Endogenous gammaretroviruses (EGVs) have been widely studied in terrestrial mammals but seldom so in marine species. A genomic mining of the bottlenose dolphin (Tursiops truncatus) genome revealed a new EGV, termed Tursiops truncatus endogenous retrovirus (TTEV), which is divergent from extant mammalian EGVs. Molecular clock dating estimated the invasion time of TTEV into the host genome to be approximately 10-19 million years ago (MYA), while a previously identified killer whale endogenous gammaretrovirus (KWERV) was estimated to have invaded the host genome approximately 3-5 MYA. Using a PCR-based technique, we then verified that similar endogenous viruses exist in nine cetacean genomes. Phylogenetic analysis revealed that these cetacean EGVs are highly divergent from their counterparts in other mammals, including KWERV from the killer whale. In sum, we conclude that there have been at least two invasion episodes of EGVs into cetaceans during their evolutionary history.

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.

The human exogenous gammaretrovirus XMRV is thought to be implicated in prostate cancer and chronic fatigue syndrome. Besides pressing epidemiologic questions, the elucidation of the tissue and cell tropism of the virus, as well as its sensitivity to retroviral restriction factors is of fundamental importance. The Apobec3 (A3) proteins, a family of cytidine deaminases, are one important group of host proteins that control primary infection and efficient viral spread.

Post-transcriptional regulatory mechanisms of several complex and simple retroviruses and retroelements have been elucidated, with the exception of the gammaretrovirus family. We found that, similar to the other retroviruses, gag gene expression of MuLV and XMRV depends on post-transcriptional regulation mediated via an RNA sequence overlapping the pro-pol open reading frame, termed the Post-Transcriptional Element (PTE). PTE function can be replaced by heterologous RNA export elements, e.g. CTE of simian type D retroviruses. Alternatively, Gag particle production is achieved using an RNA/codon optimized gag gene. PTE function is transferable and can replace HIV Rev-RRE-regulated expression of HIV gag. Analysis of PTE by SHAPE revealed a highly structured RNA comprising seven stem-loop structures, with the 5' and 3' stem-loops forming an essential bipartite signal. MuLV and XMRV PTE share 98% identity and have highly similar RNA structures, with changes mostly located to single-stranded regions. PTE identification strongly suggests that all retroviruses and retroelements share common strategies of post-transcriptional gene regulation to produce Gag. Expression depends on complex RNA structures embedded within retroviral mRNA, in coding regions or the 3' untranslated region. These specific structures serve as recognition signals for either cellular or viral proteins.

Ribonuclease L (RNase L) is an important effector of the innate antiviral response. Mutations or variants that impair function of RNase L, particularly R462Q, have been proposed as susceptibility factors for prostate cancer. Given the role of this gene in viral defense, we sought to explore the possibility that a viral infection might contribute to prostate cancer in individuals harboring the R462Q variant. A viral detection DNA microarray composed of oligonucleotides corresponding to the most conserved sequences of all known viruses identified the presence of gammaretroviral sequences in cDNA samples from seven of 11 R462Q-homozygous (QQ) cases, and in one of eight heterozygous (RQ) and homozygous wild-type (RR) cases. An expanded survey of 86 tumors by specific RT-PCR detected the virus in eight of 20 QQ cases (40%), compared with only one sample (1.5%) among 66 RQ and RR cases. The full-length viral genome was cloned and sequenced independently from three positive QQ cases. The virus, named XMRV, is closely related to xenotropic murine leukemia viruses (MuLVs), but its sequence is clearly distinct from all known members of this group. Comparison of gag and pol sequences from different tumor isolates suggested infection with the same virus in all cases, yet sequence variation was consistent with the infections being independently acquired. Analysis of prostate tissues from XMRV-positive cases by in situ hybridization and immunohistochemistry showed that XMRV nucleic acid and protein can be detected in about 1% of stromal cells, predominantly fibroblasts and hematopoietic elements in regions adjacent to the carcinoma. These data provide to our knowledge the first demonstration that xenotropic MuLV-related viruses can produce an authentic human infection, and strongly implicate RNase L activity in the prevention or clearance of infection in vivo. These findings also raise questions about the possible relationship between exogenous infection and cancer development in genetically susceptible individuals.

