FeLIX is a restriction factor for mammalian retrovirus infection.

Endogenous retroviruses (ERVs) are remnants of ancestral viral infections. Feline leukemia virus (FeLV) is an exogenous and endogenous retrovirus in domestic cats. It is classified into several subgroups (A, B, C, D, E, and T) based on viral receptor interference properties or receptor usage. ERV-derived molecules benefit animals, conferring resistance to infectious diseases. However, the soluble protein encoded by the defective envelope (env) gene of endogenous FeLV (enFeLV) functions as a co-factor in FeLV subgroup T infections. Therefore, whether the gene emerged to facilitate viral infection is unclear. Based on the properties of ERV-derived molecules, we hypothesized that the defective env genes possess antiviral activity that would be advantageous to the host because FeLV subgroup B (FeLV-B), a recombinant virus derived from enFeLV env, is restricted to viral transmission among domestic cats. When soluble truncated Env proteins from enFeLV were tested for their inhibitory effects against enFeLV and FeLV-B, they inhibited viral infection. Notably, this antiviral machinery was extended to infection with the Gibbon ape leukemia virus, Koala retrovirus A, and Hervey pteropid gammaretrovirus. Although these viruses used feline phosphate transporter 1 (fePit1) and phosphate transporter 2 as receptors, the inhibitory mechanism involved competitive receptor binding in a fePit1-dependent manner. The shift in receptor usage might have occurred to avoid the inhibitory effect. Overall, these findings highlight the possible emergence of soluble truncated Env proteins from enFeLV as a restriction factor against retroviral infection and will help in developing host immunity and antiviral defense by controlling retroviral spread.IMPORTANCERetroviruses are unique in using reverse transcriptase to convert RNA genomes into DNA, infecting germ cells, and transmitting to offspring. Numerous ancient retroviral sequences are known as endogenous retroviruses (ERVs). The soluble Env protein derived from ERVs functions as a co-factor that assists in FeLV-T infection. However, herein, we show that the soluble Env protein exhibits antiviral activity and provides resistance to mammalian retrovirus infection through competitive receptor binding. In particular, this finding may explain why FeLV-B transmission is not observed among domestic cats. ERV-derived molecules can benefit animals in an evolutionary arms race, highlighting the double-edged-sword nature of ERVs.

Multiple recombination events between endogenous retroviral elements and feline leukemia virus.

Feline leukemia virus (FeLV) is an exogenous retrovirus that causes malignant hematopoietic disorders in domestic cats, and its virulence may be closely associated with viral sequences. FeLV is classified into several subgroups, including A, B, C, D, E, and T, based on viral receptor interference properties or receptor usage. However, the transmission manner and disease specificity of the recombinant viruses FeLV-D and FeLV-B remain unclear. The aim of this study was to understand recombination events between exogenous and endogenous retroviruses within a host and elucidate the emergence and transmission of recombinant viruses. We observed multiple recombination events involving endogenous retroviruses (ERVs) in FeLV from a family of domestic cats kept in one house; two of these cats (ON-T and ON-C) presented with lymphoma and leukemia, respectively. Clonal integration of FeLV-D was observed in the ON-T case, suggesting an association with FeLV-D pathogenesis. Notably, the receptor usage of FeLV-B observed in ON-T was mediated by feline Pit1 and feline Pit2, whereas only feline Pit1 was used in ON-C. Furthermore, XR-FeLV, a recombinant FeLV containing an unrelated sequence referred to the X-region, which is homologous to a portion of the 5'-leader sequence of Felis catus endogenous gammaretrovirus 4 (FcERV-gamma4), was isolated. Genetic analysis suggested that most recombinant viruses occurred de novo; however, the possibility of FeLV-B transmission was also recognized in the family. This study demonstrated the occurrence of multiple recombination events between exogenous and endogenous retroviruses in domestic cats, highlighting the contribution of ERVs to pathogenic recombinant viruses.IMPORTANCEFeline leukemia virus subgroup A (FeLV-A) is primarily transmitted among cats. During viral transmission, genetic changes in the viral genome lead to the emergence of novel FeLV subgroups or variants with altered virulence. We isolated three FeLV subgroups (A, B, and D) and XR-FeLV from two cats and identified multiple recombination events in feline endogenous retroviruses (ERVs), such as enFeLV, ERV-DC, and FcERV-gamma4, which are present in the cat genome. This study highlights the pathogenic contribution of ERVs in the emergence of FeLV-B, FeLV-D, and XR-FeLV in a feline population.

