Generation of novel, biologically active Harvey sarcoma viruses via apparent illegitimate recombination

194

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.

Section: Resultsmentioning

confidence: 99%

McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline oncogene (v-fms)

Donner¹,

Fedele²,

Garon³

et al. 1982

194

“…Unresolved by these conclusions was the observation that these cell lines, whose HSV proviruses were partially deleted, were nevertheless able to release small amounts of particles which behaved like infectious HSV. The resolution of this paradox and a more precise characterization of the deleted proviruses are given in the accompanying report (12).…”

Section: Resultsmentioning

confidence: 99%

Structure of the provirus within NIH 3T3 cells transfected with Harvey sarcoma virus DNA

Goldfarb¹,

Weinberg²

1981

Self Cite

NIH 3T3 cells transformed with unintegrated Harvey sarcoma virus (HSV) linear DNA generally acquired a complete HSV provirus. Infection of these transformed cells with Moloney murine leukemia helper virus was followed by release of infectious particles. The HSV provirus within these transfected cells was convalently joined to nonviral DNA sequences and was terned "cell-linked" HSV DNA. The association of this cell-virus DNA sequence with the chromosomal DNA of a transfected cell was unclear. NIH 3T3 cells could also become transformed by transfection with this cell-linked HSV DNA. In this case, the recipient cells generally acquired a donor DNA fragment containing both the HSV provirus and its flanking nonviral sequences. After celLs acquired either unintegrated or cell-linked HSV DNA, the newly established provirus and flanking cellular sequences underwent amplification to between 5 and 100 copies per diploid cell. NIH 3T3 cells transfected with HSV DNA may acquire deleted proviral DNA lacking at least 1.3 kilobase pairs from the right end of full-length HSV 6-kilobase-pair DNA (corresponding to the 3'-proximal portion of wild-type HSV RNA). Cells bearing such deleted HSV genomes were transfonned, indicating that the viral transformation gene lies in the middle or 5'-proximal portion of the HSV RNA genome. However, when these cells were infected with Moloney murine leukemia helper virus, only low levels of biologically active sarcoma virus particles were released. Therefore, the 3' end of full-length HSV RNA was required for efficient transmission of the viral genome.Harvey sarcoma virus (HSV) is a retrovirus that induces fibrosarcomas in infected rats and mice and causes morphological transformation of fibroblasts cultured in vitro. HSV originated through genetic recombination between a nonsarcomagenic retrovirus, Moloney murine leukemia virus (M-MLV), and rat cellular sequences (16,24). As Fig. 1 shows, HSV genomic RNA (size, 5.4 kilobases) contains a large internal rat cellular sequence, whereas the RNA 5' and 3' ends are derived from the 5' and 3' ends of genomic M-MLV RNA (5).The HSV genome replicates within an infected cell through DNA intermediates analogous to the DNA intermediates of other retroviruses. Shortly after infection, HSV RNA is reverse transcribed to generate a linear doublestranded DNA which is slightly longer (6.0 kilobase pairs [kbp]) than genomic RNA (11,20). As Fig. 1 shows, the 6-kbp DNA bears a terminal sequence repetition; nucleotide sequences det Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.on July 6, 2020 by guest

“…7). In a second experiment (data not shown), in which 20 I1 G418' clones were obtained after Mo-MuLVs"PF infection of two different MT-neoAenvM I clones, 12 yielded neo fragments after XbaI endonuclease digestion that were different in size from their 10 clone progenitors. These results suggest that retroviral RNAs lacking a T sequence undergo frequent recombination during reverse transcription and that such events are detectable even in the absence of selection.…”

Section: Constructmentioning

confidence: 96%

Transduction of cellular neo mRNA by retrovirus-mediated recombination

Stuhlmann

Dieckmann

Berg

1990

Transduction of cellular oncogenes by retroviruses is thought to be a multistep process, involving transcriptional activation of a cellular gene by upstream proviral integration and joining of cellular DNA to retroviral transcriptional signals, followed by copackaging and recombination with a helper virus genome during reverse transcription. To examine the molecular mechanism of the reverse transcriptase-mediated recombination, we introduced into mouse fibroblast cells a variety of constructs in which the neo selectable marker was joined to flanking retroviruslike or cell-like sequences. After superinfection and copackaging with a replication-competent Mo-MuLVs"PF virus, the formation of recombinant neo transducing viruses was assessed in a second round of virus infection by the ability to confer G418 resistance to infected cells. Our results showed that recombinant neo proviruses were generated from neo RNA containing either a 5' or 3' retroviral end, implying that one recombination event with helper virus RNA was sufficient to incorporate the neo gene into proviral DNA. Recombination occurred with an apparent frequency of 10-4 to 10-5 per replication cycle in the absence of homology between the two recombining partners. This frequency, however, increased at least 100-fold if homology was provided at the site of recombination. Our results support the hypothesis that neo-transducing viruses arise via reverse transcriptase-mediated recombination of RNA rather than by recombination proceeding through DNA intermediates. Unexpectedly, removal of the retroviral packaging site 'W reduced the number of neo recombinants only slightly. Our data indicated that although RNAs lacking the 'I site are poorly packaged into virions, those RNAs that are included in the virions undergo frequent recombination, even if there is no selection for recombination. Many of the neo recombinants formed with the Jconstructs had undergone additional recombinations and often incorporated the site from the helper RNA.