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The study of the role of retroviruses in amyotrophic lateral sclerosis (ALS) dates back to the 1960s shortly after transposable elements themselves were first discovered. It was quickly realized that in wild mice both horizontal and vertical transmissions of retroviral elements were key to the development of an ALS-like syndrome leading to the postulate that endogenous retroviruses (ERVs) contribute significantly to the pathogenicity of this disease. Subsequent studies identified retroviral reverse transcriptase activity in brains of individuals with ALS from Guam. However, except for a single study from the former Soviet Union, ALS could not be transmitted to rhesus macaques. The discovery of an ALS-like syndrome in human immunodeficiency virus (HIV) and human T cell leukemia virus infected individuals led to renewed interest in the field and reverse transcriptase activity was found in the blood and cerebrospinal fluid of individuals with sporadic ALS. However, exogenous retroviruses could not be found in individuals with ALS which further reinforced the possibility of involvement of a human ERV (HERV). The first demonstration of the involvement of a HERV was the discovery of the activation of human endogenous retrovirus-K subtype HML-2 in the brains of individuals with ALS. The envelope protein of HML-2 is neurotoxic and transgenic animals expressing the envelope protein develop an ALS-like syndrome. Activation of HML-2 occurs in the context of generalized transposable element activation and is not specific for ALS. Individuals with HIV-associated ALS show a remarkable response to antiretroviral therapy; however, antiretroviral trials in ALS down-regulate HML-2 without ameliorating the disease. This highlights the need for specific drugs to be developed against HML-2 as a novel therapeutic target for ALS. Other approaches might include antisense oligonucleotides, shRNA targeted against the envelope gene or antibodies that can target the extracellular envelope protein. Future clinical trials in ALS should consider combination therapies to control these ERVs.

Vertebrate genomes contain endogenous retroviruses (ERVs) that represent remnants of past germline infections by ancient retroviruses. Despite comprising 8% of the human genome, the human ERVs (HERVs) do not encode a replication competent retrovirus. However, some HERV genes have been co-opted to serve host functions, most notably the viral envelope-derived syncytins involved in placentation. Here, we identify the oldest HERV intact gag gene with an open reading frame, gagV1. Its provirus contains an intact env, envV1, and the first open reading frame found in an HERV gag leader, pre-gagV1, which encodes a novel protein. This HERV is linked to a related gag gene, gagV3, and these three genes all show patterns of evolutionary conservation in primates. gagV1 and pre-gagV1 orthologs are present in all simian primate lineages indicating that this HERV entered the germline of the common simian primate ancestor at least 43 Ma, whereas gagV3 is found in Old and New World monkeys. gagV1 and gagV3 have undergone recurrent gene conversion events and positive selection. Expression of gagV1, gagV3, and pre-gagV1 is restricted to the placenta in humans and macaques suggesting co-option for placenta-specific host functions. Transcriptomic analysis of human tumors also found upregulated levels of gagV1 transcripts in diffuse large B-cell lymphomas. These findings suggest that these HERV-V genes may be useful markers for the most common type of non-Hodgkin’s lymphoma and that they may have contributed to the successive domestications of env and gag genes in eutherians involved in the ongoing ERV-driven evolution of the placenta.

Endogenous retroviruses (ERVs) found in vertebrate genomes are remnants of retroviral invasions of their ancestral species. ERVs thus represent molecular fossil records of ancient retroviruses and provide a unique opportunity to study viral-host interactions, including cross-species transmissions, in deep time. While most ERVs contain the mutated remains of the original retrovirus, on rare occasions evolutionary selection pressures lead to the co-option/exaptation of ERV genes for a host function. Here, we report the identification of two ancient related non-orthologous ERVenvgenes,ARTenvVandCARenvV, that are preserved with large open reading frames (ORFs) in the mammalian orders Artiodactyla and Carnivora, respectively, but are not found in other mammals. These Env proteins lack a transmembrane motif, but phylogenetic analyses show strong sequence preservation and positive selection of theenvsurface ORF in their respective orders, and transcriptomic analyses show a broad tissue expression pattern for bothARTenvVandCARenvV, suggesting that these genes may be exapted for a host function. Multiple lines of evidence indicate thatARTenvVandCARenvVwere derived from an ancient ancestral exogenous gamma-like retrovirus that was independently endogenized in two mammalian orders more than 60 million years ago, which roughly coincides with the K-Pg mass extinction event and subsequent mammalian diversification. Thus, these findings identify the oldest known retroviral cross-ordinal transmission of a gamma-like retrovirus with no known extant infectious counterpart in mammals, and the first discovery of the convergent co-option of an ERV gene derived from the same ancestral retrovirus in two different mammalian orders.

The classical laboratory mouse strains are genetic mosaics of three Mus musculus subspecies that occupy distinct regions of Eurasia. These strains and subspecies carry infectious and endogenous mouse leukemia viruses (MLVs) that can be pathogenic and mutagenic. MLVs evolved in concert with restrictive host factors with some under positive selection, including the XPR1 receptor for xenotropic/polytropic MLVs (X/P-MLVs) and the post-entry restriction factor Fv1. Since positive selection marks host-pathogen genetic conflicts, we examined MLVs for counter-adaptations at sites that interact with XPR1, Fv1, and the CAT1 receptor for ecotropic MLVs (E-MLVs). Results describe different co-adaptive evolutionary paths within the ranges occupied by these virus-infected subspecies. The interface of CAT1, and the otherwise variable E-MLV envelopes, is highly conserved, antiviral protection is afforded by the Fv4 restriction factor. XPR1 and X/P-MLVs variants show coordinate geographic distributions, with receptor critical sites in envelope, under positive selection but with little variation in envelope and XPR1 in mice carrying P-ERVs. The major Fv1 target in the viral capsid is under positive selection, and the distribution of Fv1 alleles is subspecies-correlated. These data document adaptive, spatial and temporal, co-evolutionary trajectories at the critical interfaces of MLVs and the host factors that restrict their replication.

