Methylation of high-molecular-weight subunit RNA of feline leukemia virus.

The high-molecular-weight subunit RNA of feline leukemia virus (Rickard strain) (FeLV-R) was analyzed for the presence of methyl groups. After purification of native 50-60S FeLV-R RNA on nondenaturing aqueous sucrose density gradients. FeLV-R 28S subunit RNA, doubly labeled with [14C]uridine and [methyl-3H]methionine, was isolated by centrifugation through denaturing sucrose density gradients in dimethyl sulfoxide. As calculated from their respective 3H/14C ratios. FeLV-R 28S RNA was methylated to the same degree as host cell poly(A)+ mRNA. When the 28S FeLV-R RNA was hydrolyzed to completion with RNase T2 or alkali, all of the methyl-3H chromatographed with mononucleotides on Pellionex-WAX, a weak anion exchanger. The methyl-labeled material co-chromatographed with 6-methyladenosine if the mononucleotide fraction obtained by Pellionex-WAX chromatography was hydrolyzed to nucleosides by bacterial alkaline phosphatase or with 6-methyladenine if purine bases were released from the mononucleotides by acid hydrolysis. In another experiment in which FeLV-R 28S RNA uniformly labeled with 32P was hydrolyzed and then analyzed by Pellionex-WAX chromatography, all of the 32P label again co-chromatographed with mononucleotides. Thus FeLV-R 28S RNA does not appear to contain a 5' structure, either methylated or nonmethylated similar to those recently reported for cellular and some animal virus mRNA's.     

Analysis of intracellular feline leukemia virus proteins II. Generation of feline leukemia virus structural proteins from precursor polypeptides.

The synthesis and processing of feline leukemia virus (FeLV) polypeptides were studied in a chronically infected feline thymus tumor cell line, F-422, which produces the Rickard strain of FeLV. Immune precipitation with antiserum to FeLV p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to isolate intracellular FeLV p30 and possible precursor polypeptides. SDS-PAGE of immune precipitates from cells pulse-labeled for 2.5 min with [35S]methionin revealed the presence of a 60,000-dalton precursor polypeptide (Pp60) as well as a 30,000-dalton polypeptide. When cells were grown in the presence of the proline analogue L-azetidine-2-carboxylic acid, a 70,000-dalton precursor polypeptide (Pp70) was found in addition to Pp60 after a 2.5-min pulse. The cleavage of Pp60 could be partially inhibited by the general protease inhibitor phenyl methyl sulfonyl fluoride (PMSF). This partial inhibition was found to occur only if PMSF was present during pulse-labeling. Intracellular Pp70 and Pp60 and FeLV virion p70, p30, p15, p11, and p10 were subjected to tryptic peptide analysis. The results of this tryptic peptide analysis demonstrated that intracellular Pp70 and virion p70 were identical and that both contained the tryptic peptides of FeLV p30, p15, p11, and p10. Pp60 contained the tryptic peptides of FeLV P30, P15, and P10, but lacked the tryptic peptides of P11. The results of pactamycin gene ordering experiments indicated that the small structural proteins of FeLV are ordered p11-p15-p10-p30. The data indicate that the small structural proteins of FeLV are synthesized as part of a 70,000-dalton precursor. A cleavage scheme for the generation of FeLV p70, p30, p15, p11, and p10 from precursor polypeptides is proposed.     

Analysis of intracellular feline leukemia virus proteins. I. Identification of a 60,000-dalton precursor of feline leukemia virus p30.

The synthesis and release of feline leukemia virus p30 was studied using a permanently infected feline thymus tumor cell line. Disrupted cells were divided into two subcellular fractions, a cytoplasmic extract (CE) representing cellular material soluble in 0.5% NP-40 and a particulate fraction (PF) insoluble in 0.5% NP-40 but soluble in 0.2% deoxycholate and 0.5% NP-40. Intracellular feline leukemia virus p30 was isolated from infected cells by immune precipitation with antiserum to p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the precipitated proteins. Cells labeled for 3 h with [35S]methionine contained equal amounts of p30 in both the CE and the PF. p30 synthesis was estimated to be 0.8% of the total host cell protein synthesis. Immune precipitates from cell pulse labeled for 2.5 min contained a labeled 60,000-dalton polypeptide (Pp60) in the PF and a polypeptide in the CE that comigrated with feline leukemia virus p30 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When cells were chased after a pulse label, there was a rapid loss of Pp60 in the PF and an accumulation of p30 in the CE within 30 min followed by distribution of p30 in both the PF and the CE. Estimation of intracellular and extracellular p30 levels during a 0.5- to 24-h chase period suggested that most of the newly synthesized p30 was incorporated into extracellular virus. Typtic peptide analysis of labeled Pp60 and p30 demonstrated the presence of 13 of 15 p30 peptides within the Pp60 molecule. The tryptic peptide analysis in concert with the pulse-chase labeling data provides strong evidence that Pp60 is a precursor of p30.     