Feline Foamy Virus Transmission in Tsushima Leopard Cats (Prionailurus bengalensis euptilurus) on Tsushima Island, Japan.

Tsushima leopard cats (TLC; Prionailurus bengalensis euptilurus) only inhabit Tsushima Island, Nagasaki, Japan and are critically endangered and threatened by infectious diseases. The feline foamy virus (FFV) is widely endemic in domestic cats. Therefore, its transmission from domestic cats to TLCs may threaten the TLC population. Thus, this study aimed to assess the possibility that domestic cats could transmit FFV to TLCs. Eighty-nine TLC samples were screened, and FFV was identified in seven (7.86%). To assess the FFV infection status of domestic cats, 199 domestic cats were screened; 14.07% were infected. The phylogenetic analysis revealed that the FFV partial sequence from domestic cats and TLC sequences clustered in one clade, suggesting that the two populations share the same strain. The statistical data minimally supported the association between increased infection rate and sex (p = 0.28), indicating that FFV transmission is not sex dependent. In domestic cats, a significant difference was observed in FFV detection in feline immunodeficiency virus (p = 0.002) and gammaherpesvirus1 infection statuses (p = 0.0001) but not in feline leukemia virus infection status (p = 0.21). Monitoring FFV infection in domestic cats and TLC populations is highly recommended as part of TLC surveillance and management strategies.

Bioinformatics approaches for unveiling virus-host interactions.

The coronavirus disease-2019 (COVID-19) pandemic has elucidated major limitations in the capacity of medical and research institutions to appropriately manage emerging infectious diseases. We can improve our understanding of infectious diseases by unveiling virus-host interactions through host range prediction and protein-protein interaction prediction. Although many algorithms have been developed to predict virus-host interactions, numerous issues remain to be solved, and the entire network remains veiled. In this review, we comprehensively surveyed algorithms used to predict virus-host interactions. We also discuss the current challenges, such as dataset biases toward highly pathogenic viruses, and the potential solutions. The complete prediction of virus-host interactions remains difficult; however, bioinformatics can contribute to progress in research on infectious diseases and human health.

Large-scale investigation of zoonotic viruses in the era of high-throughput sequencing.

Zoonotic diseases considerably impact public health and socioeconomics. RNA viruses reportedly caused approximately 94% of zoonotic diseases documented from 1990 to 2010, emphasizing the importance of investigating RNA viruses in animals. Furthermore, it has been estimated that hundreds of thousands of animal viruses capable of infecting humans are yet to be discovered, warning against the inadequacy of our understanding of viral diversity. High-throughput sequencing (HTS) has enabled the identification of viral infections with relatively little bias. Viral searches using both symptomatic and asymptomatic animal samples by HTS have revealed hidden viral infections. This review introduces the history of viral searches using HTS, current analytical limitations, and future potentials. We primarily summarize recent research on large-scale investigations on viral infections reusing HTS data from public databases. Furthermore, considering the accumulation of uncultivated viruses, we discuss current studies and challenges for connecting viral sequences to their phenotypes using various approaches: performing data analysis, developing predictive modeling, or implementing high-throughput platforms of virological experiments. We believe that this article provides a future direction in large-scale investigations of potential zoonotic viruses using the HTS technology.

Borna Disease Virus 1 Phosphoprotein Forms a Tetramer and Interacts with Host Factors Involved in DNA Double-Strand Break Repair and mRNA Processing.