Background Retroviruses exist as exogenous infectious agents and as endogenous retroviruses (ERVs) integrated into host chromosomes. Such endogenous retroviruses (ERVs) are grouped into three classes roughly corresponding to the seven genera of infectious retroviruses: class I (gamma-, epsilonretroviruses), class II (alpha-, beta-, delta-, lentiretroviruses) and class III (spumaretroviruses). Some ERVs have counterparts among the known infectious retroviruses, while others represent paleovirological relics of extinct or undiscovered retroviruses. Results Here we identify an intact ERV in the Anuran amphibian, Xenopus tropicalis. XtERV-S has open reading frames (ORFs) for gag, pol (polymerase) and env (envelope) genes, with a small additional ORF in pol and a serine tRNA primer binding site. It has unusual features and domain relationships to known retroviruses. Analyses based on phylogeny and functional motifs establish that XtERV-S gag and pol genes are related to the ancient env-less class III ERV-L family but the surface subunit of env is unrelated to known retroviruses while its transmembrane subunit is class I-like. LTR constructs show transcriptional activity, and XtERV-S transcripts are detected in embryos after the maternal to zygotic mid-blastula transition and before the late tailbud stage. Tagged Gag protein shows typical subcellular localization. The presence of ORFs in all three protein-coding regions along with identical 5’ and 3’ LTRs (long terminal repeats) indicate this is a very recent germline acquisition. There are older, full-length, nonorthologous, defective copies in Xenopus laevis and the distantly related African bullfrog, Pyxicephalus adspersus. Additional older, internally deleted copies in X. tropicalis carry a 300 bp LTR substitution. Conclusions XtERV-S represents a genera-spanning member of the largely env-less class III ERV that has ancient and modern copies in Anurans. This provirus has an env ORF with a surface subunit unrelated to known retroviruses and a transmembrane subunit related to class I gammaretroviruses in sequence and organization, and is expressed in early embryogenesis. Additional XtERV-S-related but defective copies are present in X. tropicalis and other African frog taxa. XtERV-S is an unusual class III ERV variant, and it may represent an important transitional retroviral form that has been spreading in African frogs for tens of millions of years.

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.

The evolutionary conflict between retroviruses and their vertebrate hosts over millions of years has led to the emergence of cellular innate immune proteins termed restriction factors as well as their viral antagonists. Evidence accumulated in the last two decades has substantially increased our understanding of the elaborate mechanisms utilized by these restriction factors to inhibit retroviral replication, mechanisms that either directly block viral proteins or interfere with the cellular pathways hijacked by the viruses. Analyses of these complex interactions describe patterns of accelerated evolution for these restriction factors as well as the acquisition and evolution of their virus-encoded antagonists. Evidence is also mounting that many restriction factors identified for their inhibition of specific retroviruses have broader antiviral activity against additional retroviruses as well as against other viruses, and that exposure to these multiple virus challenges has shaped their adaptive evolution. In this review, we provide an overview of the restriction factors that interfere with different steps of the retroviral life cycle, describing their mechanisms of action, adaptive evolution, viral targets and the viral antagonists that evolved to counter these factors.

Evolution of cellular innate immune genes in response to viral threats represents a rich area of study for understanding complex events that shape mammalian genomes. One of these genes, TRIM5, is a retroviral restriction factor that mediates a post-entry block to infection. Previous studies on the genomic cluster that contains TRIM5 identified different patterns of gene amplification and the independent birth of CypA gene fusions in various primate species. However, the evolution of Trim5 in the largest order of mammals, Rodentia, remains poorly characterized. Here, we present an expansive phylogenetic and genomic analysis of the Trim5 cluster in rodents. Our findings reveal substantial evolutionary changes including gene amplifications, rearrangements, loss and fusion. We describe the first independent evolution of TrimCyp fusion genes in rodents. We show that the TrimCyp gene found in some Peromyscus species was acquired about 2 million years ago. When ectopically expressed, the P. maniculatus TRIMCyp shows anti-retroviral activity that is reversed by cyclosporine, but it does not activate Nf-κB or AP-1 promoters, unlike the primate TRIMCyps. These results describe a complex pattern of differential gene amplification in the Trim5 cluster of rodents and identify the first functional TrimCyp fusion gene outside of primates and tree shrews.

Inbred laboratory mouse strains carry endogenous retroviruses (ERVs) classed as ecotropic, xenotropic or polytropic mouse leukemia viruses (E-, X- or P-MLVs). Some of these MLV ERVs produce infectious virus and/or contribute to the generation of intersubgroup recombinants. Analyses of selected mouse strains have linked the appearance of MLVs and virus-induced disease to the strain complement of MLV E-ERVs and to host genes that restrict MLVs, particularly Fv1. Here we screened inbred strain DNAs and genome assemblies to describe the distribution patterns of 45 MLV ERVs and Fv1 alleles in 58 classical inbred strains grouped in two ways: by common ancestry to describe ERV inheritance patterns, and by incidence of MLV-associated lymphomagenesis. Each strain carries a unique set of ERVs, and individual ERVs are present in 5-96% of the strains, often showing lineage-specific distributions. Two ERVs are alternatively present as full-length proviruses or solo long terminal repeats. High disease incidence strains carry the permissive Fv1n allele, tested strains have highly expressed E-ERVs and most have the Bxv1 X-ERV; these three features are not present together in any low-moderate disease strain. The P-ERVs previously implicated in P-MLV generation are not preferentially found in high leukemia strains, but the three Fv1 alleles that restrict inbred strain E-MLVs are found only in low-moderate leukemia strains. This dataset helps define the genetic basis of strain differences in spontaneous lymphomagenesis, describes the distribution of MLV ERVs in strains with shared ancestry, and should help annotate sequenced strain genomes for these insertionally polymorphic and functionally important proviruses.

