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.

Endogenous feline leukemia virus long terminal repeat integration site diversity is highly variable in related and unrelated domestic cats.

Endogenous retroviruses (ERV) are indicators of vertebrate evolutionary history and play important roles as homeostatic regulators. ERV long terminal repeat (LTR) elements may act as cis-activating promoters or trans-activating enhancer elements modifying gene transcription distant from LTR insertion sites. We previously documented that endogenous feline leukemia virus (FeLV)-LTR copy number variation in individual cats tracks inversely with susceptibility to virulent FeLV disease. To evaluate FeLV-LTR insertion characteristics, we assessed enFeLV-LTR integration site diversity in 20 cats from three genetically distinct populations using a baited linker-mediated PCR approach. We documented 765 individual integration sites unequally represented among individuals. Only three LTR integration sites were shared among all individuals, while 412 sites were unique to a single individual. When primary fibroblast cultures were challenged with exogenous FeLV, we found significantly increased expression of both exogenous and endogenous FeLV orthologs, supporting previous findings of potential exFeLV-enFeLV interactions; however, viral challenge did not elicit transcriptional changes in genes associated with the vast majority of integration sites. This study assesses FeLV-LTR integration sites in individual animals, providing unique transposome genotypes. Further, we document substantial individual variation in LTR integration site locations, even in a highly inbred population, and provide a framework for understanding potential endogenous retroviral element position influence on host gene transcription.

What role do endogenous retroviruses play in domestic cats infected with feline leukaemia virus?

Feline leukaemia virus (FeLV) is a retrovirus that infects domestic and wild cats around the world. FeLV infection is associated with the development of neoplasms, bone marrow disorders and immunosuppression. Viral subgroups arise from mutations in the FeLV genome or from recombination of FeLV with ancestral endogenous retroviruses in the cat genome. The retroviral endogenisation process has allowed generation of a diversity of endogenous viruses, both functional and defective. These elements may be part of the normal functioning of the feline genome and may also interact with FeLV to form recombinant FeLV subgroups, enhance pathogenicity of viral subgroups, or inhibit and/or regulate other retroviral infections. Recombination of the env gene occurs most frequently and appears to be the most significant in terms of both the quantity and diversification of pathogenic effects in the viral population, as well as affecting cell tropism and types of disease that occur in infected cats. This review focuses on available information regarding genetic diversity, pathogenesis and diagnosis of FeLV as a result of the interaction between endogenous and exogenous viruses.

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.

Adeno-Associated Vector-Delivered CRISPR/SaCas9 System Reduces Feline Leukemia Virus Production In Vitro.

Feline leukemia virus (FeLV) is a retrovirus of cats worldwide. High viral loads are associated with progressive infection and the death of the host, due to FeLV-associated disease. In contrast, low viral loads, an effective immune response, and a better clinical outcome can be observed in cats with regressive infection. We hypothesize that by lowering viral loads in progressively infected cats, using CRISPR/SaCas9-assisted gene therapy, the cat's immune system may be permitted to direct the infection towards a regressive outcome. In a step towards this goal, the present study evaluates different adeno-associated vectors (AAVs) for their competence in delivering a gene editing system into feline cells, followed by investigations of the CRISPR/SaCas9 targeting efficiency for different sites within the FeLV provirus. Nine natural AAV serotypes, two AAV hybrid strains, and Anc80L65, an in silico predicted AAV ancestor, were tested for their potential to infect different feline cell lines and feline primary cells. AAV-DJ revealed superior infection efficiency and was thus employed in subsequent transduction experiments. The introduction of double-strand breaks, using the CRISPR/SaCas9 system targeting 12 selected FeLV provirus sites, was confirmed by T7 endonuclease 1 (T7E1), as well as Tracking of Indels by Decomposition (TIDE) analysis. The highest percentage (up to 80%) of nonhomologous end-joining (NHEJ) was found in the highly conserved gag and pol regions. Subsequent transduction experiments, using AAV-DJ, confirmed indel formation and showed a significant reduction in FeLV p27 antigen for some targets. The targeting of the FeLV provirus was efficient when using the CRISPR/SaCas9 approach in vitro. Whether the observed extent of provirus targeting will be sufficient to provide progressively FeLV-infected cats with the means to overcome the infection needs to be further investigated in vivo.

