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1.  Genome-wide changes in expression profile of murine endogenous retroviruses (MuERVs) in distant organs after burn injury 
BMC Genomics  2007;8:440.
Previous studies have shown that burn-elicited stress signals alter expression of certain murine endogenous retroviruses (MuERVs) in distant organs of mice. These findings suggest that MuERVs may participate in a network of pathophysiologic events during post-burn systemic response. To gain a better understanding of the biological roles of MuERVs in post-burn systemic response, we examined the genome-wide changes in the MuERV expression profiles in distant organs and the biological properties of the putative-burn related MuERVs were characterized.
Female C57BL/6J mice were subjected to an approximately 18 % total body surface area flame burn and tissues (liver, lung, and kidney) were harvested at 3 hours and 24 hours after injury. The changes in the MuERV expression profiles in these tissues were examined by RT-PCR using a primer set flanking the non-ecotropic MuERV U3 promoter region within the 3' long terminal repeat. There were differential changes in the expression profiles of MuERV U3 regions after injury in all three tissues examined. Subsequently, a total of 31 unique U3 promoter sequences were identified from the tissues of both burn and no burn mice. An analysis of viral tropisms revealed that putative MuERVs harboring these U3 promoter sequences were presumed to be either xenotropic or polytropic. Some putative transcription regulatory elements were present predominantly in U3 promoter sequences isolated from burn and no burn mice, respectively. In addition, in silico mapping using these U3 sequences as a probe against the mouse genome database identified 59 putative MuERVs. The biological properties (coding potentials for retroviral polypeptides, primer binding sites, tropisms, branching ages, recombination events, and neighboring host genes) of each putative MuERV were characterized. In particular, 16 putative MuERVs identified in this study retained intact coding potentials for all three retroviral polypeptides (gag, pol, and env). None of the putative MuERVs identified in this study were mapped to the coding sequences of host genes.
In this study, we identified and characterized putative MuERVs whose expression might be altered in response to burn-elicited systemic stress signals. Further investigation is needed to understand the role of these MuERVs in post-burn systemic pathogenesis, in particular, via characterization of their interaction with host genes, MuERV gene products, and viral activities.
PMCID: PMC2241634  PMID: 18045489
2.  The Mouse IAPE Endogenous Retrovirus Can Infect Cells through Any of the Five GPI-Anchored EphrinA Proteins 
PLoS Pathogens  2011;7(10):e1002309.
The IAPE (Intracisternal A-type Particles elements with an Envelope) family of murine endogenous retroelements is present at more than 200 copies in the mouse genome. We had previously identified a single copy that proved to be fully functional, i.e. which can generate viral particles budding out of the cell and infectious on a series of cells, including human cells. We also showed that IAPE are the progenitors of the highly reiterated IAP elements. The latter are now strictly intracellular retrotransposons, due to the loss of the envelope gene and re-localisation of the associated particles in the course of evolution. In the present study we searched for the cellular receptor of the IAPE elements, by using a lentiviral human cDNA library and a pseudotype assay on transduced cells. We identified Ephrin A4, a GPI-anchored molecule involved in several developmental processes, as a receptor for the IAPE pseudotypes. We also found that the other 4 members of the Ephrin A family –but not those of the closely related Ephrin B family- were also able to mediate IAPE cell entry, thus significantly increasing the amount of possible cell types susceptible to IAPE infection. We show that these include mouse germline cells, as illustrated by immunohistochemistry experiments, consistent with IAPE genomic amplification by successive re-infection. We propose that the uncovered properties of the identified receptors played a role in the accumulation of IAPE elements in the mouse genome, and in the survival of a functional copy.
Author Summary
In mammals, nearly half the genome is composed of reiterated scattered sequences. Some of them, called endogenous retroviruses, have a structure similar to that observed for the integrated form of infectious retroviruses. The current theory to account for their presence is that an infectious retrovirus once infected the germline of its host. This viral genome was then transmitted to the progeny and expressed from there, producing new infectious particles, which could re-infect new germline cells and thus increase the viral genomic copy number. However no evidence has yet been provided to support this model. In this study, we identify a family of five cellular proteins, the Ephrin As, as receptors for a model mouse family of endogenous retroviruses, the IAPE elements. We analyse their expression pattern and show that both the oocytes and some male germline cells express Ephrin A proteins and can thus be infected by IAPE particles. This finding strongly supports the current model of ERVs amplification. In addition, the IAPE envelope ability to use five different cellular receptors suggests that it might be impossible for the host to evolve a resistance against this viral element, and provides a clue on how the IAPE family survived so long in the mouse genome.
PMCID: PMC3197615  PMID: 22028653
3.  Genome-Wide Assessments Reveal Extremely High Levels of Polymorphism of Two Active Families of Mouse Endogenous Retroviral Elements 
PLoS Genetics  2008;4(2):e1000007.
Endogenous retroviral elements (ERVs) in mice are significant genomic mutagens, causing ∼10% of all reported spontaneous germ line mutations in laboratory strains. The majority of these mutations are due to insertions of two high copy ERV families, the IAP and ETn/MusD elements. This significant level of ongoing retrotranspositional activity suggests that inbred mice are highly variable in content of these two ERV groups. However, no comprehensive genome-wide studies have been performed to assess their level of polymorphism. Here we compared three test strains, for which sufficient genomic sequence is available, to each other and to the reference C57BL/6J genome and detected very high levels of insertional polymorphism for both ERV families, with an estimated false discovery rate of only 0.4%. Specifically, we found that at least 60% of IAP and 25% of ETn/MusD elements detected in any strain are absent in one or more of the other three strains. The polymorphic nature of a set of 40 ETn/MusD elements found within gene introns was confirmed using genomic PCR on DNA from a panel of mouse strains. For some cases, we detected gene-splicing abnormalities involving the ERV and obtained additional evidence for decreased gene expression in strains carrying the insertion. In total, we identified nearly 700 polymorphic IAP or ETn/MusD ERVs or solitary LTRs that reside in gene introns, providing potential candidates that may contribute to gene expression differences among strains. These extreme levels of polymorphism suggest that ERV insertions play a significant role in genetic drift of mouse lines.