The laboratory mouse Fv1 gene encodes a retroviral restriction factor that mediates resistance to murine leukemia viruses (MLVs). Sequence similarity between Fv1 and the gag protein of the murine endogenous retrovirus L (MuERV-L) family of ERVs suggests that Fv1 was coopted from an ancient provirus. Previous evolutionary studies found Fv1 orthologs only in the genus Mus Here, we describe identification of orthologous Fv1 sequences in several species belonging to multiple families of rodents outside the genus Mus We show that these Fv1 orthologs are in the same region of conserved synteny, between the genes Miip and Mfn2, suggesting a minimum insertion time of 45 million years for the ancient progenitor of Fv1 Our analysis also revealed that Fv1 was not detectable or heavily mutated in some lineages in the superfamily Muroidea, while, in concert with previous findings in the genus Mus, we found strong evidence of positive selection of Fv1 in the African clade in the subfamily Muridae Residues identified as evolving under positive selection include those that have been previously found to be important for restriction of multiple retroviral lineages. Taken together, these findings suggest that the evolutionary origin of Fv1 substantially predates Mus evolution, that the rodent Fv1 has been shaped by lineage-specific differential selection pressures, and that Fv1 has long been evolving under positive selection in the rodent family Muridae, supporting a defensive role that significantly antedates exposure to MLVs.IMPORTANCE Retroviruses have adapted to living in concert with their hosts throughout vertebrate evolution. Over the years, the study of these relationships revealed the presence of host proteins called restriction factors that inhibit retroviral replication in host cells. The first of these restriction factors to be identified, encoded by the Fv1 gene found in mice, was thought to have originated in the genus Mus In this study, we utilized genome database searches and DNA sequencing to identify Fv1 copies in multiple rodent lineages. Our findings suggest a minimum time of insertion into the genome of rodents of 45 million years for the ancestral progenitor of Fv1 While Fv1 is not detectable in some lineages, we also identified full-length orthologs showing signatures of a molecular "arms race" in a family of rodent species indigenous to Africa. This finding suggests that Fv1 in these species has been coevolving with unidentified retroviruses for millions of years.

Naturally-occurring lymphomagenesis is induced by mouse leukemia viruses (MLVs) carried as endogenous retroviruses (ERVs). Replicating the ecotropic MLVs recombines with polytropic (P-ERVs) and xenotropic ERVs (X-ERVs) to generate pathogenic viruses with an altered host range. While most recovered nonecotropic recombinants have a polytropic host range, the X-MLVs are also present in the pre-leukemic tissues. We analyzed two such isolates from the AKR mice to identify their ERV progenitors and to look for evidence of recombination. AKR40 resembles the active X-ERV Bxv1, while AKR6 has a Bxv1-like backbone with substitutions that alter the long terminal repeat (LTR) enhancer and the envelope (env). AKR6 has a modified xenotropic host range, and its Env residue changes all lie outside of the domain that governs the receptor choice. The AKR6 segment spanning the two substitutions, but not the entire AKR6 env-LTR, exists as an ERV, termed Xmv67, in AKR, but not in the C57BL/6 mice. This suggests that AKR6 is the product of one, not two, recombination events. Xmv67 originated in the Asian mice. These data indicate that the recombinant X-MLVs that can be generated during lymphomagenesis, describe a novel X-ERV subtype found in the AKR genome, but not in the C57BL/6 reference genome, and identify residues in the envelope C-terminus that may influence the host range.

The laboratory mouse Fv1 gene encodes a retroviral restriction factor that mediates resistance to murine leukemia viruses (MLVs). Sequence similarity between Fv1 and the gag protein of the murine endogenous retrovirus L (MuERV-L) family of ERVs suggests that Fv1 was coopted from an ancient provirus. Previous evolutionary studies found Fv1 orthologs only in the genus Mus. Here, we describe identification of orthologous Fv1 sequences in several species belonging to multiple families of rodents outside the genus Mus. We show that these Fv1 orthologs are in the same region of conserved synteny, between the genes Miip and Mfn2, suggesting a minimum insertion time of 45 million years for the ancient progenitor of Fv1. Our analysis also revealed that Fv1 was not detectable or heavily mutated in some lineages in the superfamily Muroidea, while, in concert with previous findings in the genus Mus, we found strong evidence of positive selection of Fv1 in the African clade in the subfamily Muridae. Residues identified as evolving under positive selection include those that have been previously found to be important for restriction of multiple retroviral lineages. Taken together, these findings suggest that the evolutionary origin of Fv1 substantially predates Mus evolution, that the rodent Fv1 has been shaped by lineage-specific differential selection pressures, and that Fv1 has long been evolving under positive selection in the rodent family Muridae, supporting a defensive role that significantly antedates exposure to MLVs. IMPORTANCE Retroviruses have adapted to living in concert with their hosts throughout vertebrate evolution. Over the years, the study of these relationships revealed the presence of host proteins called restriction factors that inhibit retroviral replication in host cells. The first of these restriction factors to be identified, encoded by the Fv1 gene found in mice, was thought to have originated in the genus Mus. In this study, we utilized genome database searches and DNA sequencing to identify Fv1 copies in multiple rodent lineages. Our findings suggest a minimum time of insertion into the genome of rodents of 45 million years for the ancestral progenitor of Fv1. While Fv1 is not detectable in some lineages, we also identified full-length orthologs showing signatures of a molecular “arms race” in a family of rodent species indigenous to Africa. This finding suggests that Fv1 in these species has been coevolving with unidentified retroviruses for millions of years.

Ecotropic, xenotropic, and polytropic mouse leukemia viruses (E-, X-, and P-MLVs) exist in mice as infectious viruses and endogenous retroviruses (ERVs) inserted into mouse chromosomes. All three MLV subgroups are linked to leukemogenesis, which involves generation of recombinants with polytropic host range. Although P-MLVs are deemed to be the proximal agents of disease induction, few biologically characterized infectious P-MLVs have been sequenced for comparative analysis. We analyzed the complete genomes of 16 naturally occurring infectious P-MLVs, 12 of which were typed for pathogenic potential. We sought to identify ERV progenitors, recombinational hot spots, and segments that are always replaced, never replaced, or linked to pathogenesis or host range. Each P-MLV has an E-MLV backbone with P- or X-ERV replacements that together cover 100% of the recombinant genomes, with different substitution patterns for X- and P-ERVs. Two segments are always replaced, both coding for envelope (Env) protein segments: the N terminus of the surface subunit and the cytoplasmic tail R peptide. Viral gag gene replacements are influenced by host restriction genes Fv1 and Apobec3 . Pathogenic potential maps to the env transmembrane subunit segment encoding the N-heptad repeat (HR1). Molecular dynamics simulations identified three novel interdomain salt bridges in the lymphomagenic virus HR1 that could affect structural stability, entry or sensitivity to host immune responses. The long terminal repeats of lymphomagenic P-MLVs are differentially altered by recombinations, duplications, or mutations. This analysis of the naturally occurring, sometimes pathogenic P-MLV recombinants defines the limits and extent of intersubgroup recombination and identifies specific sequence changes linked to pathogenesis and host interactions. IMPORTANCE During virus-induced leukemogenesis, ecotropic mouse leukemia viruses (MLVs) recombine with nonecotropic endogenous retroviruses (ERVs) to produce polytropic MLVs (P-MLVs). Analysis of 16 P-MLV genomes identified two segments consistently replaced: one at the envelope N terminus that alters receptor choice and one in the R peptide at the envelope C terminus, which is removed during virus assembly. Genome-wide analysis shows that nonecotropic replacements in the progenitor ecotropic MLV genome are more extensive than previously appreciated, covering 100% of the genome; contributions from xenotropic and polytropic ERVs differentially alter the regions responsible for receptor determination or subject to APOBEC3 and Fv1 restriction. All pathogenic viruses had modifications in the regulatory elements in their long terminal repeats and differed in a helical segment of envelope involved in entry and targeted by the host immune system. Virus-induced leukemogenesis thus involves generation of complex recombinants, and specific replacements are linked to pathogenesis and host restrictions.