Most viruses infect only a few hosts, but the xenotropic and polytropic mouse leukemia viruses (X/P-MLVs) are broadly infectious in mammalian species. X/P-MLVs use the XPR1 receptor for cell entry, and tropism differences are due to polymorphisms in XPR1 and the viral envelope. To characterize these receptor variants and identify blocks to cross-species transmission, we examined the XPR1 receptors in six mammalian species that restrict different subsets of X/P-MLVs. These restrictive receptors have replacement mutations in regions implicated in receptor function, and some entry restrictions can be relieved by glycosylation inhibitors. Mutation of the cow and hamster XPR1 genes identified a shared, previously unrecognized receptor-critical site. This G/Q503N replacement dramatically improves receptor function. While this substitution introduces an N-linked glycosylation site, XPR1 receptors are not glycosylated indicating that this replacement alters the virus-receptor interface independently of glycosylation. Our data also suggest that an unidentified glycosylated cofactor may influence X/P-MLV entry.

Despite the absence of a confirmed exogenously replicating retrovirus in Canis lupus familiaris (C. familiaris), past retroviral infections are evident in the genomes of living animals via the presence of endogenous retroviruses (ERVs). Although gammaretrovirus-like transcripts and enzyme activities were previously reported to be present in canine leukemias and lymphomas, those findings were not further explored. Initial analysis of the C. familiaris reference genome revealed a minor subset of one ERV lineage, classified as CfERV-Fc1(a), or Fc1(a) here, with features characteristic of recent integration, including the presence of ORFs and identical or nearly identical LTRs. Our previous analysis of whole genome sequence data belonging to extant Canidae revealed a burst of past infections in Canis ancestors resulting in numerous young, polymorphic, and highly intact loci now segregating in dogs. Here, we demonstrate the expression of full-length Fc1(a) proviruses in tissues collected from healthy animals and from animals with cancer. We observed significantly higher expression in samples of dogs with various cancer diagnoses when compared to samples from healthy dogs. Genotyping of insertionally polymorphic Fc1(a) loci identified candidate expressed proviruses and delineated distributions over sample groups. Collectively, the data show that Fc1(a) proviruses retain biological activity in the domestic dog and provides a means to examine potential genetic links with disease states in this species.

Endogenous retroviruses (ERVs) were acquired during evolution of their host organisms after infection and mendelian inheritance in the germline by their exogenous counterparts. The ERVs can spread in the host genome and in some cases they affect the host phenotype. The cervid endogenous gammaretrovirus (CrERV) is one of only a few well-defined examples of evolutionarily recent invasion of mammalian genome by retroviruses. Thousands of insertionally polymorphic CrERV integration sites have been detected in wild ranging mule deer (Odocoileus hemionus) host populations. Here, we describe for the first time induction of replication competent CrERV by cocultivation of deer and human cells. We characterize the physical properties and tropism of the induced virus. The genomic sequence of the induced virus is phylogenetically related to the evolutionarily young endogenous CrERVs described so far. We also describe the level of replication block of CrERV on deer cells and its capacity to establish superinfection interference.

Endogenous retroviruses (ERVs) constitute a significant part of vertebrate genomes. They originated from past retroviral infections and some of them retain transcriptional activity. The key mechanism avoiding uncontrolled ERV transcription is DNA methylation-mediated epigenetic silencing. Despite numerous studies describing the involvement of ERV activity in cellular processes, epigenetic regulation of ERVs is still poorly understood. We previously described a cervid endogenous retrovirus (CrERV) in the mule deer genome. This virus exhibits massive insertional polymorphism, suggesting recent activity. Here we employed NGS-based strategy to determine the methylation pattern of CrERV integrations in four mule deer. Besides the vast majority of methylated integrations, we identified a tiny fraction of demethylated proviral copies. These copies represent evolutionary older integrations located near gene promoters. In general, our work is a first attempt to characterize the epigenetic landscape of insertionally polymorphic ERV on a whole-genome scale and offers insight into its interactions with a host.

The recent report of gammaretroviruses of probable murine origin in humans, called xenotropic murine retrovirus related virus (XMRV) and human murine leukemia virus related virus (HMRV), necessitated a bioinformatic search for this virus in genomes of the mouse and other vertebrates, and by PCR in humans.

Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectors with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription.