Author Summary
The laboratory mouse is the most widely used mammal for biological research. Hundreds of inbred mouse strains have been developed that vary in characteristics such as susceptibility to cancer or other diseases. There is much interest in uncovering differences between strains that result in different traits and, to aid this effort, millions of single nucleotide differences or polymorphisms between strains have been cataloged. To date, there has been less emphasis placed on other sources of genetic variation. In this study, we have conducted a genome-wide analysis to examine the level of polymorphism of mouse endogenous retroviral sequences (ERVs). ERVs are derived from infectious retroviruses that now exist in the genome and are inherited as part of chromosomes. Unlike in humans, genomic insertions of ERVs cause many new mutations in mice but their extent of variation between strains has been difficult to study because of their high copy numbers. By comparing genomic sequences of four common mouse strains, we found very high levels of polymorphism for two large active families of ERVs. Moreover, we documented nearly 700 polymorphic ERVs located within gene introns and found evidence that some of these affect gene transcript levels. This study demonstrates that ERV polymorphisms are a major source of genetic variability among mouse strains and likely contribute to strain-specific traits.
PMCID: PMC2265474  PMID: 18454193
4.  Evolutionary history of bovine endogenous retroviruses in the Bovidae family 
Endogenous retroviruses (ERVs) are genomic elements of retroviral origin that are present in the genomes of almost all vertebrates. In cattle, more than 13,000 elements related to ERVs have been detected, and based on the pol gene, 24 families or groups of bovine ERVs have been described. However, information about ERVs in other bovids and the presence of families of related bovine ERVs in different species of the Bovidae family is scarce.
The 24 families of bovine ERVs previously detected in cattle (Bos taurus) were also detected in zebus (Bos indicus) and yaks (Bos grunniens). In addition, six new families, named BoERV25 to BoERV30, were detected in the three Bos species. Five more ruminant species were screened for related ERVs: 26 families were detected in these species, but four families (BoERV24, BoERV26, BoERV28 and BoERV29) were specific to cattle, zebus, yaks and buffalo. An analysis of the homology of the ERVs of cattle, zebus and yaks revealed that the level of LTR divergence was similar between ERVs from cattle and zebus but was less similar between with ERVs from cattle and yaks. In addition, purifying selection was detected in the genes and retroviral regions of clusters of ERVs of cattle, zebus and yaks.
In this work, the 24 ERV families previously identified in cattle were also found in two other species in the Bos genus. In addition, six new bovine ERV families were detected. Based on LTR divergence, the most recently inserted families are from Class II. The divergence of the LTR, used as an indirect estimate of the ERV insertion time, seemed to be influenced by the differences in genome evolution since the divergence of the species. In addition, purifying selection could be acting on clusters of ERVs from different species.
PMCID: PMC3879100  PMID: 24256121
5.  Role of APOBEC3 in Genetic Diversity among Endogenous Murine Leukemia Viruses 
PLoS Genetics  2007;3(10):e183.
The ability of human and murine APOBECs (specifically, APOBEC3) to inhibit infecting retroviruses and retrotransposition of some mobile elements is becoming established. Less clear is the effect that they have had on the establishment of the endogenous proviruses resident in the human and mouse genomes. We used the mouse genome sequence to study diversity and genetic traits of nonecotropic murine leukemia viruses (polytropic [Pmv], modified polytropic [Mpmv], and xenotropic [Xmv] subgroups), the best-characterized large set of recently integrated proviruses. We identified 49 proviruses. In phylogenetic analyses, Pmvs and Mpmvs were monophyletic, whereas Xmvs were divided into several clades, implying a greater number of replication cycles between the integration events. Four distinct primer binding site types (Pro, Gln1, Gln2 and Thr) were dispersed within the phylogeny, indicating frequent mispriming. We analyzed the frequency and context of G-to-A mutations for the role of mA3 in formation of these proviruses. In the Pmv and Mpmv (but not Xmv) groups, mutations attributable to mA3 constituted a large fraction of the total. A significant number of nonsense mutations suggests the absence of purifying selection following mutation. A strong bias of G-to-A relative to C-to-T changes was seen, implying a strand specificity that can only have occurred prior to integration. The optimal sequence context of G-to-A mutations, TTC, was consistent with mA3. At least in the Pmv group, a significant 5′ to 3′ gradient of G-to-A mutations was consistent with mA3 editing. Altogether, our results for the first time suggest mA3 editing immediately preceding the integration event that led to retroviral endogenization, contributing to inactivation of infectivity.
Author Summary
Vertebrate genomes are littered with remnants from earlier retroviral infections, in the form of endogenous retroviruses (ERVs). Cellular host defenses against retroviruses, including the APOBEC3 family of cytidine deaminases, have been described previously. APOBEC3 proteins have been shown to edit some retroviruses and other retrotransposing elements during their replication by deamination of C to U during negative-strand synthesis, resulting in G-to-A mutations in the sense strand. Here, we studied the possible effects that the APOBEC-protein family might have had in the establishing ERVs. We identified 49 endogenous (nonecotropic) murine leukemia viruses, divided into three groups; polytropic, modified polytropic, and xenotropic, in the sequenced C57BL/6J mouse genome. We analyzed genetic variation within and among subgroups and found mutation patterns consistent with APOBEC3 editing of Pmv and Mpmv, but not Xmv proviruses. Evidence such as (i) significantly higher G-to-A mutation frequencies compared to controls and large fractions leading to inactivating stop mutations, (ii) optimal sequence contexts surrounding the mutation positions, and (iii) editing gradient following the time course of retroviral replication, implicate APOBEC3 as a factor contributing to inactivation of these ERVs in the mouse genome.
