Detailed restriction endonuclease maps were developed for Harvey murine sarcoma virus (Ha-MuSV) DNA (clone H-1), molecularly closed at its unique EcoRI site in pBR322, for three nonoverlapping subgenomic HindIII clones which together span the entire H-1 clone and for a molecularly cloned DNA copy of a portion of rat 30S RNA (which represents the majority of the rat genetic sequences in Ha-MuSV). Molecular hybridization of the 30S clone to small restriction fragments of clone H-1 revealed a 0.9-to-1.0-kilobase pair region in the 5' half of the Ha-MuSV genome not homologous to the 30S clone, although the 30S clone did contain related sequences in Ha-MuSV on both sides of this nonhomologous region. By using cloned sequences from a segment of the Ha-MuSV nonhomology region as a probe for hybridization to Southern blots of DNA from rat, mouse, bat, and chicken cells, one to three bands were detected in DNA of each species. By contrast, the 30S clone DNA was highly related to many sequences in rat DNA, partially related to fewer mouse DNA sequences, and homologous only to one to three bands in bat and chicken DNA. Earlier work had shown that the 5' half of the Ha-MuSV genome coded for transformation and for the viral p21 protein (Chang et al., J. Virol. 35: 76--92, 1980; Wei et al., Proc. Natl. Acad. Sci. U.S.A., in press). We used two subgenomic HindIII clones whose shared HindIII site mapped within the 5' region of clone H-1 nonhomologous to the 30S clone to test whether the nonhomologous segment might encode the transforming and p21 functions. Although neither of the subgenomic HindIII fragments by themselves induced transformation, ligation of these two nontransforming DNAs to each other did restore p21-mediated transformation. A conclusion consistent with these results is that a region in the 5' half of the Ha-MuSV genome evolutionarily distinct from and not present in rat 30S RNA is essential for transformation and for p21 encoding.

Various rat cell lines have been analyzed for expression of endogenous RNA homologous either to RT21C, a typical rat type C virus, or to Kirsten sarcoma virus. Cells have been found that express either (i) high levels of RNA homologous to RT21C rat type C virus and low levels of RNA homologous to Kirsten sarcoma virus (RT21Chigh,sarclow) or (ii) high levels of RNA homologous to Kirsten sarcoma virus and low levels of RNA homologous to typical rat type C virus (sarchigh, RT21Clow). The properties of these two classes of cell lines have been compared. Each type of cell contains an equal amount of the expressed RNA on polysomes. Cell lines that are RT21Chigh produce abundant rat p30 nad p12 structural proteins and release rat type C particles containing viral RNA and reverse transcriptase into supernatant fluids from these cultures. Cell lines that are sarchigh,RTC21Clow have no detectable rat viral p12 protein and no p30 protein immunoreactive in even broad interspecies radioimmunoassays, and do not release type C particles into the supernatant from the cultures. When the particle-negative cell lines are superinfected with heterologous mouse or wooly type C viruses or are producing typical rat type C virus particles, the endogenous sarcoma virus-specific RNA is secreted from these cells. The sarcoma virus-specific RNA can be transcribed in complementary DNA in the endogenous reverse transcriptase reactions carried out in vitro with such virus preparations. However, exposure of cells that are permissive to the helper virus with the particles containing sarcoma virus-specific RNA has not yet resulted in cell transformation or in the synthesis of these RNA sequences. The results suggest: (i) that the first step in the genesis of sarcoma viruses involves the packaging of this expressed sarcoma virus-specific RNA in helper viral particles; (ii) that efficient transmission of the sarcoma virus-specific RNA requires additional events; and (iii) that the formation of a stable sarcoma virus by recombination between the helper viral genome and part of the rescued sarcoma virus-specific RNA is much less common event than the rescue process itself.