Polymerase chain reaction-based detection of myc transduction in feline leukemia virus-infected cats.

Feline lymphomas are associated with the transduction and activation of cellular proto-oncogenes, such as c-myc, by feline leukemia virus (FeLV). We describe a polymerase chain reaction assay for detection of myc transduction usable in clinical diagnosis. The assay targets c-myc exons 2 and 3, which together result in a FeLV-specific fusion gene following c-myc transduction. When this assay was conducted on FeLV-infected feline tissues submitted for clinical diagnosis of tumors, myc transduction was detected in 14% of T-cell lymphoma/leukemias. This newly established system could become a useful diagnostic tool in veterinary medicine.

Notch2 transduction by feline leukemia virus in a naturally infected cat.

Feline leukemia virus (FeLV) induces neoplastic and nonneoplastic diseases in cats. The transduction of cellular genes by FeLV is sometimes observed and associated with neoplastic diseases including lymphoma and sarcoma. Here, we report the first natural case of feline Notch2 transduction by FeLV in an infected cat with multicentric lymphoma and hypercalcemia. We cloned recombinant FeLVs harboring Notch2 in the env gene. Notch2 was able to activate expression of a reporter gene, similar to what was previously reported in cats with experimental FeLV-induced thymic lymphoma. Our findings suggest that the transduction of Notch2 strongly correlates with FeLV-induced lymphoma.

Glycan moiety modifications of feline alpha1-acid glycoprotein in retrovirus (FIV, FeLV) affected cats.

alpha1-Acid glycoprotein (AGP) is considered one of the major acute phase proteins in cats. In humans, AGP is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. In this paper we present the feline AGPs (fAGP) glycan moiety modifications in the course of two prevalent feline diseases, the FIV (feline immunodeficiency virus) dependent feline acquired viral immunodeficiency and the feline leukemia virus (FeLV) associated lymphoma. The glycan moiety of fAGP was investigated by means of the binding of its oligosaccharides residues with specific lectins. Four lectins were used: Sambucus nigra agglutinin I and Maackia amurensis agglutinin lectins were used to detect sialic acid residues, Aleuria aurantia lectin was used to detect L-fucose residues and Concanavalin A was used to evaluate the degree of branching. It was found that fAGP undergoes several post-translational modifications of its glycan pattern: in particular the degree of sialylation is increased in FeLV-positive cats diagnosed with lymphoma, while FeLV-positive that did not presented any specific clinical signs cats do not present any increase of expression of sialic acid on the surface. Furthermore, FIV induced a modification of the glycan moiety of fAGP, which however varied widely among individuals. In order to determine the number and the position of oligosaccharide chains, the cDNA sequence of fAGP was also determined. The translation of the mature fAGP coding sequence gave rise to a sequence of 183 residues, with five potential N-glycosylation sites, but also with seven potential phosphorylation sites.

Chromosome abnormalities and oncogenesis in cat leukemias.

Chromosome abnormalities are found in feline leukemia virus (FeLV)-infected tumor cells as well as in tumor cells free of the virus. Three cell lines derived from tumors in the domestic cat (Felis catus), two of thymic origin and one of multicentric lymphoma origin, were analyzed cytogenetically to determine whether the FeLV virus was associated with chromosomal abnormalities in these tumor cell lines. One thymic tumor and the multicentric lymphoma were FeLV infected. The other thymic tumor cell line was FeLV-free. The normal diploid number in the domestic cat is 38. All three cell lines had numerical chromosome abnormalities with modal numbers of 37, 38 (pseudodiploid), and 39, respectively and had consistent structural chromosome abnormalities. Three markers in the virus-free cell line (S markers) were shared with one or the other of the virus-positive cell lines. The two FeLV-positive cell lines did not have S markers in common. The finding of chromosome abnormalities in both the virus-infected and the virus-free cell lines suggests that these abnormalities may be important in oncogenesis. The FeLV virus could not be considered the only causative agent of the abnormalities observed.