PMCID: PMC2041998  PMID: 17967065
6.  A Novel Recombinant Retrovirus in the Genomes of Modern Birds Combines Features of Avian and Mammalian Retroviruses 
Journal of Virology  2014;88(5):2398-2405.
Endogenous retroviruses (ERVs) represent ancestral sequences of modern retroviruses or their extinct relatives. The majority of ERVs cluster alongside exogenous retroviruses into two main groups based on phylogenetic analyses of the reverse transcriptase (RT) enzyme. Class I includes gammaretroviruses, and class II includes lentiviruses and alpha-, beta-, and deltaretroviruses. However, analyses of the transmembrane subunit (TM) of the envelope glycoprotein (env) gene result in a different topology for some retroviruses, suggesting recombination events in which heterologous env sequences have been acquired. We previously demonstrated that the TM sequences of five of the six genera of orthoretroviruses can be divided into three types, each of which infects a distinct set of vertebrate classes. Moreover, these classes do not always overlap the host range of the associated RT classes. Thus, recombination resulting in acquisition of a heterologous env gene could in theory facilitate cross-species transmissions across vertebrate classes, for example, from mammals to reptiles. Here we characterized a family of class II avian ERVs, “TgERV-F,” that acquired a mammalian gammaretroviral env sequence. Although TgERV-F clusters near a sister clade to alpharetroviruses, its genome also has some features of betaretroviruses. We offer evidence that this unusual recombinant has circulated among several avian orders and may still have infectious members. In addition to documenting the infection of a nongalliform avian species by a mammalian retrovirus, TgERV-F also underscores the importance of env sequences in reconstructing phylogenies and supports a possible role for env swapping in allowing cross-species transmissions across wide taxonomic distances.
IMPORTANCE Retroviruses can sometimes acquire an envelope gene (env) from a distantly related retrovirus. Since env is a key determinant of host range, such an event affects the host range of the recombinant virus and can lead to the creation of novel retroviral lineages. Retroviruses insert viral DNA into the host DNA during infection, and therefore vertebrate genomes contain a “fossil record” of endogenous retroviral sequences thought to represent past infections of germ cells. We examined endogenous retroviral sequences in avian genomes for evidence of recombination events involving env. Although cross-species transmissions of retroviruses between vertebrate classes (from mammals to birds, for example) are thought to be rare, we here characterized a group of avian retroviruses that acquired an env sequence from a mammalian retrovirus. We offer evidence that this unusual recombinant circulated among songbirds 2 to 4 million years ago and has remained active into the recent past.
PMCID: PMC3958055  PMID: 24352464
7.  Identification of a group of Mus dunni endogenous virus-like endogenous retroviruses from the C57BL/6J mouse genome: proviral genomes, strain distribution, expression characteristics, and genomic integration profile 
About 10 % of the mouse genome is occupied by sequences associated with endogenous retroviruses (ERVs). However, a comprehensive profile of the mouse ERVs and related elements has not been established yet. In this study, we identified a group of ERVs from the mouse genome and characterized their biological properties. Using a custom ERV mining protocol, 191 ERVs (159 loci reported previously and 32 new loci), tentatively named Mus dunni endogenous virus (MDEV)-like ERVs (MDL-ERVs), were mapped on the C57BL/6J mouse genome. Seven of them retained putative full coding potentials for three retroviral polypeptides (gag, pol, and env). Among the 57 mouse strains examined, all but the Mus pahari/Ei strain had PCR amplicons corresponding to a conserved MDL-ERV region. Interestingly, the Mus caroli/EiJ’s amplicon was somewhat larger than the others, coinciding with a substantial phylogenetic distance between the MDL-ERV populations of Mus caroli/EiJ and C57BL/6J strains. MDLERVs were highly expressed in the lung, spleen, and thymus of C57BL/6J mice compared to the brain, heart, kidney, and liver. Seven MDL-ERVs were mapped in the introns of six annotated genes. Of interest, some MDL-ERVs were mapped periodically on three clusters in the chromosome X. The finding that these MDL-ERVs were one of several types of retroelements, which form mosaic-repeat units of tandem arrays, suggests that the formation of the mosaicrepeat unit preceded the tandem arrangement event. Further studies are warranted to understand the biological roles of MDL-ERVs in both normal and pathologic conditions.
PMCID: PMC3535300  PMID: 23197326
8.  A Paradigm for Virus–Host Coevolution: Sequential Counter-Adaptations between Endogenous and Exogenous Retroviruses 
PLoS Pathogens  2007;3(11):e170.
Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of the host germline transmitted vertically from generation to generation. It is hypothesized that some ERVs are used by the host as restriction factors to block the infection of pathogenic retroviruses. Indeed, some ERVs efficiently interfere with the replication of related exogenous retroviruses. However, data suggesting that these mechanisms have influenced the coevolution of endogenous and/or exogenous retroviruses and their hosts have been more difficult to obtain. Sheep are an interesting model system to study retrovirus-host coevolution because of the coexistence in this animal species of two exogenous (i.e., horizontally transmitted) oncogenic retroviruses, Jaagsiekte sheep retrovirus and Enzootic nasal tumor virus, with highly related and biologically active endogenous retroviruses (enJSRVs). Here, we isolated and characterized the evolutionary history and molecular virology of 27 enJSRV proviruses. enJSRVs have been integrating in the host genome for the last 5–7 million y. Two enJSRV proviruses (enJS56A1 and enJSRV-20), which entered the host genome within the last 3 million y (before and during speciation within the genus Ovis), acquired in two temporally distinct events a defective Gag polyprotein resulting in a transdominant phenotype able to block late replication steps of related exogenous retroviruses. Both transdominant proviruses became fixed in the host genome before or around sheep domestication (∼ 9,000 y ago). Interestingly, a provirus escaping the transdominant enJSRVs has emerged very recently, most likely within the last 200 y. Thus, we determined sequentially distinct events during evolution that are indicative of an evolutionary antagonism between endogenous and exogenous retroviruses. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections.