Entry determinants in the XPR1 receptor for the xenotropic/polytropic mouse leukemia viruses (XP-MLVs) lie in its third and fourth putative extracellular loops (ECLs). The critical ECL3 receptor determinant overlies a splice donor and is evolutionarily conserved in vertebrate XPR1 genes; 2 of the 3 rare replacement mutations at this site destroy this receptor determinant. The 13 residue ECL4 is hypervariable, and replacement mutations carrying an intact ECL3 site alter but do not abolish receptor activity, including replacement of the entire loop with that of a jellyfish (Cnidaria) XPR1. Because ECL4 deletions are found in all X-MLV-infected Mus subspecies, we deleted each ECL4 residue to determine if deletion-associated restriction is residue-specific or is effected by loop size. All deletions influence receptor function, although different deletions affect different XP-MLVs. Thus, receptor usage of a constrained splice site and a loop that tolerates mutations severely limits the likelihood of host escape mutations.

The compound immunodeficiencies in nonobese diabetic (NOD) inbred mice homozygous for the Prkdc(scid) and Il2rg(null) alleles (NSG mice) permit engraftment of a wide-range of primary human cells, enabling sophisticated modeling of human disease. In studies designed to define neoplastic stem cells of primary myelofibrosis (PMF), a myeloproliferative neoplasm characterized by profound disruption of the hematopoietic microenvironment, we observed a high frequency of acute myeloid leukemia (AML) in NSG mice. AML was of mouse origin, confined to PMF-xenografted mice, and contained multiple clonal integrations of ecotropic murine leukemia virus (E-MuLV). Significantly, MuLV replication was not only observed in diseased mice, but also in nontreated NSG controls. Furthermore, in addition to the single ecotropic endogenous retrovirus (eERV) located on chromosome 11 (Emv30) in the NOD genome, multiple de novo germ-line eERV integrations were observed in mice from each of four independent NSG mouse colonies. Analysis confirmed that E-MuLV originated from the Emv30 provirus and that recombination events were not necessary for virus replication or AML induction. Pathogenicity is thus likely attributable to PMF-mediated paracrine stimulation of mouse myeloid cells, which serve as targets for retroviral infection and transformation, as evidenced by integration into the Evi1 locus, a hotspot for retroviral-induced myeloid leukemia. This study thus corroborates a role of paracrine stimulation in PMF disease progression, underlines the importance of target cell type and numbers in MuLV-induced disease, and mandates awareness of replicating MuLV in NOD immunodeficient mice, which can significantly influence experimental results and their interpretation.

Murine leukemia virus (MLV) integrase (IN) lacking the C-terminal tail peptide (TP) loses its interaction with the host bromodomain and extraterminal (BET) proteins and displays decreased integration at promoter/enhancers and transcriptional start sites/CpG islands. MLV lacking the IN TP via an altered open reading frame was used to infect tumorigenesis mouse model (MYC/Runx2) animals to observe integration patterns and phenotypic effects, but viral passage resulted in the restoration of the IN TP through small deletions. Mice subsequently infected with an MLV IN lacking the TP coding sequence (TP-) showed an improved median survival by 15 days compared to wild type (WT) MLV infection. Recombination with polytropic endogenous retrovirus (ERV), Pmv20, was identified in seven mice displaying both fast and slow tumorigenesis, highlighting the strong selection within the mouse to maintain the full-length IN protein. Mapping the genomic locations of MLV in tumors from an infected mouse with no observed recombination with ERVs, TP-16, showed fewer integrations at TSS and CpG islands, compared to integrations observed in WT tumors. However, this mouse succumbed to the tumor in relatively rapid fashion (34 days). Analysis of the top copy number integrants in the TP-16 tumor revealed their proximity to known MLV common insertion site genes while maintaining the MLV IN TP- genotype. Furthermore, integration mapping in K562 cells revealed an insertion preference of MLV IN TP- within chromatin profile states associated with weakly transcribed heterochromatin with fewer integrations at histone marks associated with BET proteins (H3K4me1/2/3, and H3K27Ac). While MLV IN TP- showed a decreased overall rate of tumorigenesis compared to WT virus in the MYC/Runx2 model, MLV integration still occurred at regions associated with oncogenic driver genes independently from the influence of BET proteins, either stochastically or through trans-complementation by functional endogenous Gag-Pol protein., Author summary Many different retroviruses, including murine leukemia virus (MLV), are used as vectors for human gene therapy and cancer immunotherapies (CAR-T) because of their stable and efficient delivery of genetic material into the host DNA. However, this process can result in activation and/or disruption of cellular genes, which has resulted in the outgrowth of tumors in previous clinical trials. Of critical importance is the preferred location within the host genome at which the retrovirus integrates. Our study presents a modified MLV virus that has lost the ability to bind to the host BET proteins, the drivers of insertion at promoter/enhancer regions of highly activated genes. This is the first direct study within an animal model to examine whether infection with this type of modified MLV virus affects tumor progression. We found that the modified virus improved the survival of the well-characterized MYC/Runx2 mouse compared to infections with the wild-type MLV. Most modified viral integrants mapped into less active regions of the genome, but some integration events still occurred near cancer-related genes. Thus, while the modified MLV virus can be a safer vector than the wildtype virus, it still maintains the potential to activate oncogenes. This study provides new insights on how to improve the safety of MLV retroviral vectors.