Determining the structural organisation of viral replication complexes and unravelling the impact of infection on cellular homeostasis represent important challenges in virology. This may prove particularly useful when confronted with viruses that pose a significant threat to human health, that appear unique within their family, or for which knowledge is scarce. Among Mononegavirales, bornaviruses (family Bornaviridae) stand out due to their compact genomes and their nuclear localisation for replication. The recent recognition of the zoonotic potential of several orthobornaviruses has sparked a surge of interest in improving our knowledge on this viral family. In this work, we provide a complete analysis of the structural organisation of Borna disease virus 1 (BoDV-1) phosphoprotein (P), an important cofactor for polymerase activity. Using X-ray diffusion and diffraction experiments, we revealed that BoDV-1 P adopts a long coiled-coil α-helical structure split into two parts by an original ß-strand twist motif, which is highly conserved across the members of whole Orthobornavirus genus and may regulate viral replication. In parallel, we used BioID to determine the proximal interactome of P in living cells. We confirmed previously known interactors and identified novel proteins linked to several biological processes such as DNA repair or mRNA metabolism. Altogether, our study provides important structure/function cues, which may improve our understanding of BoDV-1 pathogenesis.

Squalomix: shark and ray genome analysis consortium and its data sharing platform.

The taxon Elasmobranchii (sharks and rays) contains one of the long-established evolutionary lineages of vertebrates with a tantalizing collection of species occupying critical aquatic habitats. To overcome the current limitation in molecular resources, we launched the Squalomix Consortium in 2020 to promote a genome-wide array of molecular approaches, specifically targeting shark and ray species. Among the various bottlenecks in working with elasmobranchs are their elusiveness and low fecundity as well as the large and highly repetitive genomes. Their peculiar body fluid composition has also hindered the establishment of methods to perform routine cell culturing required for their karyotyping. In the Squalomix consortium, these obstacles are expected to be solved through a combination of in-house cytological techniques including karyotyping of cultured cells, chromatin preparation for Hi-C data acquisition, and high fidelity long-read sequencing. The resources and products obtained in this consortium, including genome and transcriptome sequences, a genome browser powered by JBrowse2 to visualize sequence alignments, and comprehensive matrices of gene expression profiles for selected species are accessible through https://github.com/Squalomix/info.

Convergent evolution of antiviral machinery derived from endogenous retrovirus truncated envelope genes in multiple species.

Host genetic resistance to viral infection controls the pathogenicity and epidemic dynamics of infectious diseases. Refrex-1 is a restriction factor against feline leukemia virus subgroup D (FeLV-D) and an endogenous retrovirus (ERV) in domestic cats (ERV-DC). Refrex-1 is encoded by a subset of ERV-DC loci with truncated envelope genes and secreted from cells as a soluble protein. Here, we identified the copper transporter CTR1 as the entry receptor for FeLV-D and genotype I ERV-DCs. We also identified CTR1 as a receptor for primate ERVs from crab-eating macaques and rhesus macaques, which were found in a search of intact envelope genes capable of forming infectious viruses. Refrex-1 counteracted infection by FeLV-D and ERV-DCs via competition for the entry receptor CTR1; the antiviral effects extended to primate ERVs with CTR1-dependent entry. Furthermore, truncated ERV envelope genes found in chimpanzee, bonobo, gorilla, crab-eating macaque, and rhesus macaque genomes could also block infection by feline and primate retroviruses. Genetic analyses showed that these ERV envelope genes were acquired in a species- or genus-specific manner during host evolution. These results indicated that soluble envelope proteins could suppress retroviral infection across species boundaries, suggesting that they function to control retroviral spread. Our findings revealed that several mammalian species acquired antiviral machinery from various ancient retroviruses, leading to convergent evolution for host defense.

An endogenous bornavirus-like nucleoprotein in miniopterid bats retains the RNA-binding properties of the original viral protein.

Endogenous bornavirus-like nucleoprotein elements (EBLNs) are sequences derived from bornaviral N genes in vertebrate genomes. Some EBLNs have been suggested to encode functional proteins in host cells; however, little is known about their evolution and functional relationship to the viral genes from which EBLNs originate. Here, we predicted functionality of EBLNs based on the properties of N as an RNA-binding protein. We showed an EBLN in miniopterid bats (miEBLN-1) has evolved under purifying selection and encodes an RNA-binding protein (miEBLN-1p) with biochemical properties similar to bornaviral N. Furthermore, we revealed miEBLN-1p interacts with host RNA-binding proteins, such as MOV10. These data suggest that miEBLN-1p has been exapted as an RNA-binding protein with similar properties to exogenous bornaviral N in miniopterid bats.

Hidden Viral Sequences in Public Sequencing Data and Warning for Future Emerging Diseases.

