No abstract

Extrachromosomal DNA purified from mink cells acutely infected with the Snyder-Theilen strain of feline sarcoma virus (FeSV) was digested with restriction endonucleases, and the DNA fragments were electrophoretically separated, transferred to a solid substrate, and hybridized with radiolabeled DNA transcripts complementary to different portions of the FeSV RNA genome. Major DNA species 8.4 and 5.0 kilobase pairs (kbp) long represent the linear, unintegrated proviruses of Snyder-Theilen feline leukemia virus and FeSV, respectively. Transfection experiments performed with electroeluted DNAs showed that the 8.4-kbp form led to the production of replicating nontransforming virus in mink and cat cells; in contrast, the 5.0-kbp DNA produced helper virus-independent foci of transformation in mouse NIH/3T3 cells and helper virus-dependent foci in mink cells at an efficiency comparable to that obtained with unfractionated extrachromosomal DNA. Sites of restriction endonuclease cleavage for six enzymes were oriented with respect to one another within the FeSV provirus. EcoRI recognized cleavage sites at 0.3 to 0.4 kbp from each terminus of FeSV DNA, reducing the 5.0-kbp DNA to molecules 4.3 kbp long; this enzyme excised a large internal proviral DNA fragment of corresponding size from the DNA of FeSV-transformed mink nonproducer cells. By using DNA transcripts complementary to different portions of the FeSV genome, sarcoma-specific sequences (the FeSV src gene) were positioned within 2.1 and 3.4 kbp from the 5' end of the proviral DNA with respect to the viral RNA genome. The src gene is flanked at both ends by sequences shared in common with feline leukemia virus. The localization of src sequences to this region suggests that a portion of an FeSV polyprotein which contains feline oncornavirus-associated cell membrane antigen (FOCMA-S) is the major product of this gene.

The major internal structural proteins (p30) of type C viruses isolated from several mammalian species were studied by radioimmunoprecipitation and competitive radioimmunoassays. Three antigenically distinguishable sets of interspecies determinants could be demonstrated by both methods. One set of determinants shared by viruses of rodent origin (mouse and rat) can be detected readily in feline leukemia viruses but not in other type C viral groups. The p30 proteins of murine viruses also contain a second discrete set of antigenic determinants related to those in infectious primate viruses and endogenous porcine viruses, but not detected in the feline leukemia virus group. The p30 proteins of endogenous viruses of baboons and domestic cats share yet a third set of cross-reactive determinants not detected in type C viruses isolated from other species of animals. Enzyme inhibition studies performed with antisera raised toward the reverse transcriptases of these same groups of type C viruses showed the same patterns of immunological cross-reactions as observed with p30 proteins. The antigenic cross-reactions between the homologous proteins of type C virus isolated from genetically distant animals may reflect transmission of type C viruses across species barriers.

The genetic structure of the McDonough strain of feline sarcoma virus (SM-FeSV) was deduced by analysis of molecularly cloned, transforming proviral DNA. The 8.2-kilobase pair SM-FeSV provirus is longer than those of other feline sarcoma viruses and contains a transforming gene (v-fms) flanked by sequences derived from feline leukemia virus. The order of genes with respect to viral RNA is 5'-gag-fms-env-3', in which the entire feline leukemia virus env gene and an almost complete gag sequence are represented. Transfection of NIH/3T3 cells with cloned SM-FeSV proviral DNA induced foci of morphologically transformed cells which expressed SM-FeSV gene products and contained rescuable sarcoma viral genomes. Cells transformed by viral infection or after transfection with cloned proviral DNA expressed the polyprotein (P1700ag-fms) characteristic of the SM-FeSV strain. Two proteolytic cleavage products (Pl2OfmS and pp55sag) were also found in immunoprecipitates from metabolically labeled, transformed cells. An additional polypeptide, detected at comparatively low levels in SM-FeSV transformants, was indistinguishable in size and antigenicity from the envelope precursor (gPr85env) of feline leukemia virus. The complexity of the v-fms gene (3.1 ± 0.3 kilobase pairs) is approximately twofold greater than the viral oncogene sequences (v-fes) of Snyder-Theilen and Gardner-Arnstein FeSV. By heteroduplex, restriction enzyme, and nucleic acid hybridization analyses, v-fms and v-fes sequences showed no detectable homology to one another. Radiolabeled DNA fragments representing portions of the two viral oncogenes hybridized to different EcoRl and HindIII fragments of normal cat cellular DNA. Cellular sequences related to v-fms (designated c-fms) were much more complex than c-fes and were distributed segmentally over more than 40 kilobase pairs in cat DNA. Comparative structural studies of the molecularly cloned proviruses of Snyder-Theilen, Gardner-Arnstein, and SM-FeSV showed that a region of the feline leukemia virus genome derived from the pol-env junction is represented adjacent to v-onc sequences in each FeSV strain and may have provided sequences preferred for recombination with cellular genes.

Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions.