CTCF induces histone variant incorporation, erases the H3K27me3 histone mark and opens chromatin

Insulators functionally separate active chromatin domains frominactive ones. The insulator factor, CTCF, has been found to bind to boundaries and to mediate insulator function. CTCF binding sites are depleted for the histone modification H3K27me3 and are enriched for the histone variant H3.3. In order to determine whether demethylation of H3K27me3 and H3.3 incorporation are a requirement for CTCF binding at domain boundaries or whether CTCF causes these changes, we made use of the LacI DNA binding domain to control CTCF binding by the Lac inducer IPTG. Here we show that, in contrast to the related factor CTCFL, the N-terminus plus zinc finger domain of CTCF is sufficient to open compact chromatin rapidly. This is preceded by incorporation of the histone variant H3.3, which thereby removes the H3K27me3 mark. This demonstrates the causal role for CTCF in generating the chromatin features found at insulators. Thereby, spreading of a histone modification from one domain through the insulator into the neighbouring domain is inhibited

Absence of XMRV and Closely Related Viruses in Primary Prostate Cancer Tissues Used to Derive the XMRV-Infected Cell Line 22Rv1

The 22Rv1 cell line is widely used for prostate cancer research and other studies throughout the world. These cells were established from a human prostate tumor, CWR22, that was serially passaged in nude mice and selected for androgen independence. The 22Rv1 cells are known to produce high titers of xenotropic murine leukemia virus-related virus (XMRV). Recent studies suggested that XMRV was inadvertently created in the 1990's when two murine leukemia virus (MLV) genomes (pre-XMRV1 and pre-XMRV-2) recombined during passaging of the CWR22 tumor in mice. The conclusion that XMRV originated from mice and not the patient was based partly on the failure to detect XMRV in early CWR22 xenografts. While that deduction is certainly justified, we examined the possibility that a closely related virus could have been present in primary tumor tissue. Here we report that we have located the original prostate tumor tissue excised from patient CWR22 and have assayed the corresponding DNA by PCR and the tissue sections by fluorescence in situ hybridization for the presence of XMRV or a similar virus. The primary tumor tissues lacked mouse DNA as determined by PCR for intracisternal A type particle DNA, thus avoiding one of the limitations of studying xenografts. We show that neither XMRV nor a closely related virus was present in primary prostate tissue of patient CWR22. Our findings confirm and reinforce the conclusion that XMRV is a recombinant laboratory-generated mouse virus that is highly adapted for human prostate cancer cells

Detection of Murine Leukemia Virus or Mouse DNA in Commercial RT-PCR Reagents and Human DNAs

The xenotropic murine leukemia virus (MLV)-related viruses (XMRV) have been reported in persons with prostate cancer, chronic fatigue syndrome, and less frequently in blood donors. Polytropic MLVs have also been described in persons with CFS and blood donors. However, many studies have failed to confirm these findings, raising the possibility of contamination as a source of the positive results. One PCR reagent, Platinum Taq polymerase (pol) has been reported to contain mouse DNA that produces false-positive MLV PCR results. We report here the finding of a large number of PCR reagents that have low levels of MLV sequences. We found that recombinant reverse-transcriptase (RT) enzymes from six companies derived from either MLV or avian myeloblastosis virus contained MLV pol DNA sequences but not gag or mouse DNA sequences. Sequence and phylogenetic analysis showed high relatedness to Moloney MLV, suggesting residual contamination with an RT-containing plasmid. In addition, we identified contamination with mouse DNA and a variety of MLV sequences in commercially available human DNAs from leukocytes, brain tissues, and cell lines. These results identify new sources of MLV contamination and highlight the importance of careful pre-screening of commercial specimens and diagnostic reagents to avoid false-positive MLV PCR results

Sensitivity of PCR Assays for Murine Gammaretroviruses and Mouse Contamination in Human Blood Samples

Gammaretroviruses related to murine leukemia virus (MLV) have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs) or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples

Long-Term Infection and Vertical Transmission of a Gammaretrovirus in a Foreign Host Species

Increasing evidence has indicated natural transspecies transmission of gammaretroviruses; however, viral-host interactions after initial xeno-exposure remain poorly understood. Potential association of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and chronic fatigue syndrome has attracted broad interests in this topic. Although recent studies have indicated that XMRV is unlikely a human pathogen, further understanding of XMRV xenoinfection would allow in vivo modeling of the initial steps of gammaretroviral interspecies transmission, evolution and dissemination in a new host population. In this study, we monitored the long-term consequences of XMRV infection and its possible vertical transmission in a permissive foreign host, wild-derived Mus pahari mice. One year post-infection, XMRV-infected mice showed no notable pathological changes, while proviral DNA was detected in three out of eight mice. XMRV-infected mice remained seropositive throughout the study although the levels of gp70 Env- and p30 capsid-specific antibodies gradually decreased. When vertical XMRV transmission was assessed, no viremia, humoral immune responses nor endogenization were observed in nine offspring from infected mothers, yet one offspring was found PCR-positive for XMRV-specific sequences. Amplified viral sequences from the offspring showed several mutations, including one amino acid deletion in the receptor binding domain of Env SU. Our results therefore demonstrate long-term asymptomatic infection, low incidence of vertical transmission and limited evolution of XMRV upon transspecies infection of a permissive new host, Mus pahari

No Evidence for XMRV in German CFS and MS Patients with Fatigue Despite the Ability of the Virus to Infect Human Blood Cells In Vitro