RNA viruses cause numerous emerging diseases, mostly due to transmission from mammalian and avian reservoirs. Large-scale surveillance of RNA viral infections in these animals is a fundamental step for controlling viral infectious diseases. Metagenomic analysis is a powerful method for virus identification with low bias and has contributed substantially to the discovery of novel viruses. Deep-sequencing data have been collected from diverse animals and accumulated in public databases, which can be valuable resources for identifying unknown viral sequences. Here, we screened for infections of 33 RNA viral families in publicly available mammalian and avian sequencing data and found approximately 900 hidden viral infections. We also discovered six nearly complete viral genomes in livestock, wild, and experimental animals: hepatovirus in a goat, hepeviruses in blind mole-rats and a galago, astrovirus in macaque monkeys, parechovirus in a cow, and pegivirus in tree shrews. Some of these viruses were phylogenetically close to human-pathogenic viruses, suggesting the potential risk of causing disease in humans upon infection. Furthermore, infections of five novel viruses were identified in several different individuals, indicating that their infections may have already spread in the natural host population. Our findings demonstrate the reusability of public sequencing data for surveying viral infections and identifying novel viral sequences, presenting a warning about a new threat of viral infectious disease to public health. IMPORTANCE Monitoring the spread of viral infections and identifying novel viruses capable of infecting humans through animal reservoirs are necessary to control emerging viral diseases. Massive amounts of sequencing data collected from various animals are publicly available, and these data may contain sequences originating from a wide variety of viruses. Here, we analyzed more than 46,000 public sequencing data and identified approximately 900 hidden RNA viral infections in mammalian and avian samples. Some viruses discovered in this study were genetically similar to pathogens that cause hepatitis, diarrhea, or encephalitis in humans, suggesting the presence of new threats to public health. Our study demonstrates the effectiveness of reusing public sequencing data to identify known and unknown viral infections, indicating that future continuous monitoring of public sequencing data by metagenomic analyses would help prepare and mitigate future viral pandemics.

100-My history of bornavirus infections hidden in vertebrate genomes.

Although viruses have threatened our ancestors for millions of years, prehistoric epidemics of viruses are largely unknown. Endogenous bornavirus-like elements (EBLs) are ancient bornavirus sequences derived from the viral messenger RNAs that were reverse transcribed and inserted into animal genomes, most likely by retrotransposons. These elements can be used as molecular fossil records to trace past bornaviral infections. In this study, we systematically identified EBLs in vertebrate genomes and revealed the history of bornavirus infections over nearly 100 My. We confirmed that ancient bornaviral infections have occurred in diverse vertebrate lineages, especially in primate ancestors. Phylogenetic analyses indicated that primate ancestors were infected with various bornaviral lineages during evolution. EBLs in primate genomes formed clades according to their integration ages, suggesting that bornavirus lineages infected with primate ancestors had changed chronologically. However, some bornaviral lineages may have coexisted with primate ancestors and underwent repeated endogenizations for tens of millions of years. Moreover, a bornaviral lineage that coexisted with primate ancestors also endogenized in the genomes of some ancestral bats. The habitats of these bat ancestors have been reported to overlap with the migration route of primate ancestors. These results suggest that long-term virus-host coexistence expanded the geographic distributions of the bornaviral lineage along with primate migration and may have spread their infections to these bat ancestors. Our findings provide insight into the history of bornavirus infections over geological timescales that cannot be deduced from research using extant viruses alone, thus broadening our perspective on virus-host coevolution.

Identification of novel avian and mammalian deltaviruses provides new insights into deltavirus evolution.

Hepatitis delta virus (HDV) is a satellite virus that requires hepadnavirus envelope proteins for its transmission. Although recent studies identified HDV-related deltaviruses in certain animals, the evolution of deltaviruses, such as the origin of HDV and the mechanism of its coevolution with its helper viruses, is unknown, mainly because of the phylogenetic gaps among deltaviruses. Here, we identified novel deltaviruses of passerine birds, woodchucks, and white-tailed deer by extensive database searches and molecular surveillance. Phylogenetic and molecular epidemiological analyses suggest that HDV originated from mammalian deltaviruses and the past interspecies transmission of mammalian and passerine deltaviruses. Further, metaviromic and experimental analyses suggest that the satellite-helper relationship between HDV and hepadnavirus was established after the divergence of the HDV lineage from non-HDV mammalian deltaviruses. Our findings enhance our understanding of deltavirus evolution, diversity, and transmission, indicating the importance of further surveillance for deltaviruses.

