Transformation by subgenomic fragments of Rous sarcoma virus DNA

Subgenomic fragments of Rous sarcoma virus (RSV) DNA, generated by Eco RI digestion of DNA of RSV-infected chicken cells, induced transformation of NIH/3T3 mouse cells with efficiencies that were 100–1000 fold lower than the efficiency of transformation by intact RSV DNA. Analysis of the DNAs of NIH cells transformed by Eco RI-digested RSV DNA indicated that these cells contained no more than 2 × 10⁶ daltons of RSV DNA, and did not contain sequences from the 5′ terminus of RSV RNA which are included in the leader sequence of subgenomic src mRNA of RSV-infected cells. The product of the RSV src gene (pp60^src), however, was produced in apparently similar quantities by NIH cells transformed by Eco RI fragments of RSV DNA and by intact RSV DNA. Thus expression of the src gene of RSV in NIH cells transformed by subgenomic fragments of RSV DNA did not require the terminal sequences of the RSV genome, which appear to be involved in synthesis and processing of src mRNA in RSV-infected cells. DNAs of NIH cells transformed by Eco RI-digested RSV DNA were found to induce transformation in secondary transfection assays with efficiencies that were similar to the efficiency of transformation by intact RSV DNA. These results suggest that transformation by subgenomic fragments of RSV DNA may be a consequence of integration of src gene-containing DNA fragments in the vicinity of a promoter site in the recipient cell genome, leading to efficient expression of the RSV src gene.     

Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging.

Site-directed mutagenesis has shown that the nucleocapsid (NC) protein of Rous sarcoma virus (RSV) is required for packaging and dimerization of viral RNA. However, it has not been possible to demonstrate, in vivo or in vitro, specific binding of viral RNA sequences by NC. To determine whether specific packaging of viral RNA is mediated by NC in vivo, we have constructed RSV mutants carrying sequences of Moloney murine leukemia virus (MoMuLV). Either the NC coding region alone, the psi RNA packaging sequence, or both the NC and psi sequences of MoMuLV were substituted for the corresponding regions of a full-length RSV clone to yield chimeric plasmid pAPrcMNC, pAPrc psi M, or pAPrcM psi M, respectively. In addition, a mutant of RSV in which the NC is completely deleted was tested as a control. Upon transfection, each of the chimeric mutants produced viral particles containing processed core proteins but were noninfectious. Thus, MoMuLV NC can replace RSV NC functionally in the assembly and release of mature virions but not in infectivity. Surprisingly, the full-deletion mutant showed a strong block in virus release, suggesting that NC is involved in virus assembly. Mutant PrcMNC packaged 50- to 100-fold less RSV RNA than did the wild type; in cotransfection experiments, MoMuLV RNA was preferentially packaged. This result suggests that the specific recognition of viral RNA during virus assembly involves, at least in part, the NC protein.     

Integration of Rous sarcoma virus DNA during transfection

We have investigated the organization and integration sites of Rous sarcoma virus (RSV) DNA in NIH 3T3 mouse cells transformed by transfection with unintegrated and integrated donor RSV DNAs. RSV DNAs of different cell lines transformed by unintegrated donor DNA were flanked by different cellular DNA sequences, indicating that RSV DNA integrates at multiple sites during transfection. The RSV genomes of cells transformed by transfection were colinear with unintegrated RSV DNA, except that deletions within the terminal repeat units of RSV DNA were detected in some cell lines. These results suggested that the terminal repeat sequences of RSV DNA did not necessarily provide a specific integration site for viral DNA during transfection. In addition, cell lines transformed by integrated RSV DNAs contained both the RSV genomes and flanking cellular sequences of the parental cell lines, indicating that integration of integrated viral DNA during transfection occurred by recombinational events within flanking cellular DNA sequences rather than at the terminal of viral DNA. Integration of RSV DNA during transfection thus appears to differ from integration of RSV DNA in virus-infected cells, where the terminal repeat units of viral DNA provide a highly specific integration site. Integration of donor DNA during transfection of NIH 3T3 cells instead appears to proceed by a pathway which is nonspecific for both donor and recipient DNA sequences.     

Inhibition of Rous sarcoma virus replication by antisense RNA.

Previous results have indicated that Rous sarcoma virus env gene expression is specifically inhibited by antisense RNA (L.-J. Chang and C. M. Stoltzfus, Mol. Cell. Biol. 5:2341-2348, 1985). In this study, we compare the extents of inhibition by antisense RNA derived from different parts of the Rous sarcoma virus genome, and we show that antisense constructs containing the 3'-end noncoding region inhibit env expression to a similar extent as those containing the 5'-end noncoding region or coding region. Furthermore, we show that antisense RNA inhibits virus replication at other levels in addition to translation.     

RNA-directed DNA polymerase of Rous sarcoma virus: initiation of synthesis with 70 S viral RNA as template

DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus with 70 S viral RNA as template initiates by the covalent attachment of dAMP to the 3′ terminal adenosine of an RNA molecule. Initiation continues throughout the course of a 90-minute enzymatic reaction, and chain propagation occurs on most if not all of the dAMP residues attached to primer RNA. The nature of the primer molecules was established in two ways. First, the RNA was tagged by attachment of radioactive mono- and oligodeoxynucleotides. Second, primers were isolated directly from their covalent complexes with nascent DNA. The results of both procedures indicate that DNA synthesis initiates on the 3′ termini of 4 S RNA molecules hydrogen-bonded to 70 S RNA. Purified primer RNA has a nucleotide composition (G + C = 64%) different from that (G + C = 60%) of other 4 S RNAs found hydrogen-bonded to the 70 S RNA of Rous sarcoma virus.     

