutations were fixed in mammalian and avian species exposed to similar virus challenges.

Cholesterol depletion inactivates XMRV and leads to viral envelope protein release from virions: evidence for role of cholesterol in XMRV infection.

Membrane cholesterol plays an important role in replication of HIV-1 and other retroviruses. Here, we report that the gammaretrovirus XMRV requires cholesterol and lipid rafts for infection and replication. We demonstrate that treatment of XMRV with a low concentration (10 mM) of 2-hydroxypropyl-ß-cyclodextrin (2OHpßCD) partially depleted virion-associated cholesterol resulting in complete inactivation of the virus. This effect could not be reversed by adding cholesterol back to treated virions. Further analysis revealed that following cholesterol depletion, virus-associated Env protein was significantly reduced while the virions remained intact and retained core proteins. Increasing concentrations of 2OHpßCD (≥20 mM) resulted in loss of the majority of virion-associated cholesterol, causing disruption of membrane integrity and loss of internal Gag proteins and viral RNA. Depletion of cholesterol from XMRV-infected cells significantly reduced virus release, suggesting that cholesterol and intact lipid rafts are required for the budding process of XMRV. These results suggest that unlike glycoproteins of other retroviruses, the association of XMRV glycoprotein with virions is highly dependent on cholesterol and lipid rafts.

The role of the structural domains of human BST-2 in inhibiting the release of xenotropic murine leukemia virus-related virus.

BST-2 (bone marrow stromal cell antigen 2) is an interferon-inducible protein that inhibits the release of a variety of enveloped viruses by tethering viral particles to the cell surface. Xenotropic murine leukemia virus-related virus (XMRV) is a gamma-retrovirus that was derived from the recombination of two endogenous murine leukemia viruses during the production of a prostate cell line in mice. In this study, we observed that XMRV was highly sensitive to the inhibition by human BST-2. We were able to determine the structural domains of BST-2 that are essential to restrict XMRV, including the transmembrane domain, the coiled-coil ectodomain, the C-terminal glycosylphosphatidylinositol (GPI) anchor, the two putative N-linked glycosylation sites, and the three extracellular cysteine residues. Protease treatment effectively released XMRV particles into the supernatant, supporting the notion that BST-2 tethered nascent particles to the cell surface. These data suggest that BST-2 poses a strong restriction toward XMRV production.

XMRV induces cell migration, cytokine expression and tumor angiogenesis: are 22Rv1 cells a suitable prostate cancer model?

22Rv1 is a common prostate cancer cell line used in xenograft mouse experiments as well as in vitro cell culture assays to study aspects of prostate cancer tumorigenesis. Recently, this cell line was shown to harbor multiple copies of a gammaretrovirus, called XMRV, integrated in its genome. While the original prostate cancer xenograft CWR22 is free of any retrovirus, subsequently generated cell lines 22Rv1 and CWR-R1, carry this virus and additionally shed infectious gammaretroviral particles in their supernatant. Although XMRV most likely was generated by recombination events in cell culture this virus has been demonstrated to infect human cells in vitro and 22Rv1 as well as CWR-R1 cells are now considered biosafety 2 reagents. Here, we demonstrate that 22Rv1 cells with reduced retroviral transcription show reduced tumor angiogenesis and increased necrosis of the primary tumor derived from xenografted cells in scid mice when compared to the parental cell line. The presence of XMRV transcripts significantly increases secretion of osteopontin (OPN), CXCL14, IL13 and TIMP2 in 22Rv1 cells. Furthermore, these data are supported by in vitro cell invasion and differentiation assays. Collectively, our data suggest that the presence of XMRV transcripts at least partially contributes to 22Rv1 characteristics observed in vitro and in vivo with regard to migration, invasion and tumor angiogenesis. We propose that data received with 22Rv1 cells or equivalent cells carrying xenotropic gammaretroviruses should be carefully controlled including other prostate cancer cell lines tested for viral sequences.

Moloney murine leukemia virus glyco-gag facilitates xenotropic murine leukemia virus-related virus replication through human APOBEC3-independent mechanisms.