Laboratory mice carry three host range groups of gammaretroviruses all of which are linked to leukemia induction. Although polytropic mouse leukemia viruses (P-MLVs) are generally recognized as the proximate cause of MLV-induced leukemias in laboratory mice, wild mice that carry only endogenous P-MLVs do not produce infectious virus and are not prone to disease; these mice carry the permissive XPR1 retroviral receptor and an attenuated variant of the retroviral restriction factor, APOBEC3. In contrast, Eurasian mice carrying ecotropic and xenotropic MLVs have evolved multiple restrictive XPR1 variants, other factors that interfere with MLV entry, and more effectively antiviral variants of APOBEC3. These different antiviral restrictions in Mus musculus subspecies suggest that the different virus types found in these natural populations may pose different but largely uncharacterized survival risks in their host subspecies.

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.

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 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 Xpr1 n 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.

Xenotropic/polytropic mouse leukemia viruses (X/P-MLVs) use the XPR1 gammaretrovirus receptor for entry. X/P-MLV host range is defined by usage of naturally occurring restrictive XPR1 receptors, and is governed by polymorphisms in the virus envelope glycoprotein and in XPR1. Here, we examined receptors of four mammalian species permissive to all X/P-MLVs (Mus dunni, human, rabbit, mink). Interference assays showed the four to be functionally distinct. Preinfection with X-MLVs consistently blocked all nine XPR1-dependent viruses, while preinfection with P-MLVs and wild mouse X/P-MLVs produced distinctive interference patterns in the four cells. These patterns indicate shared usage of independent, but not always fully functional, receptor sites. XPR1 sequence comparisons identified candidate sites in receptor-determining regions that correlate with some interference patterns. The evolutionary record suggests that the X/P-MLV tropism variants evolved to adapt to host receptor polymorphisms, to circumvent blocks by competing viruses or to avoid host-encoded envelope glycoproteins acquired for defense.

Spl574 MLV (murine leukemia virus) is a variant of Moloney ecotropic MLV (MoMLV) that is cytopathic in Mus dunni cells and restricted by other mouse cells. Its host range and cytopathicity are due to a mutation, S82F, at a site critical for binding to the CAT-1 receptor. To identify residues that affect affinity for receptor variants, virus with S82F was passed in restrictive cells. The env genes of the adapted viruses contained 18 novel mutations, including one, E114G, present in 6 of 30 sequenced envs. MoMLV-E114G efficiently infected all mouse cells as well as ecotropic MLV resistant Chinese hamster cells. Virus with E114G and S82F induced large multinucleated syncytia in NIH 3T3 and SC-1 cells as well as M. dunni cells. Inoculation of Mo-S82F,E114G into mice produced lymphomas typical of MoMLV. Residues at env position 114 are thus important determinants of host range, and E114G suppresses host range restriction due to S82F, but does not affect S82F-governed cytopathicity.

Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIH 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.

The Fv1 virus resistance gene is a coopted endogenous retrovirus (ERV) sequence related to the gag gene of the MuERV-L ERV family. Three major Fv1 resistance alleles have been identified in laboratory mice, and they target virus capsid genes to produce characteristic patterns of resistance to mouse leukemia viruses (MLVs). We identified Fv1 in 3 of the 4 Mus subgenera; its absence from Coelomys and 1 of 3 species of Pyromys indicate Fv1 was acquired shortly after the origin of the Mus genus. We sequenced Fv1 genes from 21 mice representative of the major taxonomic groups of Mus . Two lines of evidence indicate that Fv1 has had antiviral function for 7 million years of evolution. First, 2 species of African pygmy mice (subgenus Nannomys ) show an Fv1 -like MLV resistance, and transduced cells expressing the Nannomys Fv1 gene reproduce this resistance pattern. Second, sequence comparisons suggest that Fv1 has been involved in genetic conflicts throughout Mus evolution. We found evidence for strong positive selection of Fv1 and identified 6 codons that show evidence of positive selection: 3 codons in the C-terminal region including 2 previously shown to contribute to Fv1 restriction in laboratory mice, and 3 codons in a 10-codon segment overlapping the major homology region of Fv1 ; this segment is known to be involved in capsid multimerization. This analysis suggests that Fv1 has had an antiviral role throughout Mus evolution predating exposure of mice to the MLVs restricted by laboratory mouse Fv1 , and suggests a mechanism for Fv1 restriction.

Mouse leukemia viruses (MLVs) are found in the common inbred strains of laboratory mice and in the house mouse subspecies of Mus musculus . Receptor usage and envelope ( env ) sequence variation define three MLV host range subgroups in laboratory mice: ecotropic, polytropic, and xenotropic MLVs (E-, P-, and X-MLVs, respectively). These exogenous MLVs derive from endogenous retroviruses (ERVs) that were acquired by the wild mouse progenitors of laboratory mice about 1 million years ago. We analyzed the genomes of seven MLVs isolated from Eurasian and American wild mice and three previously sequenced MLVs to describe their relationships and identify their possible ERV progenitors. The phylogenetic tree based on the receptor-determining regions of env produced expected host range clusters, but these clusters are not maintained in trees generated from other virus regions. Colinear alignments of the viral genomes identified segmental homologies to ERVs of different host range subgroups. Six MLVs show close relationships to a small xenotropic ERV subgroup largely confined to the inbred mouse Y chromosome. env variations define three E-MLV subtypes, one of which carries duplications of various sizes, sequences, and locations in the proline-rich region of env . Outside the env region, all E-MLVs are related to different nonecotropic MLVs. These results document the diversity in gammaretroviruses isolated from globally distributed Mus subspecies, provide insight into their origins and relationships, and indicate that recombination has had an important role in the evolution of these mutagenic and pathogenic agents. IMPORTANCE Laboratory mice carry mouse leukemia viruses (MLVs) of three host range groups which were acquired from their wild mouse progenitors. We sequenced the complete genomes of seven infectious MLVs isolated from geographically separated Eurasian and American wild mice and compared them with endogenous germ line retroviruses (ERVs) acquired early in house mouse evolution. We did this because the laboratory mouse viruses derive directly from specific ERVs or arise by recombination between different ERVs. The six distinctively different wild mouse viruses appear to be recombinants, often involving different host range subgroups, and most are related to a distinctive, largely Y-chromosome-linked MLV ERV subtype. MLVs with ecotropic host ranges show the greatest variability with extensive inter- and intrasubtype envelope differences and with homologies to other host range subgroups outside the envelope. The sequence diversity among these wild mouse isolates helps define their relationships and origins and emphasizes the importance of recombination in their evolution.