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV), a novel human retrovirus originally identified in prostate cancer tissues, has recently been associated with chronic fatigue syndrome (CFS), a disabling disease of unknown etiology affecting millions of people worldwide. However, several subsequent studies failed to detect the virus in patients suffering from these illnesses or in healthy subjects. Here we report the results of efforts to detect antibody responses and viral sequences in samples from a cohort of German CFS and relapsing remitting multiple sclerosis (MS) patients with fatigue symptoms. METHODOLOGY: Blood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors. Fatigue severity in MS patients was assessed using the Fatigue Severity Scale (FSS). Validated Gag- and Env-ELISA assays were used to screen sera for XMRV antibodies. PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene. In addition, PBMC cultures were exposed to 22Rv1-derived XMRV to assess infectivity and virus production. CONCLUSION: None of the screened sera from CFS and MS patients or healthy blood donors tested positive for XMRV specific antibodies and all PBMC (and PBMC plus LNCaP) cultures remained negative for XMRV sequences by nested PCR. These results argue against an association between XMRV infection and CFS and MS in Germany. However, we could confirm that PBMC cultures from healthy donors and from CFS patients can be experimentally infected by XMRV, resulting in the release of low levels of transmittable virus

Studies on the Restriction of Murine Leukemia Viruses by Mouse APOBEC3

APOBEC3 proteins function to restrict the replication of retroviruses. One mechanism of this restriction is deamination of cytidines to uridines in (−) strand DNA, resulting in hypermutation of guanosines to adenosines in viral (+) strands. However, Moloney murine leukemia virus (MoMLV) is partially resistant to restriction by mouse APOBEC3 (mA3) and virtually completely resistant to mA3-induced hypermutation. In contrast, the sequences of MLV genomes that are in mouse DNA suggest that they were susceptible to mA3-induced deamination when they infected the mouse germline. We tested the possibility that sensitivity to mA3 restriction and to deamination resides in the viral gag gene. We generated a chimeric MLV in which the gag gene was from an endogenous MLV in the mouse germline, while the remainder of the viral genome was from MoMLV. This chimera was fully infectious but its response to mA3 was indistinguishable from that of MoMLV. Thus, the Gag protein does not seem to control the sensitivity of MLVs to mA3. We also found that MLVs inactivated by mA3 do not synthesize viral DNA upon infection; thus mA3 restriction of MLV occurs before or at reverse transcription. In contrast, HIV-1 restricted by mA3 and MLVs restricted by human APOBEC3G do synthesize DNA; these DNAs exhibit APOBEC3-induced hypermutation

Absence of XMRV in Peripheral Blood Mononuclear Cells of ARV-Treatment Naïve HIV-1 Infected and HIV-1/HCV Coinfected Individuals and Blood Donors

<div><h3>Background</h3><p>Xenotropic murine leukemia virus-related virus (XMRV) has been found in the prostatic tissue of prostate cancer patients and in the blood of chronic fatigue syndrome patients. However, numerous studies have found little to no trace of XMRV in different human cohorts. Based on evidence suggesting common transmission routes between XMRV and HIV-1, HIV-1 infected individuals may represent a high-risk group for XMRV infection and spread.</p> <h3>Methodology/Principal Findings</h3><p>DNA was isolated from the peripheral blood mononuclear cells (PBMCs) of 179 HIV-1 infected treatment naïve patients, 86 of which were coinfected with HCV, and 54 healthy blood donors. DNA was screened for XMRV provirus with two sensitive, published PCR assays targeting XMRV <em>gag</em> and <em>env</em> and one sensitive, published nested PCR assay targeting <em>env</em>. Detection of XMRV was confirmed by DNA sequencing. One of the 179 HIV-1 infected patients tested positive for <em>gag</em> by non-nested PCR whereas the two other assays did not detect XMRV in any specimen. All healthy blood donors were negative for XMRV proviral sequences. Sera from 23 HIV-1 infected patients (15 HCV⁺) and 12 healthy donors were screened for the presence of XMRV-reactive antibodies by Western blot. Thirteen sera (57%) from HIV-1⁺ patients and 6 sera (50%) from healthy donors showed reactivity to XMRV-infected cell lysate.</p> <h3>Conclusions/Significance</h3><p>The virtual absence of XMRV in PBMCs suggests that XMRV is not associated with HIV-1 infected or HIV-1/HCV coinfected patients, or blood donors. Although we noted isolated incidents of serum reactivity to XMRV, we are unable to verify the antibodies as XMRV specific.</p> </div

Lack of Detection of XMRV in Seminal Plasma from HIV-1 Infected Men in The Netherlands

Background: Xenotropic murine leukaemia virus-related virus (XMRV) is a recently discovered human gammaretrovirus with yet unknown prevalence and transmission route(s). Its presence in prostate stromal fibroblasts and prostatic secretions suggests that XMRV might be sexually transmitted. We chose to study a compartment closely connected to the prostate, a location where XMRV was detected in independent studies. Seminal plasma samples from HIV-1 infected men were examined as they have an increased probability of acquiring sexually transmitted pathogens. Methodology/Principal Findings: We studied the prevalence of XMRV in 93 seminal plasma samples of 54 HIV-1 infected men living in The Netherlands with a nested PCR amplification specifically targeting the XMRV gag gene. As a control for the presence and integrity of retrovirus particles, HIV-1 was amplified from the same samples with a PCR amplification targeting the env gene of the virus, or HIV-1 was quantified with a real-time PCR amplifying part of the pol gene. Conclusions/Significance: Although HIV-1 was amplified from 25 % of the seminal plasma samples, no XMRV was detected, suggesting that either the prevalence of XMRV is very low in The Netherlands, or that XMRV is not naturally present in th