Distribution of infectious endogenous retroviruses in mixed-breed and purebred cats.

Endogenous retroviruses of domestic cats (ERV-DCs) are members of the genus Gammaretrovirus that infect domestic cats (Felis silvestris catus). Uniquely, domestic cats harbor replication-competent proviruses such as ERV-DC10 (ERV-DC18) and ERV-DC14 (xenotropic and nonecotropic viruses, respectively). The purpose of this study was to assess invasion by two distinct infectious ERV-DCs, ERV-DC10 and ERV-DC14, in domestic cats. Of a total sample of 1646 cats, 568 animals (34.5%) were positive for ERV-DC10 (heterozygous: 377; homozygous: 191), 68 animals (4.1%) were positive for ERV-DC14 (heterozygous: 67; homozygous: 1), and 10 animals (0.6%) were positive for both ERV-DC10 and ERV-DC14. ERV-DC10 and ERV-DC14 were detected in domestic cats in Japan as well as in Tanzania, Sri Lanka, Vietnam, South Korea and Spain. Breeding cats, including Singapura, Norwegian Forest and Ragdoll cats, showed high frequencies of ERV-DC10 (60-100%). By contrast, ERV-DC14 was detected at low frequency in breeding cats. Our results suggest that ERV-DC10 is widely distributed while ERV-DC14 is maintained in a minor population of cats. Thus, ERV-DC10 and ERV-DC14 have invaded cat populations independently.

Tracking the Fate of Endogenous Retrovirus Segregation in Wild and Domestic Cats.

Endogenous retroviruses (ERVs) of domestic cats (ERV-DCs) are one of the youngest feline ERV groups in domestic cats (Felis silvestris catus); some members are replication competent (ERV-DC10, ERV-DC18, and ERV-DC14), produce the antiretroviral soluble factor Refrex-1 (ERV-DC7 and ERV-DC16), or can generate recombinant feline leukemia virus (FeLV). Here, we investigated ERV-DC in European wildcats (Felis silvestris silvestris) and detected four loci: ERV-DC6, ERV-DC7, ERV-DC14, and ERV-DC16. ERV-DC14 was detected at a high frequency in European wildcats; however, it was replication defective due to a single G → A nucleotide substitution, resulting in an E148K substitution in the ERV-DC14 envelope (Env). This mutation results in a cleavage-defective Env that is not incorporated into viral particles. Introduction of the same mutation into feline and murine infectious gammaretroviruses resulted in a similar Env dysfunction. Interestingly, the same mutation was found in an FeLV isolate from naturally occurring thymic lymphoma and a mouse ERV, suggesting a common mechanism of virus inactivation. Refrex-1 was present in European wildcats; however, ERV-DC16, but not ERV-DC7, was unfixed in European wildcats. Thus, Refrex-1 has had an antiviral role throughout the evolution of the genus Felis, predating cat exposure to feline retroviruses. ERV-DC sequence diversity was present across wild and domestic cats but was locus dependent. In conclusion, ERVs have evolved species-specific phenotypes through the interplay between ERVs and their hosts. The mechanism of viral inactivation may be similar irrespective of the evolutionary history of retroviruses. The tracking of ancestral retroviruses can shed light on their roles in pathogenesis and host-virus evolution.IMPORTANCE Domestic cats (Felis silvestris catus) were domesticated from wildcats approximately 9,000 years ago via close interaction between humans and cats. During cat evolution, various exogenous retroviruses infected different cat lineages and generated numerous ERVs in the host genome, some of which remain replication competent. Here, we detected several ERV-DC loci in Felis silvestris silvestris Notably, a species-specific single nucleotide polymorphism in the ERV-DC14 env gene, which results in a replication-defective product, is highly prevalent in European wildcats, unlike the replication-competent ERV-DC14 that is commonly present in domestic cats. The presence of the same lethal mutation in the env genes of both FeLV and murine ERV provides a common mechanism shared by endogenous and exogenous retroviruses by which ERVs can be inactivated after endogenization. The antiviral role of Refrex-1 predates cat exposure to feline retroviruses. The existence of two ERV-DC14 phenotypes provides a unique model for understanding both ERV fate and cat domestication.