Author Summary
The genome of all vertebrates is heavily colonized by “endogenous” retroviruses (ERVs). ERVs derive from retrovirus infections of the germ cells of the host during evolution, leading to permanent integration of the viral genome into the host DNA. Because ERVs are integrated in the host genome, they are transmitted to subsequent generations like any other host gene. The function of endogenous retroviruses is not completely clear, but some ERVs can block the replication cycle of horizontally transmitted “exogenous” pathogenic retroviruses. These observations lead to the hypothesis that ERVs have protected the host during evolution against incoming pathogenic retroviruses. Here, by characterizing the evolutionary history and molecular virology of a particular group of endogenous betaretroviruses of sheep (enJSRVs) we show a fascinating series of events unveiling the endless struggle between host and retroviruses. In particular, we discovered that: (i) two enJSRV loci that entered the host genome before speciation within the genus Ovis (∼ 3 million y ago) acquired, after their integration, a mutated defective viral protein capable of blocking exogenous related retroviruses; (ii) both these transdominant enJSRV loci became fixed in the host genome before or around sheep domestication (∼ 10,000 y ago); (iii) the invasion of the sheep genome by ERVs of the JSRV/enJSRVs group is still in progress; and (iv) new viruses have recently emerged (less than 200 y ago) that can escape the transdominant enJSRV loci. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections.
PMCID: PMC2065879  PMID: 17997604
9.  Resurrection of endogenous retroviruses in antibody-deficient mice 
Nature  2012;491(7426):774-778.
The mammalian host has developed a long-standing symbiotic relationship with a considerable number of microbial species. These include the microbiota on environmental surfaces, such as the respiratory and gastrointestinal tracks1, and also endogenous retroviruses (ERVs), comprising a substantial fraction of the mammalian genome2,3. The long-term consequences for the host of interaction with these microbial species can range from mutualism to parasitism and are not always completely understood. The potential impact of one microbial symbiont on another is even less clear. We have studied the control of ERVs in the commonly-used C57BL/6 (B6) mouse strain, which lacks endogenous murine leukaemia viruses (MLVs) able to replicate in murine cells. We demonstrate the spontaneous emergence of fully infectious ecotropic4 MLV (eMLV) in B6 mice with a range of distinct immune deficiencies affecting antibody production. These recombinant retroviruses establish infection of immunodeficient mouse colonies, and ultimately result in retrovirus-induced lymphomas. Notably, ERV activation in immune-deficient mice is prevented in husbandry conditions associated with reduced or absent intestinal microbiota. Our results shed light onto a previously unappreciated role for immunity in the control of ERVs and provide a potential mechanistic link between immune activation by microbial triggers and a range of pathologies associated with ERVs, including cancer.
PMCID: PMC3511586  PMID: 23103862
10.  Negative Selection by an Endogenous Retrovirus Promotes a Higher-Avidity CD4+ T Cell Response to Retroviral Infection 
PLoS Pathogens  2012;8(5):e1002709.
Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4+ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4+ T cells and diminished the response to FV infection. Surprisingly, however, CD4+ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4+ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4+ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4+ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity.
Author Summary
Our immune systems defend against viral infection. However, the immune response to a virus often differs substantially between individuals, as does the outcome of infection. The antiviral immune response relies on recognition of viral proteins by T lymphocytes using T cell antigen receptors (TCRs). TCRs are created randomly in an individual and each T lymphocyte has a different TCR. T lymphocytes with TCRs that recognize our own (self) proteins are removed during development, to prevent autoimmunity. Our cells can also make proteins that belong to endogenous retroviruses (ERVs), relics of ancestral retroviral infection that accumulated during evolution to constitute a large proportion of our genomes. The impact of ERVs on lymphocyte development and the subsequent influence on antiviral immunity is incompletely understood. Here, we use a mouse model to investigate this link and show that the T lymphocyte response to exogenous retrovirus infection is heavily influenced by an ERV. Surprisingly, we find that ERVs do not always have a negative impact on immunity, and in our model they improve the sensitivity with which T lymphocytes react to retroviral infection. This work may thus provide a basis for the understanding of the heterogeneity in immunity to retroviral infections in genetically diverse individuals.
PMCID: PMC3349761  PMID: 22589728
11.  Strong purifying selection in endogenous retroviruses in the saltwater crocodile (Crocodylus porosus) in the Northern Territory of Australia 
Mobile DNA  2012;3:20.
Endogenous retroviruses (ERVs) are remnants of exogenous retroviruses that have integrated into the nuclear DNA of a germ-line cell. Here we present the results of a survey into the ERV complement of Crocodylus porosus, the saltwater crocodile, representing 45 individuals from 17 sampling locations in the Northern Territory of Australia. These retroelements were compared with published ERVs from other species of Crocodylia (Crocodilians; alligators, caimans, gharials and crocodiles) as well as representatives from other vertebrates. This study represents one of the first in-depth studies of ERVs within a single reptilian species shedding light on the diversity of ERVs and proliferation mechanisms in crocodilians.