Cells from the Asian wild mouse species Mus castaneus are resistant to infection by the polytropic host range group of mouse gammaretroviruses. Two factors are responsible for this resistance: a defective XPR1 cell surface receptor for polytropic murine leukemia viruses (P-MLVs), and a resistance factor detectable only in interspecies hybrids between M. castaneus and mice with an XPR1 variant that permits infection by xenotropic MLVs (X-MLVs) as well as P-MLVs. This second novel virus resistance phenotype has been associated with expression of viral Env glycoprotein; Northern blotting with specific hybridization probes identified a spliced X-MLV env message unique to virus-resistant mice. These observations suggest that resistance is due to expression of one or more endogenous X-MLV envelope genes that interfere with infection by exogenous P-MLVs. M. castaneus contains multiple X-MLV proviruses, but serial backcrosses reduced this proviral content and permitted identification of a single proviral env sequence inherited with resistance. The resistance phenotype and the provirus were mapped to the same site on distal chromosome 18. The provirus was shown to be a full-length provirus highly homologous to previously described X-MLVs. Use of viral pseudotypes confirmed that this resistance gene, termed Rmcf2 , prevents entry of P-MLVs. Rmcf2 resembles the virus resistance genes Fv4 and Rmcf in that it produces Env glycoprotein but fails to produce infectious virus; the proviruses associated with all three resistance genes have fatal defects. This type of provirus Env-mediated resistance represents an important defense mechanism in wild mouse populations exposed to endemic infections.

A variant ecotropic Friend murine leukemia virus, F-S MLV, is capable of inducing the formation of large multinucleated syncytia in Mus dunni cells. This cytopathicity resembles that of Spl574 MLV, a novel variant recently isolated from the spleen of a Mus spicilegus mouse neonatally inoculated with Moloney MLV. F-S MLV is an N-tropic Friend MLV that also has the unusual ability to infect hamster cells, which are normally resistant to mouse ecotropic MLVs. Syncytium induction by both F-S MLV and Spl574 is accompanied by the accumulation of large amounts of unintegrated viral DNA, a hallmark of pathogenic retroviruses, but not previously reported for mouse ecotropic gammaretroviruses. Sequencing and site-specific mutagenesis determined that the syncytium-inducing phenotype of F-S MLV can be attributed to a single amino acid substitution (S84A) in the VRA region of the viral env gene. This site corresponds to that of the single substitution previously shown to be responsible for the cytopathicity of Spl574, S82F. The S84A substitution in F-S MLV also contributes to the ability of this virus to infect hamster cells, but Spl574 MLV is unable to infect hamster cells. Because this serine residue is one of the critical amino acids that form the CAT-1 receptor binding site, and because M. dunni and hamster cells have variant CAT-1 receptors, these results suggest that syncytium formation as well as altered host range may be a consequence of altered interaction between virus and receptor.

A novel member of the Krox family of proteins, designated Krox-25, was identified by screening clones from the cDNA libraries of a rat incisor and mouse embryo craniofacial tissue. Rat and mouse Krox-25 mRNAs are about 2.4 kb long, encoding 225 and 224 amino acids, respectively. Krox-25 consists of five zinc finger motifs homologous to the Drosophila Krüppel segmentation gene and also contains several consensus amino acid sequences for a protein kinase C binding domain. Northern blot analysis revealed an intense expression of Krox-25 mRNA in rat and mouse teeth, although it was expressed weakly in other tissues, including calvaria, brain, lung, thymus, kidney, and submandibular gland of mouse. In situ hybridization showed that Krox-25 mRNA began to be expressed weakly in the early odontogenic mesenchyme and primitive enamel epithelium located at the apical end of the rat incisor, and Krox-25 expression increased in the presecretory ameloblasts and became intense in the secretory ameloblasts. This expression was also similar to the results of immunohistochemistry and Western blot, especially the Krox-25 localization in the nuclei of enamel epithelial cells. These results suggest that Krox-25 plays an important role as a transcription factor for the cytodifferentiation and amelogenesis of enamel epithelium.

Mus spicilegus is an Eastern European wild mouse species that has previously been reported to harbor an unusual infectious ecotropic murine leukemia virus (MLV) and proviral envelope genes of a novel MLV subgroup. In the present study, M. spicilegus neonates were inoculated with Moloney ecotropic MLV (MoMLV). All 17 inoculated mice produced infectious ecotropic virus after 8 to 14 weeks, and two unusual phenotypes distinguished the isolates from MoMLV. First, most of the M. spicilegus isolates grew to equal titers on M. dunni and SC-1 cells, although MoMLV does not efficiently infect M. dunni cells. The deduced amino acid sequence of a representative clone differed from MoMLV by insertion of two serine residues within the VRA of SU env . Modification of a molecular clone of MoMLV by the addition of these serines produced a virus that grows to high titer in M. dunni cells, establishing a role for these two serine residues in host range. A second unusual phenotype was found in only one of the M. spicilegus isolates, Spl574. Spl574 produces large syncytia of multinucleated giant cells in M. dunni cells, but its replication is restricted in other mouse cell lines. Sequencing and mutagenesis demonstrated that syncytium formation could be attributed to a single amino acid substitution within VRA, S82F. Thus, viruses with altered growth properties are selected during growth in M. spicilegus . The mutations associated with the host range and syncytium-inducing variants map to a key region of VRA known to govern interactions with the cell surface receptor, suggesting that the associated phenotypes may result from altered interactions with the unusual ecotropic virus mCAT1 receptor carried by M. dunni .

The Graffi murine leukemia virus is a nondefective ecotropic retrovirus that was originally reported to induce myeloid leukemia in some strains of mice (A. Graffi, Ann. N.Y. Acad. Sci. 68: 540-558, 1957). Using provirus-flanking sequences as DNA probes, we identified a new common retroviral integration site called Gris1 (for Graffi integration site 1). Viral integrations in Gris1 were detected in 13% of the tumors analyzed. The Gris1 locus was mapped to the distal region of mouse chromosome 6, 85 kb upstream of the cyclin D2 gene. Such viral integration in Gris1 causes overexpression of the normal 6.5-kb major transcript of cyclin D2 but also induces the expression of a new, alternatively spliced 1.1-kb transcript from the cyclin D2 gene that encodes a truncated cyclin D2 of 17 kDa. The expression of this 1.1-kb transcript is specific to tumors in which Gris1 is rearranged but is also detected at low levels in normal tissue.