Reduced Folate Carrier: an Entry Receptor for a Novel Feline Leukemia Virus Variant.

Feline leukemia virus (FeLV) is horizontally transmitted among cats and causes a variety of hematopoietic disorders. Five subgroups of FeLV, A to D and T, each with distinct receptor usages, have been described. Recently, we identified a new FeLV Env (TG35-2) gene from a pseudotyped virus that does not belong to any known subgroup. FeLV-A is the primary virus from which other subgroups have emerged via mutation or recombination of the subgroup A env gene. Retrovirus entry into cells is mediated by the interaction of envelope protein (Env) with specific cell surface receptors. Here, phenotypic screening of a human/hamster radiation hybrid panel identified SLC19A1, a feline reduced folate carrier (RFC) and potential receptor for TG35-2-phenotypic virus. RFC is a multipass transmembrane protein. Feline and human RFC cDNAs conferred susceptibility to TG35-2-pseudotyped virus when introduced into nonpermissive cells but did not render these cells permissive to other FeLV subgroups or feline endogenous retrovirus. Moreover, human cells with genomic deletion of RFC were nonpermissive for TG35-2-pseudotyped virus infection, but the introduction of feline and human cDNAs rendered them permissive. Mutation analysis of FeLV Env demonstrated that amino acid substitutions within variable region A altered the specificity of the Env-receptor interaction. We isolated and reconstructed the full-length infectious TG35-2-phenotypic provirus from a naturally FeLV-infected cat, from which the FeLV Env (TG35-2) gene was previously isolated, and compared the replication of the virus in hematopoietic cell lines with that of FeLV-A 61E by measuring the viral RNA copy numbers. These results provide a tool for further investigation of FeLV infectious disease.IMPORTANCE Feline leukemia virus (FeLV) is a member of the genus Gammaretrovirus, which causes malignant diseases in cats. The most prevalent FeLV among cats is FeLV subgroup A (FeLV-A), and specific binding of FeLV-A Env to its viral receptor, thiamine transporter feTHTR1, is the first step of infection. In infected cats, novel variants of FeLV with altered receptor specificity for viral entry have emerged by mutation or recombination of the env gene. A novel FeLV variant arose from a subtle mutation of FeLV-A Env, which altered the specific interaction of the virus with its receptor. RFC, a folate transporter, is a potential receptor for the novel FeLV variant. The perturbation of specific retrovirus-receptor interactions under selective pressure by the host results in the emergence of novel viruses.

Tracking the Continuous Evolutionary Processes of an Endogenous Retrovirus of the Domestic Cat: ERV-DC.

An endogenous retrovirus (ERV) is a remnant of an ancient retroviral infection in the host genome. Although most ERVs have lost their viral productivity, a few ERVs retain their replication capacity. In addition, partially inactivated ERVs can present a potential risk to the host via their encoded virulence factors or the generation of novel viruses by viral recombination. ERVs can also eventually acquire a biological function, and this ability has been a driving force of host evolution. Therefore, the presence of an ERV can be harmful or beneficial to the host. Various reports about paleovirology have revealed each event in ERV evolution, but the continuous processes of ERV evolution over millions of years are mainly unknown. A unique ERV family, ERV-DC, is present in the domestic cat (Felis silvestriscatus) genome. ERV-DC proviruses are phylogenetically classified into three genotypes, and the specific characteristics of each genotype have been clarified: their capacity to produce infectious viruses; their recombination with other retroviruses, such as feline leukemia virus or RD-114; and their biological functions as host antiviral factors. In this review, we describe ERV-DC-related phenomena and discuss the continuous changes in the evolution of this ERV in the domestic cat.

Presence of a Shared 5'-Leader Sequence in Ancestral Human and Mammalian Retroviruses and Its Transduction into Feline Leukemia Virus.