BACKGROUND: One of the unique features of gammaretroviruses is that they contain an additional extended form of Gag, glyco-gag, which initiates in the leader sequence. MuLV glyco-gag, gPr80Gag, promotes retrovirus replication and disease progression. Although virtually all infectious MuLVs encode glyco-gag, XMRV (xenotropic murine leukemia virus-related virus) lacks the classical gPr80Gag sequence. We examined XMRV to determine if its leader sequence contains glyco-gag activity, whether the presence of conventional gPr80Gag affects replication of XMRV, and we describe the evolution of glyco-gag-deficient MuLVs in Mus. RESULTS: We introduced several mutations disrupting two putative but noncanonical glyco-gag proteins in the leader sequence region in XMRV and found that those mutations did not affect virus release nor susceptibility to the antiviral activity of hA3G (human APOBEC3G). A chimeric XMRV encoding the Moloney MuLV (M-MuLV) leader sequence (MXMRV) demonstrated that M-MuLV glyco-gag facilitated MXMRV release and increased infectivity. Infectivity assays with several cell lines showed that glyco-gag increases XMRV infectivity in all cell lines tested, but the level of this increase varies in different cell lines. Because MuLV glyco-gag counteracts mouse APOBEC3, we investigated whether M-MuLV glyco-gag enhances XMRV infection by counteracting human APOBEC3. Comparison of hAPOBEC3 isoforms expressed in different cell lines indicated that hA3B was the most likely candidate for a restrictive hA3. However over-expression of hA3B showed no enhanced restriction of infection by XMRV compared to MXMRV. Endogenous MuLVs in the sequenced mouse genome were screened for canonical glyco-gag, which was identified in two clades of xenotropic MuLVs (X-MuLVs) and ecotropic MuLVs, but not in other X-MuLVs or in any polytropic MuLVs. CONCLUSIONS: M-MuLV glyco-gag facilitates XMRV replication, and the leader sequence region in XMRV does not encode proteins equivalent to M-MuLV glyco-gag. The fact that the ability of glyco-gag to enhance XMRV infection varies in different cell lines suggests a glyco-gag sensitive restrictive factor that further reduces XMRV infectivity. The M-MuLV glyco-gag enhancement for XMRV replication is through a hAPOBEC3 independent mechanism. The absence of glyco-gag in MuLVs carried by western European mice suggests that loss of this sequence is a relatively recent event with limited subspecies distribution.

Recombinant origin, contamination, and de-discovery of XMRV.

The discovery and de-discovery of the xenotropic murine leukemia virus-related virus (XMRV) has been a tumultuous roller-coaster ride for scientists and patients. The initial associations of XMRV with chronic fatigue syndrome and prostate cancer, while providing much hope and optimism, have now been discredited and/or retracted following overwhelming evidence that (1) numerous patient cohorts from around the world are XMRV-negative, (2) the initial reports of XMRV-positive patients were due to contamination with mouse DNA, XMRV plasmid DNA, or virus from the 22Rv1 cell line and (3) XMRV is a laboratory-derived virus generated in the mid 1990s through recombination during passage of a prostate tumor xenograft in immuno-compromised mice. While these developments are disappointing to scientists and patients, they provide a valuable road map of potential pitfalls to the would-be microbe hunters.

Lack of evidence for a role of xenotropic murine leukemia virus-related virus in the pathogenesis of prostate cancer and/or chronic fatigue syndrome.

Since the discovery of xenotropic murine leukemia virus-related virus (XMRV) in 2006, one of the most controversial topics is whether it contributes to the pathogenesis of prostate cancer (PCa) and/or chronic fatigue syndrome (CFS). The debate began with the failure to detect XMRV in clinical PCa samples. Concerns about the potential health risk of XMRV exposure were reinforced by a study demonstrating the presence of XMRV in patients with CFS. However, serious concerns on whether XMRV plays a role in the development of PCa and/or CFS have been raised. However, inconsistent reports linking XMRV with PCa and/or CFS have led to conflicting views about the potential of XMRV as a human pathogen. Several recent studies suggest that contamination could account for the positive correlations between XMRV and PCa and/or CFS to date. At present, evidence does not indicate that XMRV plays any role in the pathogenesis of PCa or CFS.