Many studies have explored the premature aging of accelerated senescence-prone (SAMP8) mice. However, the cause of premature aging in this strain remains unknown. We analyzed the expression of ecotropic, xenotropic, and polytropic murine leukemia viruses (MuLVs) in the brains of accelerated senescence-resistant (SAMR1) and SAMP8 mice. No ecotropic mRNA was detected in SAMR1 mice, and only Akv-type ecotropic MuLV mRNA was detected in SAMP8 mice. Restriction mapping of the full-length infectious E-MuLV genome from SAMP8 confirmed its identity as Akv. mRNAs corresponding to a prototypical polytropic MuLV and to an unusual xenotropic MuLV were detected at equal levels in SAMP8 and SAMR1 mice, but no infectious virus of either host range type was detected. In order to determine the cellular localization of Akv expression in SAMP8 mice, we used immunohistochemistry and electron microscopy to detect expression of the E-MuLV capsid gag (CA gag ) gene in striatum, brainstem, hippocampus, and cerebellum of 12-month-old SAMR1 and SAMP8 mice. The CA gag antigen was seen in the neurons, oligodendroglia, and vascular endothelium of these brain regions of SAMP8 mice, but not in SAMR1 mice. To evaluate the correlation between activation of astrocytes and expression of Akv, we performed double-immunohistochemical staining for both glial fibrillary acidic protein (GFAP) and CA gag in SAMR1 and SAMP8 mice. Strong astrocytic activation and extensive vacuolation were observed around CA gag -positive neurons in SAMP8 mice, whereas in SAMR1 mice neither astrocytosis nor vacuolation were present. CA gag antigen was also localized in astrocytes of the hippocampus region of SAMP8 mice. Electron micrography showed that a number of vacuoles were found in the cytoplasm of MuLV-positive neurons and the extracellular space surrounding these neurons showed lytic changes. These results suggest that endogenous Akv provirus is expressed in neurons, astrocytes, vascular endothelium, and oligodendroglia in the brains of SAMP8 and that this virus could play an important role in the brain aging processes in this mouse strain.

Type XI collagen is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the -530-bp promoter segment of the alpha 2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Kruppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB-zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.

Mice contain a serum factor capable of inactivating some subgroups of murine leukemia viruses. This leukemia virus-inactivating factor (LVIF) is distinct from immunoglobulin and complement; it has been associated with lipoprotein serum fractions and may be an apolipoprotein. The present study demonstrates that some Swiss-derived inbred strains are LVIF negative. Genetic crosses show this factor to be under control of a single gene that maps to distal chromosome 10 at or near the gene encoding a minor serum apolipoprotein, apolipoprotein F (ApoF). To evaluate this gene as a potential candidate for LVIF, the mouse ApoF gene was cloned and sequenced and its expression was assessed in LVIF-positive and -negative mice; no obvious differences were detected, suggesting that LVIF is under the control of a distinct linked gene.

The regulation of gene expression in thymic epithelial cells is critical for T cell development. The mouse thymic epithelial gene Tscot encodes a protein with weak homology to bacterial 12 transmembrane co-transporters. Using competitive reverse transcription-polymerase chain reaction (RT-PCR), we show that low level Tscot expression is detectable in several other tissues. Tscot was mapped to chromosome 4 and was also detected in other mammalian species by Southern blotting. The human cDNA clone showed 77% amino acid identity with the mouse sequence. The highest conservation was in the TM regions and in a small segment of the central cytoplasmic loop. Genomic clones spanning 17 164 bases of the Tscot gene revealed four exons with nine of the TM domains encoded in the first exon. The major transcriptional start site in mouse was identified by a primer extension analysis and confirmed by RT-PCR. Comparison of 1.7 kb of the human and mouse promoters identified six conserved possible regulatory elements, one containing a potential binding site for an interferon α inducible factor. Finally, as a functional test, 3 kb of the murine promoter was used to create a transgenic mouse that expresses enhanced green fluorescent protein message strongly in the thymus, weakly in the kidney and undetectably in the spleen, liver and heart.

The rd7 mouse, an animal model for hereditary retinal degeneration, has some characteristics similar to human flecked retinal disorders. Here we report the identification of a deletion in a photoreceptor-specific nuclear receptor (mPNR) mRNA that is responsible for hereditary retinal dysplasia and degeneration in the rd7 mouse. mPNR was isolated from a pool of photoreceptor-specific cDNAs originally created by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. Localization of the gene corresponding to mPNR to mouse Chr 9 near the rd7 locus made it a candidate for the site of the rd7 mutation. Northern analysis of total RNA isolated from rd7 mouse retinas revealed no detectable signal after hybridization with the mPNR cDNA probe. However, with reverse transcription–PCR, we were able to amplify different fragments of mPNR from rd7 retinal RNA and to sequence them directly. We found a 380-nt deletion in the coding region of the rd7 mPNR message that creates a frame shift and produces a premature stop codon. This deletion accounts for more than 32% of the normal protein and eliminates a portion of the DNA-binding domain. In addition, it may result in the rapid degradation of the rd7 mPNR message by the nonsense-mediated decay pathway, preventing the synthesis of the corresponding protein. Our findings demonstrate that mPNR expression is critical for the normal development and function of the photoreceptor cells.

The BL/VL(3) Kaplan radiation leukemia virus (RadLV-VL(3)) is a nondefective retrovirus that induces T cell lymphomas in several strains of mice. By using DNA probes derived from RadLV/VL(3) provirus-flanking sequences cloned from the BL/VL(3) cell line, we identified a DNA region rearranged in 5 of 19 tumors analysed (25%). All proviruses were integrated in the same 5'-to-3' orientation in a small DNA region called Kis1 (Kaplan integration site 1). This region was localized on distal mouse chromosome 2 in a region not previously identified as important to lymphomagenesis. The cells rearranged at the Kis1 locus represent a clonal subpopulation of the clonal tumor masses examined, indicating a probable role of Kis1 in tumor progression.