The recent recognition that recombinant retrovirus vectors can induce oncogenic transformation has stimulated much interest in the pattern of vector integration sites. We report here on the integration pattern of a gammaretrovirus reporter vector following transduction and ex vivo culture of primary mouse bone marrow progenitor cells in the absence and presence of drug selection. Using a novel method of cloning junction fragments, we observed no bias for integrations within genes, but did observe a bias for integrations within gene-dense regions and especially near transcriptional start sites of highly active genes, similar to previous reports in other cell types. We also document a novel bias for integrations within or near a class of genes that encode nuclear-localized proteins. We found that drug selection resulted in an increase in the frequency of recovered integration events that were located within the beginning of genes, integration events that were located in less gene-dense regions, and integration events that were oriented in an antisense direction relative to flanking gene transcription. Taken together, these studies provide new insights into the nature of retrovirus vector integration patterns in primary cells and demonstrate that selection based on vector expression can bias the integration site repertoire.

Although novel retroviral vectors for use in gene-therapy products are reducing the potential for formation of replication-competent retrovirus (RCR), it remains crucial to screen products for RCR for both research and clinical purposes. For clinical-grade gammaretrovirus-based vectors, RCR screening is achieved by an extended S(+)L(-) or marker-rescue assay, whereas standard methods for replication-competent lentivirus detection are still in development. In this report, we describe a rapid and sensitive method for replication-competent gammaretrovirus detection. We used this assay to detect three members of the gammaretrovirus family and compared the sensitivity of our assay with well-established methods for retrovirus detection, including the extended S(+)L(-) assay. Results presented here demonstrate that this assay should be useful for gene-therapy product testing.

Murine Leukemia Virus (MLV) is a rodent gammaretrovirus that serves as the backbone for common gene delivery tools designed for experimental and therapeutic applications. Recently, an infectious gammaretrovirus designated XMRV has been identified in prostate cancer patients. The similarity between the MLV and XMRV genomes suggests a possibility that the two viruses may interact when present in the same cell.

Develop an engineered cell line containing two flexible gene expression systems enabling the continuous production of tailor-made recombinant gammaretrovirus with predictable productivities through targeted integration.

Several studies have implicated a recently discovered gammaretrovirus, XMRV (Xenotropic murine leukaemia virus-related virus), in chronic fatigue syndrome and prostate cancer, though whether as causative agent or opportunistic infection is unclear. It has also been suggested that the virus can be found circulating amongst the general population. The discovery has been controversial, with conflicting results from attempts to reproduce the original studies.

All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated.

Xenotropic Murine Leukemia Virus-related Virus (XMRV) is a human gammaretrovirus recently identified in prostate cancer tissue and in lymphocytes of patients with chronic fatigue syndrome. To establish the etiologic role of XMRV infection in human disease requires large scale epidemiologic studies. Development of assays to detect XMRV-specific antibodies would greatly facilitate such studies. However, the nature and kinetics of the antibody response to XMRV infection have yet to be determined.

Bats are reservoirs of emerging viruses that are highly pathogenic to other mammals, including humans. Despite the diversity and abundance of bat viruses, to date they have not been shown to harbor exogenous retroviruses. Here we report the discovery and characterization of a group of koala retrovirus-related (KoRV-related) gammaretroviruses in Australian and Asian bats. These include the Hervey pteropid gammaretrovirus (HPG), identified in the scat of the Australian black flying fox (Pteropus alecto), which is the first reproduction-competent retrovirus found in bats. HPG is a close relative of KoRV and the gibbon ape leukemia virus (GALV), with virion morphology and Mn2+-dependent virion-associated reverse transcriptase activity typical of a gammaretrovirus. In vitro, HPG is capable of infecting bat and human cells, but not mouse cells, and displays a similar pattern of cell tropism as KoRV-A and GALV. Population studies reveal the presence of HPG and KoRV-related sequences in several locations across northeast Australia, as well as serologic evidence for HPG in multiple pteropid bat species, while phylogenetic analysis places these bat viruses as the basal group within the KoRV-related retroviruses. Taken together, these results reveal bats to be important reservoirs of exogenous KoRV-related gammaretroviruses.

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus reported to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS). While the association of XMRV with CFS and PC has recently been discredited, no studies have been performed in Australian patients to investigate the association between PC and XMRV or related murine leukemia virus (MLV) in matched PC and normal tissue.