Analyses of the retroviral pro-pol gene region have corroborated the presence of two major clades of ERVs in C. porosus and revealed 18 potentially functional fragments out of the 227 recovered that encode intact pro-pol ORFs. Interestingly, we have identified some patterns of diversification among those ERVs as well as a novel sequence that suggests the presence of an additional retroviral genus in C. porosus. In addition, considerable diversity but low genetic divergence within one of the C. porosus ERV lineages was identified.
We propose that the ERV complement of C. porosus has come about through a combination of recent infections and replication of ancestral ERVs. Strong purifying selection acting on these clades suggests that this activity is recent or still occurring in the genome of this species. The discovery of potentially functional elements is an interesting development that warrants further investigation.
PMCID: PMC3531266  PMID: 23217152
Crocodylia; Endogenous retrovirus; Crocodylus porosus
12.  Origins of the Endogenous and Infectious Laboratory Mouse Gammaretroviruses 
Viruses  2014;7(1):1-26.
The mouse gammaretroviruses associated with leukemogenesis are found in the classical inbred mouse strains and in house mouse subspecies as infectious exogenous viruses (XRVs) and as endogenous retroviruses (ERVs) inserted into their host genomes. There are three major mouse leukemia virus (MuLV) subgroups in laboratory mice: ecotropic, xenotropic, and polytropic. These MuLV subgroups differ in host range, pathogenicity, receptor usage and subspecies of origin. The MuLV ERVs are recent acquisitions in the mouse genome as demonstrated by the presence of many full-length nondefective MuLV ERVs that produce XRVs, the segregation of these MuLV subgroups into different house mouse subspecies, and by the positional polymorphism of these loci among inbred strains and individual wild mice. While some ecotropic and xenotropic ERVs can produce XRVs directly, others, especially the pathogenic polytropic ERVs, do so only after recombinations that can involve all three ERV subgroups. Here, I describe individual MuLV ERVs found in the laboratory mice, their origins and geographic distribution in wild mouse subspecies, their varying ability to produce infectious virus and the biological consequences of this expression.
PMCID: PMC4306825  PMID: 25549291
mouse endogenous retroviruses; mouse leukemia viruses; house mouse subspecies; ecotropic/xenotropic/polytropic gammaretroviruses; retrovirus restriction factors; recombinant mouse gammaretroviruses
13.  GRB2 Interaction with the Ecotropic Murine Leukemia Virus Receptor, mCAT-1, Controls Virus Entry and Is Stimulated by Virus Binding 
Journal of Virology  2012;86(3):1421-1432.
For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2–mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2–mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking.
PMCID: PMC3264351  PMID: 22090132
14.  Multiple invasions of an infectious retrovirus in cat genomes 
Scientific Reports  2015;5:8164.
Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of host germ-line cells. While most ERVs are defective, some are active and express viral proteins. The RD-114 virus is a replication-competent feline ERV, and several feline cell lines produce infectious RD-114 viral particles. All domestic cats are considered to have an ERV locus encoding a replication-competent RD-114 virus in their genomes; however, the locus has not been identified. In this study, we investigated RD-114 virus-related proviral loci in genomes of domestic cats, and found that none were capable of producing infectious viruses. We also found that all domestic cats have an RD-114 virus-related sequence on chromosome C2, termed RDRS C2a, but populations of the other RDRSs are different depending on the regions where cats live or breed. Our results indicate that RDRS C2a, the oldest RD-114-related provirus, entered the host genome before an ancestor of domestic cats started diverging and the other new RDRSs might have integrated into migrating cats in Europe. We also show that infectious RD-114 virus can be resurrected by the recombination between two non-infectious RDRSs. From these data, we conclude that cats do not harbor infectious RD-114 viral loci in their genomes and RD-114-related viruses invaded cat genomes multiple times.
PMCID: PMC4313119  PMID: 25641657
15.  Repeated Recruitment of LTR Retrotransposons as Promoters by the Anti-Apoptotic Locus NAIP during Mammalian Evolution 
PLoS Genetics  2007;3(1):e10.
Neuronal apoptosis inhibitory protein (NAIP, also known as BIRC1) is a member of the conserved inhibitor of apoptosis protein (IAP) family. Lineage-specific rearrangements and expansions of this locus have yielded different copy numbers among primates and rodents, with human retaining a single functional copy and mouse possessing several copies, depending on the strain. Roles for this gene in disease have been documented, but little is known about transcriptional regulation of NAIP. We show here that NAIP has multiple promoters sharing no similarity between human and rodents. Moreover, we demonstrate that multiple, domesticated long terminal repeats (LTRs) of endogenous retroviral elements provide NAIP promoter function in human, mouse, and rat. In human, an LTR serves as a tissue-specific promoter, active primarily in testis. However, in rodents, our evidence indicates that an ancestral LTR common to all rodent genes is the major, constitutive promoter for these genes, and that a second LTR found in two of the mouse genes is a minor promoter. Thus, independently acquired LTRs have assumed regulatory roles for orthologous genes, a remarkable evolutionary scenario. We also demonstrate that 5′ flanking regions of IAP family genes as a group, in both human and mouse are enriched for LTR insertions compared to average genes. We propose several potential explanations for these findings, including a hypothesis that recruitment of LTRs near NAIP or other IAP genes may represent a host-cell adaptation to modulate apoptotic responses.