Analysis of cytoplasmic RNA and polyribosmomes from feline leukemia virus-infected cells.

Cytoplasmic virus-specific RNA and polyribosomes from a chronically infected feline thymus tumor cell line, F-422, were analyzed by using in vitro-synthesized feline leukemia virus (Rickard strain) (R-FeLV) complementary DNA (cDNA) probe. By hybridization kinetics analysis, cytoplasmic, polyribosomat, and nuclear RNAs were found to be 2.1, 2.6, and 0.7% virus specific, respectively. Size classes within subcellular fractions were determined by sucrose gradient centrifugation in the presence of dimethyl sulfoxide followed by hybridization. The cytoplasmic fraction contained a 28S size class, which corresponds to the size of virion subunit RNA, and 36S, 23S, and 15 to 18S RNA species. The virus-specific 36S, 23S, and 15 to 18S species but not the 28S RNA were present in both the total and polyadenylic acid-containing polyribosomal RNA. Anti-FeLV gamma globulin bound to rapidly sedimenting polyribosomes, with the peak binding at 400S. The specificity of the binding for nascent virus-specific protein was determined in control experiments that involved mixing polyribosomes with soluble virion proteins, absorption of specific gamma globulin with soluble virion proteins, and puromycin-induced nascent protein release. The R-FeLV cDNA probe hybridized to RNA in two polyribosomal regions (approximately 400 to 450S and 250S) within the polyribosomal gradients before but not after EDTA treatment. The 400 to 450S polyribosomes contained three major peaks of virus-specific RNA at 36S, 23S, and 15 to 18S, whereas the 250S polyribosomes contained predominantly 36S and 15 to 18S RNA. Further experiments suggest that an approximately 36S minor subunit is present in virion RNA.     

Prevalence of feline immunodeficiency virus and feline leukemia virus infections in random-source cats.

Retroviral serologic profiles were generated for 506 random-source cats (Felis catus) that were received by our facility during a twenty-month period. Feline leukemia virus antigens were detected in plasma samples from 26 (5.1%) of the cats. Antibodies to feline immunodeficiency virus were present in 24 (4.7%) of the samples tested. A single cat (0.2%) was positive for both viruses. Neither gender nor vendor correlation with retroviral seropositivity could be demonstrated.     

Recombinant feline leukemia virus genes detected in naturally occurring feline lymphosarcomas.

Using a polymerase chain reaction strategy aimed at detecting recombinant feline leukemia virus (FeLV) genomes with 5' env sequences originating from an endogenous source and 3' env sequences resulting from FeLV subgroup A (FeLV-A), we detected recombinant proviruses in approximately three-fourths of naturally occurring thymic and alimentary feline lymphosarcomas (LSAs) and one-third of the multicentric LSAs from cats determined to be FeLV capsid antigen positive by immunofluorescence assay. In contrast, only 1 of 22 naturally arising FeLV-negative feline LSAs contained recombinant proviruses, and no recombinant env gene was detected in seven samples from normal tissues or tissues from FeLV-positive animals that died from other diseases. Four preferred structural motifs were identified in the recombinants; one is FeLV-B like (recognizing that FeLV-B itself is a product of recombination between FeLV-A and endogenous env genes), and three contain variable amounts of endogenous-like env gene before crossing over to FeLV-A-related sequences: (i) a combination of full-length and deleted env genes with recombination at sites in the middle of the surface glycoprotein (SU), (ii) the entire SU encoded by endogenous-like sequences, and (iii) the entire SU and approximately half of the transmembrane protein encoded by endogenous-like sequences. Additionally, three of the thymic tumors contained recombinant proviruses with mutations in the vicinity of the major neutralizing determinant for the SU protein. These molecular genetic analyses of the LSA DNAs correspond to our previous results in vitro and support the occurrence and association of viral recombinants and mutants in vivo in FeLV-induced leukemogenesis.     

Recombinant feline herpesviruses expressing feline leukemia virus envelope and gag proteins.

We constructed recombinant feline herpesviruses (FHVs) expressing the envelope (env) and gag genes of feline leukemia virus (FeLV). Expression cassettes, utilizing the human cytomegalovirus immediate-early promoter, were inserted within the thymidine kinase gene of FHV. The FeLV env glycoprotein expressed by recombinant FHV was processed and transported to the cell surface much as in FeLV infection, with the exception that proteolytic processing to yield the mature gp70 and p15E proteins was less efficient in the context of herpesvirus infection. Glycosylation of the env protein was not affected; modification continued in the absence of efficient proteolytic processing to generate terminally glycosylated gp85 and gp70 proteins. A recombinant FHV containing the FeLV gag and protease genes expressed both gag and gag-protease precursor proteins. Functional protease was produced which mediated the proteolytic maturation of the FeLV gag proteins as in authentic FeLV infection. Use of these recombinant FHVs as live-virus vaccines may provide insight as to the role of specific retroviral proteins in protective immunity. The current use of conventional attenuated FHV vaccines speaks to the wider potential of recombinant FHVs for vaccination in cats.     