No evidence of cross-species transmission of mouse retroviruses to animal workers exposed to mice.

BACKGROUND: Although recent data have brought into question the association between xenotropic murine leukemia virus-related virus (XMRV) and chronic fatigue syndrome, one group has reported evidence of human infection with distinct polytropic murine leukemia viruses (MLVs). Occult retroviral infection among humans poses a significant public health risk should it be introduced into the blood supply. To explore the possibility of cross-species transmission of MLVs to humans, we sought molecular and serologic evidence of XRMV/MLV infection among a cohort of animal workers highly exposed to mice. STUDY DESIGN AND METHODS: Before the commencement of the study, the laboratory and equipment were demonstrated to be free of XMRV/MLV DNA sequences. DNA extracted from 43 animal workers was tested using nested polymerase chain reaction (PCR) with published primer sets, targeting regions of XMRV and MLV gag. Negative controls were assayed in a 1:1 ratio with specimens. Serum specimens were tested using a validated immunoblot assay containing cross-reactive XMRV antigens. RESULTS: Initial molecular assays demonstrated that the physical space and laboratory equipment were free of MLV and XMRV DNA sequences. Nested PCR assays using multiple primer sets successfully amplified XMRV and MLV sequences from positive controls with high sensitivity. A single, nonreproducible, false-positive result from one specimen was shown to be the result of subsequent contamination. Immunoblotting of all subjects' sera failed to demonstrate any evidence of seroreactivity to XMRV proteins. CONCLUSIONS: There was no evidence of human infection with XMRV/MLV among a cohort of individuals highly exposed to mice. These data suggest that the likelihood of cross-species transmission events of MLV from mice to humans is low.

Restricted replication of xenotropic murine leukemia virus-related virus in pigtailed macaques.

Although xenotropic murine leukemia virus-related virus (XMRV) has been previously linked to prostate cancer and myalgic encephalomyelitis/chronic fatigue syndrome, recent data indicate that results interpreted as evidence of human XMRV infection reflect laboratory contamination rather than authentic in vivo infection. Nevertheless, XMRV is a retrovirus of undefined pathogenic potential that is able to replicate in human cells. Here we describe a comprehensive analysis of two male pigtailed macaques (Macaca nemestrina) experimentally infected with XMRV. Following intravenous inoculation with >10(10) RNA copy equivalents of XMRV, viral replication was limited and transient, peaking at ≤2,200 viral RNA (vRNA) copies/ml plasma and becoming undetectable by 4 weeks postinfection, though viral DNA (vDNA) in peripheral blood mononuclear cells remained detectable through 119 days of follow-up. Similarly, vRNA was not detectable in lymph nodes by in situ hybridization despite detectable vDNA. Sequencing of cell-associated vDNA revealed extensive G-to-A hypermutation, suggestive of APOBEC-mediated viral restriction. Consistent with limited viral replication, we found transient upregulation of type I interferon responses that returned to baseline by 2 weeks postinfection, no detectable cellular immune responses, and limited or no spread to prostate tissue. Antibody responses, including neutralizing antibodies, however, were detectable by 2 weeks postinfection and maintained throughout the study. Both animals were healthy for the duration of follow-up. These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro, human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread.

XMRV accelerates cellular proliferation, transformational activity, and invasiveness of prostate cancer cells by downregulating p27(Kip1).