Author Summary
When retroviruses infect cells, the viral DNA inserts into the cellular genome. If this happens in gametes (egg or sperm), the viral DNA will be transmitted from parent to offspring, like all chromosomal DNA. Through evolutionary time, such infections of gametes have been so prevalent that 8%–10% of the normal human and mouse genomes are now composed of ancient viral DNA, termed endogenous retroviruses (ERVs). In human, these ERVs are mutated or “dead” but it has been shown that ERV regulatory regions can be employed by the host to help control expression of cellular genes. Here, we report on a remarkable example of this phenomenon. We demonstrate that both the human and rodent neuronal apoptosis inhibitory protein (NAIP) genes, involved in preventing cell death, use different ERV sequences to drive gene expression. Moreover, in each of the primate and rodent lineages, two separate ERVs contribute to NAIP gene expression. This repeated ERV recruitment by NAIP genes throughout evolution is very unlikely to have occurred by chance. We offer a number of potential explanations, including the intriguing possibility that it may be advantageous for anti-cell death genes like NAIP to use ERVs to control their expression. These results support the view that not all retroviral remnants in our genome are simply junk DNA.
PMCID: PMC1781489  PMID: 17222062
16.  Multiple Gag Domains Contribute to Selective Recruitment of Murine Leukemia Virus (MLV) Env to MLV Virions 
Journal of Virology  2013;87(3):1518-1527.
Retroviruses, like all enveloped viruses, must incorporate viral glycoproteins to form infectious particles. Interactions between the glycoprotein cytoplasmic tail and the matrix domain of Gag are thought to direct recruitment of glycoproteins to native virions for many retroviruses. However, retroviruses can also incorporate glycoproteins from other viruses to form infectious virions known as pseudotyped particles. The glycoprotein murine leukemia virus (MLV) Env can readily form pseudotyped particles with many retroviruses, suggesting a generic mechanism for recruitment. Here, we sought to identify which components of Gag, particularly the matrix domain, contribute to recruitment of MLV Env into retroviral particles. Unexpectedly, we discovered that the matrix domain of HIV-1 Gag is dispensable for generic recruitment, since it could be replaced with a nonviral membrane-binding domain without blocking active incorporation of MLV Env into HIV virions. However, MLV Env preferentially assembles with MLV virions. When MLV and HIV particles are produced from the same cell, MLV Env is packaged almost exclusively by MLV particles, thus preventing incorporation into HIV particles. Surprisingly, the matrix domain of MLV Gag is not required for this selectivity, since MLV Gag containing the matrix domain from HIV is still able to outcompete HIV particles for MLV Env. Although MLV Gag is sufficient for selective incorporation to occur, no single Gag domain dictates the selectivity. Our findings indicate that Env recruitment is more complex than previously believed and that Gag assembly/budding sites have fundamental properties that affect glycoprotein incorporation.
PMCID: PMC3554149  PMID: 23152533
17.  Lipopolysaccharide stress induces cell-type specific production of murine leukemia virus type-endogenous retroviral virions in primary lymphoid cells 
The Journal of General Virology  2011;92(Pt 2):292-300.
Some murine-endogenous retroviruses, making up ∼10 % of the mouse genome, are induced during the course of experimental sepsis in which lipopolysaccharide (LPS), a pathogenic component of Gram-negative bacteria, often plays a critical role. In this study, we investigated whether LPS stress induces the production of murine leukemia virus type-endogenous retrovirus (MuLV-ERV) virions in primary lymphoid cells. LPS treatment of cells (single-cell suspensions and sorted B- and T-cells) isolated from seven lymphoid organs of C57BL/6J mice resulted in a differential increase in the production of MuLV-ERV virions in most cells examined. Interestingly, among the 34 unique MuLV-ERV U3 sequences cloned from the viral genomic RNAs, the nuclear respiratory factor 1 (transcription factor) element was present only in the 20 U3 sequences that were derived from the LPS-induced MuLV-ERV U3 bands. Using the U3 sequences as a probe, 55 putative MuLV-ERV loci were mapped onto the C57BL/6J mouse genome and 15 of them retained full coding potential. Furthermore, one full-length recombinant MuLV-ERV originating from a locus on chromosome 13 was determined to be responsive to LPS stress. The findings from this study suggest that LPS stress differentially activates MuLV-ERV virion production in lymphoid organs in a cell type- and MuLV-ERV-specific manner. Further investigation is needed to define the role of MuLV-ERVs in the LPS signalling pathway(s) in general, as well as in the pathogenesis of sepsis.
PMCID: PMC3081078  PMID: 20965985
18.  Cerebellum-specific and age-dependent expression of an endogenous retrovirus with intact coding potential 
Retrovirology  2011;8:82.
Endogenous retroviruses (ERVs), including murine leukemia virus (MuLV) type-ERVs (MuLV-ERVs), are presumed to occupy ~10% of the mouse genome. In this study, following the identification of a full-length MuLV-ERV by in silico survey of the C57BL/6J mouse genome, its distribution in different mouse strains and expression characteristics were investigated.
Application of a set of ERV mining protocols identified a MuLV-ERV locus with full coding potential on chromosome 8 (named ERVmch8). It appears that ERVmch8 shares the same genomic locus with a replication-incompetent MuLV-ERV, called Emv2; however, it was not confirmed due to a lack of relevant annotation and Emv2 sequence information. The ERVmch8 sequence was more prevalent in laboratory strains compared to wild-derived strains. Among 16 different tissues of ~12 week-old female C57BL/6J mice, brain homogenate was the only tissue with evident expression of ERVmch8. Further ERVmch8 expression analysis in six different brain compartments and four peripheral neuronal tissues of C57BL/6J mice revealed no significant expression except for the cerebellum in which the ERVmch8 locus' low methylation status was unique compared to the other brain compartments. The ERVmch8 locus was found to be surrounded by genes associated with neuronal development and/or inflammation. Interestingly, cerebellum-specific ERVmch8 expression was age-dependent with almost no expression at 2 weeks and a plateau at 6 weeks.
The ecotropic ERVmch8 locus on the C57BL/6J mouse genome was relatively undermethylated in the cerebellum, and its expression was cerebellum-specific and age-dependent.