Molecular analysis and pathogenesis of the feline aplastic anemia retrovirus, feline leukemia virus C-Sarma.

We describe the molecular cloning of an anemogenic feline leukemia virus (FeLV), FeLV-C-Sarma, from the productively infected human rhabdomyosarcoma cell line RD(FeLV-C-S). Molecularly cloned FeLV-C-S proviral DNA yielded infectious virus (mcFeLV-C-S) after transfection of mammalian cells, and virus interference studies using transfection-derived virus demonstrated that our clone encodes FeLV belonging to the C subgroup. mcFeLV-C-S did not induce viremia in eight 8-week-old outbred specific-pathogen-free (SPF) cats. It did, however, induce viremia and a rapid, fatal aplastic anemia due to profound suppression of erythroid stem cell growth in 9 of 10 inoculated newborn, SPF cats within 3 to 8 weeks (21 to 58 days) postinoculation. Thus, the genome of mcFeLV-C-S encodes the determinants responsible for the genetically dominant induction of irreversible erythroid aplasia in outbred cats. A potential clue to the pathogenic determinants of this virus comes from previous work indicating that all FeLV isolates belonging to the C subgroup, an envelop-gene-determined property, and only those belonging to the C subgroup, are potent, consistent inducers of aplastic anemia in cats. To approach the molecular mechanism underlying the induction of this disease, we first determined the nucleotide sequence of the envelope genes and 3' long terminal repeat of FeLV-C-S and compared it with that of FeLV-B-Gardner-Arnstein (mcFeLV-B-GA), a subgroup-B feline leukemia virus that consistently induces a different disease, myelodysplastic anemia, in neonatal SPF cats. Our analysis revealed that the p15E genes and long terminal repeats of the two FeLV strains are highly homologous, whereas there are major differences in the gp70 proteins, including five regions of significant amino acid differences and apparent sequence substitution. Some of these changes are also reflected in predicted glycosylation sites; the gp70 protein of FeLV-B-GA has 11 potential glycosylation sites, only 8 of which are present in FeLV-C-S.     

Identification of a putative receptor for subgroup A feline leukemia virus on feline T cells.

Retrovirus infection is initiated by the binding of virus envelope glycoprotein to a receptor molecule present on cell membranes. To characterize a receptor for feline leukemia virus (FeLV), we extensively purified the viral envelope glycoprotein, gp70, from culture supernatants of FeLV-61E (subgroup A)-infected cells by immunoaffinity chromatography. Binding of purified 125I-labeled gp70 to the feline T-cell line 3201 was specific and saturable, and Scatchard analysis revealed a single class of receptor binding sites with an average number of 1.6 x 10(5) receptors per cell and an apparent affinity constant (Ka) of 1.15 x 10(9) M-1. Cross-linking experiments identified a putative gp70-receptor complex of 135 to 140 kDa. Similarly, coprecipitation of 125I-labeled cell surface proteins with purified gp70 and a neutralizing but noninterfering anti-gp70 monoclonal antibody revealed a single cell surface protein of approximately 70 kDa. These results indicate that FeLV-A binds to feline T cells via a 70-kDa cell surface protein, its presumptive receptor.     

Transforming potential of a myc-containing variant of feline leukemia virus in vitro in early-passage feline cells.

We studied a naturally occurring variant of feline leukemia virus (FeLV) in which the oncogene myc has substituted for a portion of the viral structural genes (myc-FeLV). myc-FeLV was rescued by replication in the presence of FeLV as helper, and its biological activity was examined in early-passage feline cells in vitro. Infection of leukocytes from peripheral blood, spleen, or thymus, or of kitten fibroblasts did not immortalize these cells or alter them morphologically. Northern blot (RNA blot) analysis of virion RNA prepared from the supernatant of infected cells demonstrated the 8.2-kilobase genome of FeLV, but did not demonstrate the 5.0-kilobase genome of myc-FeLV. Apparently, the myc-FeLV genome was lost in the absence of the selective pressure of transformation. In contrast, infection of embryonic fibroblasts with myc-FeLV(FeLV) rendered these cells capable of greatly increased, if not infinite, proliferative potential. The cells were morphologically altered compared with controls and were only loosely adherent to the substrate. The cells failed to proliferate in semisolid medium and did not form tumors when inoculated subcutaneously into athymic mice. Blot analyses demonstrated the presence and expression of integrated proviral DNAs of both FeLV and myc-FeLV in these cells. They appear, then, to represent cells partially transformed by infection with myc-FeLV(FeLV). The action of feline v-myc in early-passage cells in vitro was compared to that of avian v-myc.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Reevaluation of host ranges of feline leukemia virus subgroups