BACKGROUND: Xenotropic murine leukemia virus-related retrovirus (XMRV) is a recently discovered gammaretrovirus that was originally detected in prostate tumors. However, a causal relationship between XMRV and prostate cancer remains controversial due to conflicting reports on its etiologic occurrence. Even though gammaretroviruses are known to induce cancer in animals, a mechanism for XMRV-induced carcinogenesis remains unknown. Several mechanisms including insertional mutagenesis, proinflammatory effects, oncogenic viral proteins, immune suppression, and altered epithelial/stromal interactions have been proposed for a role of XMRV in prostate cancer. However, biochemical data supporting any of these mechanisms are lacking. Therefore, our aim was to evaluate a potential role of XMRV in prostate carcinogenesis. METHODS: Growth kinetics of prostate cancer cells are conducted by MTT assay. In vitro transformation and invasion was carried out by soft agar colony formation, and Matrigel cell invasion assay, respectively. p27(Kip1) expression was determined by Western blot and MMP activation was evaluated by gelatin-zymography. Up-regulation of miR221 and miR222 expression was examined by real-time PCR. RESULTS: We demonstrate that XMRV infection can accelerate cellular proliferation, enhance transformation, and increase invasiveness of slow growing prostate cancer cells. The molecular basis of these viral induced activities is mediated by the downregulation of cyclin/cyclin dependent kinase inhibitor p27(Kip1) . Downstream analyses illustrated that XMRV infection upregulates miR221 and miR222 expression that target p27(Kip1) mRNA. CONCLUSIONS: We propose that downregulation of p27(Kip1) by XMRV infection facilitates transition of G1 to S, thereby accelerates growth of prostate cancer cells. Our findings implicate that if XMRV is present in humans, then under appropriate cellular microenvironment it may serve as a cofactor to promote cancer progression in the prostate.

Characterization, mapping, and distribution of the two XMRV parental proviruses.

Xenotropic murine leukemia virus-related virus (XMRV) was previously reported to be associated with human prostate cancer and chronic fatigue syndrome. Our groups recently showed that XMRV was created through recombination between two endogenous murine retroviruses, PreXMRV-1 and PreXMRV-2, during the passaging of a prostate tumor xenograft in nude mice. Here, multiple approaches that led to the identification of PreXMRV-2, as well as the distribution of both parental proviruses among different mouse species, are described. The chromosomal loci of both proviruses were determined in the mouse genome, and integration site information was used to analyze the distribution of both proviruses in 48 laboratory mouse strains and 46 wild-derived strains. The strain distributions of PreXMRV-1 and PreXMRV-2 are quite different, the former being found predominantly in Asian mice and the latter in European mice, making it unlikely that the two XMRV ancestors could have recombined independently in the wild to generate an infectious virus. XMRV was not present in any of the mouse strains tested, and among the wild-derived mouse strains analyzed, not a single mouse carried both parental proviruses. Interestingly, PreXMRV-1 and PreXMRV-2 were found together in three laboratory strains, Hsd nude, NU/NU, and C57BR/cd, consistent with previous data that the recombination event that led to the generation of XMRV could have occurred only in the laboratory. The three laboratory strains carried the Xpr1(n) receptor variant nonpermissive to XMRV and xenotropic murine leukemia virus (X-MLV) infection, suggesting that the xenografted human tumor cells were required for the resulting XMRV recombinant to infect and propagate.

Xenotropic murine leukemia virus-related virus does not pose a risk to blood recipient safety.

BACKGROUND: When xenotropic murine leukemia virus-related virus (XMRV) was first reported in association with chronic fatigue syndrome, it was suggested that it might offer a risk to blood safety. Thus, the prevalence of the virus among blood donors and, if present, its transmissibility by transfusion need to be defined. STUDY DESIGN AND METHODS: Two populations of routine blood donor samples (1435 and 13,399) were obtained for prevalence evaluations; samples from a linked donor-recipient repository were also evaluated. Samples were tested for the presence of antibodies to XMRV-related recombinant antigens and/or for XMRV RNA, using validated, high-throughput systems. RESULTS: The presence of antibodies to XMRV could not be confirmed among a total of 17,249 blood donors or recipients (0%; 95% confidence interval [CI], 0%-0.017%); 1763 tested samples were nonreactive for XMRV RNA (0%; 95% CI, 0%-0.17%). Evidence of infection was absent from 109 recipients and 830 evaluable blood samples tested after transfusion of a total of 3741 blood components. CONCLUSIONS: XMRV and related murine leukemia virus (MLV) markers are not present among a large population of blood donors and evidence of transfusion transmission could not be detected. Thus, these viruses do not currently pose a threat to blood recipient safety and further actions relating to XMRV and MLV are not justified.