PMCID: PMC3207890  PMID: 21992658
19.  Endogenous Gammaretrovirus Acquisition in Mus musculus Subspecies Carrying Functional Variants of the XPR1 Virus Receptor 
Journal of Virology  2013;87(17):9845-9855.
The xenotropic and polytropic mouse leukemia viruses (X-MLVs and P-MLVs, respectively) have different host ranges but use the same functionally polymorphic receptor, XPR1, for entry. Endogenous retroviruses (ERVs) of these 2 gammaretrovirus subtypes are largely segregated in different house mouse subspecies, but both MLV types are found in the classical strains of laboratory mice, which are genetic mosaics of 3 wild mouse subspecies. To describe the subspecies origins of laboratory mouse XP-MLV ERVs and their coevolutionary trajectory with their XPR1 receptor, we screened the house mouse subspecies for known and novel Xpr1 variants and for the individual full-length XP-MLV ERVs found in the sequenced C57BL mouse genome. The 12 X-MLV ERVs predate the origins of laboratory mice; they were all traced to Japanese wild mice and are embedded in the 5% of the laboratory mouse genome derived from the Asian Mus musculus musculus and, in one case, in the <1% derived from M. m. castaneus. While all 31 P-MLV ERVs map to the 95% of the laboratory mouse genome derived from P-MLV-infected M. m. domesticus, no C57BL P-MLV ERVs were found in wild M. m. domesticus. All M. m. domesticus mice carry the fully permissive XPR1 receptor allele, but all of the various restrictive XPR1 receptors, including the X-MLV-restricting laboratory mouse Xpr1n and a novel M. m. castaneus allele, originated in X-MLV-infected Asian mice. Thus, P-MLV ERVs show more insertional polymorphism than X-MLVs, and these differences in ERV acquisition and fixation are linked to subspecies-specific and functionally distinct XPR1 receptor variants.
PMCID: PMC3754108  PMID: 23824809
20.  Genome-Wide Characterization of Endogenous Retroviruses in the Bat Myotis lucifugus Reveals Recent and Diverse Infections 
Journal of Virology  2013;87(15):8493-8501.
Bats are increasingly recognized as reservoir species for a variety of zoonotic viruses that pose severe threats to human health. While many RNA viruses have been identified in bats, little is known about bat retroviruses. Endogenous retroviruses (ERVs) represent genomic fossils of past retroviral infections and, thus, can inform us on the diversity and history of retroviruses that have infected a species lineage. Here, we took advantage of the availability of a high-quality genome assembly for the little brown bat, Myotis lucifugus, to systematically identify and analyze ERVs in this species. We mined an initial set of 362 potentially complete proviruses from the three main classes of ERVs, which were further resolved into 13 major families and 86 subfamilies by phylogenetic analysis. Consensus or representative sequences for each of the 86 subfamilies were then merged to the Repbase collection of known ERV/long terminal repeat (LTR) elements to annotate the retroviral complement of the bat genome. The results show that nearly 5% of the genome assembly is occupied by ERV-derived sequences, a quantity comparable to findings for other eutherian mammals. About one-fourth of these sequences belong to subfamilies newly identified in this study. Using two independent methods, intraelement LTR divergence and analysis of orthologous loci in two other bat species, we found that the vast majority of the potentially complete proviruses identified in M. lucifugus were integrated in the last ∼25 million years. All three major ERV classes include recently integrated proviruses, suggesting that a wide diversity of retroviruses is still circulating in Myotis bats.
PMCID: PMC3719839  PMID: 23720713
21.  Mus spicilegus Endogenous Retrovirus HEMV Uses Murine Sodium-Dependent Myo-Inositol Transporter 1 as a Receptor 
Journal of Virology  2012;86(11):6341-6344.
We sought to determine the relationship between two recent additions to the murine leukemia virus (MLV) ecotropic subgroup: Mus cervicolor isolate M813 and Mus spicilegus endogenous retrovirus HEMV. Though divergent in sequence, the two viruses share an Env protein with similarly curtailed VRA and VRB regions, and infection by both is restricted to mouse cells. HEMV and M813 displayed reciprocal receptor interference, suggesting that they share a receptor. Expression of the M813 receptor murine sodium-dependent myo-inositol transporter 1 (mSMIT1) allowed previously nonpermissive cells to be infected by HEMV, indicating that mSMIT1 also serves as a receptor for HEMV. Our findings add HEMV as a second member to the MLV subgroup that uses mSMIT1 to gain entry into cells.
PMCID: PMC3372175  PMID: 22457525
Virology  2008;373(2):263-273.
Endogenous retroviruses (ERVs) constitute approximately 8−10% of the human and mouse genome. Some autoimmune diseases are attributed to the altered expression of ERVs. In this study, we examined the ERV expression profiles in lymphoid tissues and analyzed their biological properties. Tissues (spleen, thymus, and lymph nodes [axillary, inguinal, and mesenteric]) from C57BL/6J mice were analyzed for differential murine ERV (MuERV) expression by RT-PCR examination of polymorphic U3 sequences. Each tissue had a unique profile of MuERV expression. A genomic map identifying 60 putative MuERVs was established using 22 unique U3s as probes and their biological properties (primer binding site, coding potential, transcription regulatory element, tropism, recombination event, and integration age) were characterized. Interestingly, 12 putative MuERVs retained intact coding potentials for all three polypeptides essential for virus assembly and replication. We suggest that MuERV expression is differentially regulated in conjunction with the transcriptional environment of individual lymphoid tissues.
PMCID: PMC2427371  PMID: 18187179
endogenous retrovirus; spleen; thymus; lymph node; coding potential; transcription; genome
23.  Identification of the Myelin Protein Plasmolipin as the Cell Entry Receptor for Mus caroli Endogenous Retrovirus▿  
Journal of Virology  2008;82(14):6862-6868.