We reevaluated the host ranges of feline leukemia virus (FeLV) subgroups A, B and C using pseudotype assays based on recombinant NB-tropic murine leukemia virus, which is not usually blocked after viral entry in mammalian cells. Pseudotype viruses of FeLV-B and -C infected a variety of cell lines from many mammalian species. Unexpectedly, FeLV-A pseudotype viruses of two independent isolates from the UK and US also infected a variety of non-feline cell lines including cells from humans, rabbits, pigs and minks. Moreover, both isolates of FeLV-A productively infected human embryonic kidney 293 and mink Mv-1-Lu cells. We conclude that FeLV-A is not strictly ecotropic.     

Posttranslational modifications distinguish the envelope glycoprotein of the immunodeficiency disease-inducing feline leukemia virus retrovirus.

The envelope glycoprotein (gp70) of a molecularly cloned, replication-defective feline leukemia virus (FeLV-FAIDS clone 61C) carries determinants for induction of fatal immunodeficiency disease, whereas the gp70 of its companion replication-competent, probably parent virus (clone 61E) does not. Immunoprecipitation analysis of the extracellular glycoproteins of 61E and EECC, a replication-competent viral construct composed of the 61C env and 3' long terminal repeat fused to the 61E gag-pol genes, demonstrated that the gp70 of EECC could be distinguished from that of 61E by both feline immune serum and a murine monoclonal antibody. Molecular weights of both the envelope precursor polyprotein (gp80) and the mature extracellular glycoprotein (gp70) of 61E were smaller than the corresponding proteins from the pathogenic EECC. Both the molecular weight disparity and monoclonal antibody discrimination of the two gp80s were abolished by inhibition of envelope protein glycosylation with tunicamycin, whereas the apparent gp70 size differences were resolved by enzymatic removal of N-linked oligosaccharides. Pulse-chase studies in EECC-infected cells demonstrated that processing of gp80 to gp70 was delayed and that this retardation of envelope glycoprotein processing could be simulated in 61E-infected cells by treatment with the glucosidase inhibitor N-methyldeoxynojirimycin, a compound that causes retention of oligosaccharides in the high-mannose form. The resultant 61E gp70 then could be recognized by sera from EECC-immunized cats. The presence of a higher content of sialic acid on the apathogenic 61E gp70 indicated that oligosaccharides of 61E and EECC gp70 were processed differently. These data suggested that the unique biochemical properties which distinguish the envelope glycoproteins of the FeLV-FAIDS variant from its companion apathogenic parent virus were responsible for T-cell cytopathicity and induction of immunodeficiency disease. Further biochemical characterization of these glycoproteins should be useful in understanding the pathogenic mechanisms of immunodeficiency disease induced by retroviruses.     

Epizootiology and management of feline leukemia virus in the Florida puma

Feline leukemia virus (FeLV) was not detected in Florida pumas (Puma concolor coryi) in almost 20 yr of surveillance; however, the finding of two FeLV antigen-positive pumas during the 2002-2003 capture season led to an investigation of FeLV in the population. Between January 1990 and April 2007, the proportion of pumas testing FeLV antibody positive increased, with antibody-positive pumas concentrated in the northern portion of puma range. Five of 131 (4%) pumas sampled between July 2000 and April 2007 were viremic, with all cases clustered in Okaloacoochee Slough (OKS). Clinical signs and clinical pathology at capture were absent or included lymphadenopathy, moderate-to-severe anemia, and lymphopenia. All viremic pumas died; causes of death were septicemia (n=2), intraspecific aggression (n=2), and anemia/dehydration (n=1). Outcome after FeLV exposure in pumas was similar to that in domestic cats, with evidence of regressive, latent, and persistent infections. Management of the epizootic included vaccination, and as of April 2007, 52 free-ranging pumas had received one or more inoculations. Vaccinations were concentrated in OKS and in a band between OKS and the remainder of the puma population. There have been no new cases since July 2004; however, the potential for reintroduction of the virus remains.     

Effects of cobra venom factor treatment on latent feline leukemia virus infection.

The role of the complement system in containment of feline leukemia virus infection was studied by cobra venom factor treatment of feline leukemia virus-immune cats. One to three weeks after cobra venom factor treatment, an increase in viral antigen in marrow myelomonocytic cells and circulating immune complexes was noted. Prevention of reactivation of feline leukemia virus infection may in part depend on an intact complement system.