DNA transcripts from V-NRK and RT21c rat type-C viruses were found to differ in their sequence homology to Kirsten and Harvey sarcoma viruses. V-NRK DNA transcripts consistently had homology to Kirsten and Harvey sarcoma virus, whereas RT21c DNA transcripts did not. To explain the differences, the nucleic acids and structural proteins of the two type-C viruses, released from each of two cell lines derived from Osborne-Mendel rats, were analyzed by molecular hybridization and competition radioimmunoassays. The p30 and p12 structural proteins of the two viruses were found to be highly related immunologically. In the V-NRK virus preparation, two sets of distinct RNA sequences were found in approximately equal amounts. One set is homologous to Ki-SV, and the other homologous to RT21c. In contrast, the RT21c virus preparation was found to contain a different ratio of these sequences. In this case the RT21c-like RNA sequences are present in 100-fold excess as compared to the additional Ki-SV specific sequences. Both NRK and RT21c cells contain in their DNA the full complement of Ki-SV homologous sequences, but NRK cells express much higher levels of these Ki-SV sequences in their RNA. These additional sequences, not homologous to RT21c, which are detected in uninfected NRK cellular RNA or V-NRK rat virus, could also be detected in the 60-70S RNA from a Moloney mouse type-C virus released from the NRK cells infected with the Moloney type-C virus. The results suggest that type-C viruses released from NRK cells incorporate species of RNA present in NRK cells which are homologous to Kirsten and Harvey sarcoma viruses. Either these sequences are of cellular origin, or rat cells contain two endogenous viruses with completely distinct nucleic acid sequences.

Current studies were undertaken to compare the genomes of Kirsten murine sarcoma virus (Ki-MuSV), Harvey murine sarcoma virus (Ha-MuSV), and the replication-defective endogenous rat virus to understand the function of these viral RNAs. Genome organization and sequence homology were studied by fingerprinting large RNase Tl-resistant oligonucleotides and by cross-protecting homologous oligonucleotides against RNase A and Ti digestion with complementary DNA prepared from each of the other viral RNA. Ki-MuSV and Ha-MuSV were found to share an extensive series of rat-derived oligonucleotides begining ca. 1 kilobase (kb) from the 3' end and extending to within 1.5 kb of the 5' end of Ki-MuSV RNA. The total map distance covered is ca. 5.5 kb. The eight oligonucleotides covering the 1.5 kb at the 5' end of Ki-MuSV RNA were not found in Ha-MuSV RNA. Five out of these eight oligonucleotides, however, could be designated with certainty to be of rat virus origin. Since Ha-MuSV is 6.5 kb in size and Ki-MuSV is 8 kb in size, the major difference between them is the 1.5 kb from the replication-defective endogenous rat virus sequences at the 5' end of Ki-MuSV not present in Ha-MuSV. Consistent with the difference in the genome structure, these two sarcoma viral RNAs yielded distinct major translation products in cell-free systems, i.e., a 50,000-dalton polypeptide (p50) from Ki-MuSV and a 22,000-dalton polypeptide (p22) from Ha-MuSV. These polypeptides may provide the necessary protein markers for identifying in vivo virus-coded proteins.

The comparative infectivity of Harvey murine sarcoma virus (Ha-MuSV) DNA for NIH 3T3 cells was determined for supercoiled Ha-MuSV DNA molecularly cloned in lambda phage and pBR322 at its unique EcoRI site (which is located near the middle of the 6-kilobase pair [kbp] unintegrated linear viral DNA) and for two cloned subgenomic fragments: one was 3.8 kbp and lacked about 1 kbp from each side of the EcoRI site, and the second did not contain the 3 kbp of the unintegrated linear viral DNA located on the 3' side of the EcoRI site. Each subgenomic DNA induced foci of transformed cells, but with a lower relative efficiency then genomic DNA. Transfection with intact vector Ha-MuSV DNA yielded results similar to those obtained after separation of Ha-MuSV DNA from vector DNA. Cells lines were then derived from individual foci transformed with each type of viral DNA. Focus-forming virus was recovered from transformed cells after superinfection with a helper-independent virus, but the efficiency varied by several orders of magnitude. For several transformed lines, the efficiency of recovery of focus-forming virus was correlated with the structure of the Ha-MuSV DNA in the cells before superinfection. When 32P-labeled Ha-MuSV DNA probes specific for sequences on either the 3' or 5' side of the EcoRI site were used to analyze the viral RNA in the transformed cell lines, all lines were found to hybridize with the 5' probe, but some lines did not hybridize with the 3' probe. The transformed lines contained high levels of the Ha-MuSV-coded p21 or its associated GDP-binding activity. We conclude that the transforming region and the sequences that code for the viral p21 protein are both located within the 2 kilobases closest to the 5' end of the Ha-MuSV genome.