The mammalian Daxx gene has been identified in a diverse set of yeast interaction trap experiments. Although a facilitating role for Daxx in Fas-induced apoptosis has been suggested, Daxx's physiologic function remains unknown. To elucidate the in vivo role of Daxx, we have generated Daxx-deficient mice. Surprisingly, rather than a hyperproliferative disorder expected from the loss of a pro-apoptotic gene, mutation of Daxx results in extensive apoptosis and embryonic lethality. These findings argue against a role for Daxx in promoting Fas-induced cell death and suggest that Daxx either directly or indirectly suppresses apoptosis in the early embryo.

Retinitis pigmentosa (RP) represents a group of inherited human retinal diseases which involve degeneration of photoreceptor cells resulting in visual loss and often leading to blindness. In order to identify candidate genes for the causes of these diseases, we have been studying a pool of photoreceptor-specific cDNAs isolated by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. One of these cDNAs was of interest because it mapped to proximal mouse chromosome 1 in a region homo-logous to human 8q11-q13, the locus of autosomal dominant RP1. Therefore, using the mouse cDNA as probe, we cloned the human cDNA (hG28) and its corresponding gene and mapped it near to D8S509, which lies in the RP1 locus. This gene consists of four exons with an open reading frame of 6468 nt encoding a protein of 2156 amino acids with a predicted mass of 240 kDa. Given its chromosomal localization, we screened this gene for mutations in a large family affected with autosomal dominant RP previously linked to the RP1 locus. We found an R677X mutation that co-segregated with disease in the family and is absent from unaffected members and 100 unrelated controls. This mutation is predicted to lead to rapid degradation of hG28 mRNA or to the synthesis of a truncated protein lacking approximately 70% of its original length. Our results suggest that R677X is responsible for disease in this family and that the gene corresponding to hG28 is the RP1 gene.

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed differentially in testis was isolated from mouse and human. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, including an EF, a Ca2+ binding motif, and a central coiled-coil structure, as well as a nucleotide binding consensus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was designated EHD1 (EH domain-containing 1). In cells in tissue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, endocytic vesicles. Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes, and in specific retinal layers. Results of in situ hybridization to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches and at day 10.5 in sclerotomes, at various elements of the branchial apparatus (mandible and hyoid), and in the occipital region. At day 15.5 EHD1 expression peaked in cartilage, preceding hypertrophy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly conserved between nematode, Drosophila, mouse, and human. Its predicted protein structure and cellular localization point to the possibility that EHD1 participates in ligand-induced endocytosis.

A new mouse connexin gene has been isolated that codes for a connexin protein of 505 amino acid residues. Based on the predicted molecular mass of 57.115 kDa, it has been designated connexin-57. Similar to most other mouse connexin genes, the coding region of connexin-57 is not interrupted by introns and exists in the mouse genome as a single-copy gene. Within the connexin family, this new gene shows highest sequence identity to porcine connexin-60 in the alpha group of connexins. The connexin-57 gene was mapped to a position on mouse chromosome 4, 30 centimorgans proximal to a cluster of previously mapped connexin genes. Low levels of connexin-57 mRNA were detected in skin, heart, kidney, testis, ovary, intestine, and in the mouse embryo after 8 days post coitum, but expression was not detected in brain, sciatic nerve or liver. In order to analyze gene function, the connexin-57 coding region was expressed by transfection in human HeLa cells, where it restored homotypic intercellular transfer of microinjected neurobiotin. Heterotypic transfer was observed between HeLa connexin-57 transfectants and HeLa cells, expressing murine connexin-43, -37, or -30.3. Double whole-cell voltage clamp analyses revealed that HeLa-connexin-57 transfectants expressed about 10 times more channels than parental HeLa cells. Voltage gating by transjunctional and transmembrane voltages as well as unitary conductance ( approximately 27 picosiemens) were different from intrinsic connexin channels in parental HeLa cells.

A genomic clone encoding the mouse Hsc70 gene has been isolated and characterized by DNA sequence analysis. The gene is approximately 3. 9 kb in length and contains eight introns, the fifth, sixth and eighth of which encode the three U14 snoRNAs. The gene has been located on Chr 9 in the order Fli1-Itm1-Olfr7-Hsc70(Rnu14)-Cbl by genetic analysis. Expression of Hsc70 is universal in all tissues of the mouse, but is slightly elevated in liver, skeletal muscle and kidney tissue, while being depressed in testes. In cultured mouse NIH 3T3 cells or human HeLa cells, Hsc70 mRNA levels are low under normal conditions, but can be induced 8-fold higher in both lines by treatment with the amino acid analog azetidine. A similar induction is seen in cells treated with the proteosome inhibitor MG132 suggesting that elevated Hsc70 expression may be coupled to protein degradation. Surprisingly, expression of the human Hsc70 gene is also regulated by cell-cycle position being 8-10-fold higher in late G1/S-phase cells as opposed to the levels in early G1-phase cells.

Retinal photoreceptor cells are particularly vulnerable to degenerations that can eventually lead to blindness. Our purpose is to identify and characterize genes expressed specifically in photoreceptors in order to increase our understanding of the biochemistry and function of these cells, and then to use these genes as candidates for the sites of mutations responsible for degenerative retinal diseases. We have characterized a cDNA, a fragment of which (SR3.1) was originally isolated by subtractive hybridization of adult, photoreceptorless rd mouse retinal cDNAs from the cDNAs of normal mouse retina. The full-length sequence of this cDNA was determined from clones obtained by screening mouse retinal and eye cDNA libraries and by using the 5'- and 3'-RACE methods. Both Northern blot analysis and in situ hybridization showed that the corresponding mRNA is expressed in rod and cone photoreceptors. The gene encoding this cDNA was mapped to the X chromosome using an interspecific cross. Based on the nucleotide and amino acid sequences, as well as chromosome mapping, we determined that this gene is the mouse ortholog (Xlrs1) of the human X-linked juvenile retinoschisis gene (XLRS1). Analysis of the predicted amino acid sequence indicates that the Xlrs1 mRNA may encode a secretable, adhesion protein. Therefore, our data suggest that X-linked juvenile retinoschisis originates from abnormalities in a photoreceptor-derived adhesion protein.