Recombination events induce significant genetic changes, and this process can result in virus genetic diversity or in the generation of novel pathogenicity. We discovered a new recombinant feline leukemia virus (FeLV) gag gene harboring an unrelated insertion, termed the X region, which was derived from Felis catus endogenous gammaretrovirus 4 (FcERV-gamma4). The identified FcERV-gamma4 proviruses have lost their coding capabilities, but some can express their viral RNA in feline tissues. Although the X-region-carrying recombinant FeLVs appeared to be replication-defective viruses, they were detected in 6.4% of tested FeLV-infected cats. All isolated recombinant FeLV clones commonly incorporated a middle part of the FcERV-gamma4 5'-leader region as an X region. Surprisingly, a sequence corresponding to the portion contained in all X regions is also present in at least 13 endogenous retroviruses (ERVs) observed in the cat, human, primate, and pig genomes. We termed this shared genetic feature the commonly shared (CS) sequence. Despite our phylogenetic analysis indicating that all CS-sequence-carrying ERVs are classified as gammaretroviruses, no obvious closeness was revealed among these ERVs. However, the Shannon entropy in the CS sequence was lower than that in other parts of the provirus genome. Notably, the CS sequence of human endogenous retrovirus T had 73.8% similarity with that of FcERV-gamma4, and specific signals were detected in the human genome by Southern blot analysis using a probe for the FcERV-gamma4 CS sequence. Our results provide an interesting evolutionary history for CS-sequence circulation among several distinct ancestral viruses and a novel recombined virus over a prolonged period.IMPORTANCE Recombination among ERVs or modern viral genomes causes a rapid evolution of retroviruses, and this phenomenon can result in the serious situation of viral disease reemergence. We identified a novel recombinant FeLV gag gene that contains an unrelated sequence, termed the X region. This region originated from the 5' leader of FcERV-gamma4, a replication-incompetent feline ERV. Surprisingly, a sequence corresponding to the X region is also present in the 5' portion of other ERVs, including human endogenous retroviruses. Scattered copies of the ERVs carrying the unique genetic feature, here named the commonly shared (CS) sequence, were found in each host genome, suggesting that ancestral viruses may have captured and maintained the CS sequence. More recently, a novel recombinant FeLV hijacked the CS sequence from inactivated FcERV-gamma4 as the X region. Therefore, tracing the CS sequences can provide unique models for not only the modern reservoir of new recombinant viruses but also the genetic features shared among ancient retroviruses.

Existence of Two Distinct Infectious Endogenous Retroviruses in Domestic Cats and Their Different Strategies for Adaptation to Transcriptional Regulation.

UNLABELLED: Endogenous retroviruses (ERVs) are the remnants of ancient retroviral infections of germ cells. Previous work identified one of the youngest feline ERV groups, ERV-DC, and reported that two ERV-DC loci, ERV-DC10 and ERV-DC18 (ERV-DC10/DC18), can replicate in cultured cells. Here, we identified another replication-competent provirus, ERV-DC14, on chromosome C1q32. ERV-DC14 differs from ERV-DC10/DC18 in its phylogeny, receptor usage, and, most notably, transcriptional activities; although ERV-DC14 can replicate in cultured cells, it cannot establish a persistent infection owing to its low transcriptional activity. Furthermore, we examined ERV-DC transcription and its regulation in feline tissues. Quantitative reverse transcription-PCR (RT-PCR) detected extremely low ERV-DC10 expression levels in feline tissues, and bisulfite sequencing showed that 5' long terminal repeats (LTRs) of ERV-DC10/DC18 are significantly hypermethylated in feline blood cells. Reporter assays found that the 5'-LTR promoter activities of ERV-DC10/DC18 are high, whereas that of ERV-DC14 is low. This difference in promoter activity is due to a single substitution from A to T in the LTR, and reverse mutation at this nucleotide in ERV-DC14 enhanced its replication and enabled it to persistently infect cultured cells. Therefore, ERV-DC LTRs can be divided into two types based on this nucleotide, the A type or T type, which have strong or attenuated promoter activity, respectively. Notably, ERV-DCs with T-type LTRs, such as ERV-DC14, have expanded in the cat genome significantly more than A-type ERV-DCs, despite their low promoter activities. Our results provide insights into how the host controls potentially infectious ERVs and, conversely, how ERVs adapt to and invade the host genome. IMPORTANCE: The domestic cat genome contains many endogenous retroviruses, including ERV-DCs. These ERV-DCs have been acquired through germ cell infections with exogenous retroviruses. Some of these ERV-DCs are still capable of producing infectious virions. Hosts must tightly control these ERVs because replication-competent viruses in the genome pose a risk to the host. Here, we investigated how ERV-DCs are adapted by their hosts. Replication-competent viruses with strong promoter activity, such as ERV-DC10 and ERV-DC18, were suppressed by promoter methylation in LTRs. On the other hand, replication-competent viruses with weak promoter activity, such as ERV-DC14, seemed to escape strict control via promoter methylation by the host. Interestingly, ERV-DCs with weak promoter activity, such as ERV-DC14, have expanded in the cat genome significantly more than ERV-DCs with strong promoter activity. Our results improve the understanding of the host-virus conflict and how ERVs adapt in their hosts over time.