The Asian wild mouse species Mus caroli harbors an endogenous retrovirus (McERV) that is closely related to but distinct from the endogenous retrovirus family defined by the Mus dunni endogenous virus and the Mus musculus endogenous retrovirus. McERV could infect some cell types from humans, dogs, and rats, but not all, and did not infect any mouse cell line tested. Because of its interesting host range and proposed ancestral relationship to primate retroviruses and because none of the entry receptors for this family of retroviruses had been identified, we began a search for the McERV receptor. We determined the chromosomal location of the receptor gene in the human genome by phenotypic screening of the G3 human-hamster radiation hybrid cell line panel and confirmed the localization by assaying for receptor activity conferred by bacterial artificial chromosome (BAC) clones spanning the region. We next localized the gene more precisely in one positive BAC by assaying for receptor activity following BAC digestion with several restriction enzymes that cleaved different sets of genes, and we confirmed that the final candidate gene, plasmolipin (PLLP; TM4SF11), is the novel receptor by showing that the expression of the human PLLP cDNA renders hamster and mouse cells susceptible to McERV infection. PLLP functions as a voltage-dependent potassium ion channel and is expressed primarily in kidney and brain, helping to explain the limited range of cell types that McERV can infect. Interestingly, mouse PLLP also functioned well as a receptor for McERV but was simply not expressed in the mouse cell types that we originally tested.
PMCID: PMC2446966  PMID: 18463156
24.  Retrovirus-like particles released from the human breast cancer cell line T47-D display type B- and C-related endogenous retroviral sequences. 
Journal of Virology  1995;69(10):6408-6416.
The human mammary carcinoma cell line T47-D releases retrovirus-like particles of type B morphology in a steroid-dependent manner (I. Keydar, T. Ohno, R. Nayak, R. Sweet, F. Simoni, F. Weiss, S. Karby, R. Mesa-Tejada, and S. Spiegelman, Proc. Natl. Acad. Sci. USA 81:4188-4192, 1984). Furthermore, reverse transcriptase (RT) activity is found to be associated with particle preparations. Using a set of degenerate primers derived from a conserved region of retroviral pol genes, we repeatedly amplified three different retroviral sequences (MLN, FRD, and FTD) from purified T47-D particles in several RT-PCR experiments. Screening of a human genomic library and Southern blot analysis revealed that these sequences are of endogenous origin. ERV-MLN represents a multicopy family of human endogenous retroviral elements (HERVs) with two closely related copies and up to 20 more distantly related members. In contrast, ERV-FRD and ERV-FTD comprise only one copy and five to seven related elements per haploid human genome. DNA sequence analysis of the proviral pol region of ERV-MLN revealed an uninterrupted stretch of 241 amino acids that shows 65% identity with the RT of the type B-related HERV designated HERV-K10. ERV-FRD and ERV-FTD are defective type C-related HERVs. The pol gene of ERV-FRD displays a nucleotide homology of 54% to the gibbon ape leukemia virus, and the pol gene of ERV-FTD is about 67% homologous to members of the RTVL-I family of HERVs. Our results thus indicate that the retroviral particles released by the breast cancer cell line T47-D are probably generated by complementation of several endogenous proviruses and can package retroviral transcripts of different origins.
PMCID: PMC189540  PMID: 7545247
25.  Misfolding of CasBrE SU is reversed by interactions with 4070A Env: implications for gammaretroviral neuropathogenesis 
Retrovirology  2010;7:93.
CasBrE is a neurovirulent murine leukemia virus (MLV) capable of inducing paralytic disease with associated spongiform neurodegeneration. The neurovirulence of this virus has been genetically mapped to the surface expressed subunit (SU) of the env gene. However, CasBrE SU synthesized in the absence of the transmembrane subunit (TM) does not retain ecotropic receptor binding activity, indicating that folding of the receptor binding domain (RBD) requires this domain. Using a neural stem cell (NSC) based viral trans complementation approach to examine whether misfolded CasBrE SU retained neurovirulence, we observed CasBrE SU interaction with the "non-neurovirulent" amphotropic helper virus, 4070A which restored functional activity of CasBrE SU.
Herein, we show that infection of NSCs expressing CasBrE SU with 4070A (CasES+4070A-NSCs) resulted in the redistribution of CasBrE SU from a strictly secreted product to include retention on the plasma membrane. Cell surface cross-linking analysis suggested that CasBrE SU membrane localization was due to interactions with 4070A Env. Viral particles produced from CasES+4070A-NSCS contained both CasBrE and 4070A gp70 Env proteins. These particles displayed ecotropic receptor-mediated infection, but were still 100-fold less efficient than CasE+4070A-NSC virus. Infectious center analysis showed CasBrE SU ecotropic transduction efficiencies approaching those of NSCs expressing full length CasBrE Env (CasE; SU+TM). In addition, CasBrE SU-4070A Env interactions resulted in robust ecotropic superinfection interference indicating near native intracellular SU interaction with its receptor, mCAT-1.
In this report we provided evidence that 4070A Env and CasBrE SU physically interact within NSCs leading to CasBrE SU retention on the plasma membrane, incorporation into viral particles, restoration of mCAT-1 binding, and capacity for initiation of TM-mediated fusion events. Thus, heterotropic Env-SU interactions facilitates CasBrE SU folding events that restore Env activity. These findings are consistent with the idea that one protein conformation acts as a folding scaffold or nucleus for a second protein of similar primary structure, a process reminiscent of prion formation. The implication is that template-based protein folding may represent an inherent feature of neuropathogenic proteins that extends to retroviral Envs.
PMCID: PMC2998453  PMID: 21054857

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