Novel Feline Leukemia Virus Interference Group Based on the env Gene.

Feline leukemia virus (FeLV) subgroups have emerged in infected cats via the mutation or recombination of the env gene of subgroup A FeLV (FeLV-A), the primary virus. We report the isolation and characterization of a novel env gene, TG35-2, and report that the TG35-2 pseudotype can be categorized as a novel FeLV subgroup. The TG35-2 envelope protein displays strong sequence identity to FeLV-A Env, suggesting that selection pressure in cats causes novel FeLV subgroups to emerge.

Ancestral Mutations Acquired in Refrex-1, a Restriction Factor against Feline Retroviruses, during its Cooption and Domestication.

UNLABELLED: Endogenous retroviruses (ERVs) are remnants of ancestral retroviral infections of germ cells. Retroviral endogenization is an adaptation process for the host genome, and ERVs are gradually attenuated or inactivated by mutation. However, some ERVs that have been "domesticated" by their hosts eventually gain physiological functions, such as placentation or viral resistance. We previously reported the discovery of Refrex-1, a soluble antiretroviral factor in domestic cats that specifically inhibits infection by feline leukemia virus subgroup D (FeLV-D), a chimeric virus of FeLV, and a feline ERV, ERV-DC. Refrex-1 is a truncated envelope protein (Env) encoded by both ERV-DC7 and ERV-DC16 proviral loci. Here, we reconstituted ancestral and functional Env from ERV-DC7 and ERV-DC16 envelope genes (env) by inducing reverse mutations. Unexpectedly, ERV-DC7 and ERV-DC16 full-length Env (ERV-DC7 fl and ERV-DC16 fl), reconstructed by removing stop codons, did not produce infectious viral particles. ERV-DC7 fl and ERV-DC16 fl were highly expressed in cells but were not cleaved into surface subunits (SU) and transmembrane subunits, nor were they incorporated into virions. G407R/N427I-A429T and Y431D substitutions within the SU C-terminal domain of ERV-DC7 fl and ERV-DC16 fl, respectively, caused these dysfunctions. The residues glycine 407 and tyrosine 431 are relatively conserved among infectious gammaretroviruses, and their substitution causes the same dysfunctions as the tested retroviruses. Our results reveal that specific mutations within the SU C-terminal domain suppressed Env cleavage and incorporation into virions and indicate that these mutations contributed to the domestication of Refrex-1 through multistep events that occurred in the postintegration period. IMPORTANCE: Domestic cats are colonized with various exogenous retroviruses (exRVs), such as feline leukemia virus (FeLV), and their genomes contain numerous ERVs, some of which are replication-competent proviruses. The feline hosts, exRVs, and ERVs have complicated genetic interactions and provide an interesting field model for triangular relationships: recombination between FeLV and ERV-DC, which is a feline ERV, generated FeLV-D, a chimeric virus, and FeLV-D is restricted by Refrex-1, an antiretroviral factor corresponding to truncated Env of ERV-DC7 and ERV-DC16. Here, we reconstructed ancestral, functional Env from ERV-DC7 and ERV-DC16 env by inducing reverse mutations to elucidate how Refrex-1 was generated from its ancestor. Our results reveal that they were repeatedly inactivated by mutations preventing Env maturation. Our results provide insights into how ERVs were "domesticated" by their hosts and identify the mutations that mediated these evolutions. Notably, experiments that restore inactivated ERVs might uncover previously unrecognized features or properties of retroviruses.