Regulation of Rous sarcoma virus RNA-dependent DNA polymerase isoenzymes by in vitro phosphorylation-dephosphorylation

The activity of the αβ form of Rous sarcoma virus RNA-dependent DNA polymerase was stimulated upon treatment with the protein kinase purified from the same virus. This enhancement was observed for both DNA-dependent and RNA-dependent DNA polymerase activities, whereas the RNase H activity associated with the polymerase was not affected. On the other hand, the protein kinase did not induce detectable changes in the activities of the α-polymerase isoenzyme. Treatment with Escherichia coli alkaline phosphatase resulted in a reduction of the polymerase activities of the αβ isoenzyme with no effects on RNase H as well as on the α form of the DNA polymerase. Preincubations of the αβ- and α-oncornaviral polymerase isoenzymes with two other protein kinases—from avian myeloblastosis virus and from beef heart (catalytic subunit)—had no substantial effects on DNA polymerase and RNase H activities of both polymerase isoenzymes. Both α and β subunits of the polymerase isoenzymes were phosphorylated in vitro by all three protein kinases employed, although only the β subunit was shown previously to be phosphorylated in vivo.     

Monomer and multimer covalently closed circular forms of Rous sarcoma virus DNA.

Covalently closed circular molecules of viral DNA synthesized in virus-infected cells are composed mainly of monomers sedimenting at 22 to 27S in neutral sucrose gradients. These monomers are detected by annealing with complementary DNA or transfection assay. However, 11% of the infectious circles sediment faster than monomers. There is a peak at 32S which may correspond to dimer molecules. Traces of infectivity (about 3%) found between 32S and 65S suggest the presence of higher oligomers. In alkaline sucrose gradients, covalently closed monomers are found at 64 to 71S. Infectivity of these monomers is reduced by alkali treatment to less than one-tenth, and, perhaps for this reason, no infectious dimers or higher oligomers are observed. It has been shown that upon resedimentation the dimers of 95 can be separated from monomers and detected by hybridization.     

Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus

To detect Rous sarcoma virus-specific DNA in mammalian cells, we have measured the capacity of unlabeled cell DNA to accelerate the reassociation of labeled double-stranded DNA synthesized by the Rous sarcoma virus RNA directed DNA polymerase. Two populations of double-stranded polymerase products are identified by their reassociation kinetics and represent approximately 5% and 30% of the viral 70 S RNA genome. Using two strains of Rous sarcoma virus and four lines of transformed mammalian cells, we found two copies of DNA homologous to both DNA populations in Rous sarcoma virustransformed rat and mouse cells, but not in normal cells. The Rous sarcoma viruslike DNA can be demonstrated in the non-repeated fraction of transformed cell DNA and in nuclear DNA. The results are supported by evidence that the techniques employed detect the formation of extensive well-matched duplexes of cell DNA and viral polymerase products.     

Infection of nondividing cells by Rous sarcoma virus

A direct comparison demonstrates that Rous sarcoma virus is capable of infecting aphidicolin-arrested cells 10-fold more efficiently than murine leukemia virus but less efficiently than human immunodeficiency virus. The efficiency of infection of nondividing cells by the three viruses correlates with the respective ability of each viral DNA to enter the nucleus.     

Inhibition of infectious Rous sarcoma virus production by rifamycin derivative.

A new rifamycin derivative, rifazone-82 (R-82), an inhibitor of viral RNA-dependent DNA polymerase, is selectively toxic to transformed chicken cells in culture. R-82 has now been shown to possess antiviral activity as well. The relatively nontoxic properties of R-82 to nontransformed cells have permitted the execution of experiments examining the effect of a rifamycin derivative on virus reproduction. Addition of low concentrations of R-82 (15 mug/ml) to cultures soon after Rous sarcoma virus infection prevents the spread of infection thoroughout the culture. This inhibition is not dependent on concomitant cellular transformation as identical results were obtained with cells infected with a transformation-defective Rous sarcoma virus. Addition of R-82 to cultures in which all the cells are infected does not substantially affect the yield of physical particles as measured by RNA-dependent DNA polymerase activity and by (3H) uridine incorporation into viral RNA. However, the infectivity of the progeny virus, as measured by focus-forming ability, is decrreased 95 to 99% by R-82 treatment.     

Rous sarcoma virus glycoproteins contain hybrid-type oligosaccharides.

Examination of [3H]mannose-labeled glycopeptides from Prague C Rous sarcoma virus gp85 with gel filtration and sequential glycosidase digestions demonstrated the presence of hybrid-type asparaginyl-oligosaccharides. The major hybrid species had an oligomannosyl core (Man5GlcNAc2-ASN) characteristic of neutral structures, plus "branch" sugars (NeuNAc-Gal-GlcNAc-) characteristic of complex, acidic structures.     

Neural crest cells: temperature-dependent transformation by Rous sarcoma virus.

Cranial neural crest cells from chick embryos, when cultured under appropriate conditions, differentiate after approx. 1 week into pigmented cells. Neurol crest cells were infested with a mutant (RSV-BH-Ta) of the Bryan 'high titer' strain of Rous sarcoma virus on the second day of culture before the cells were morphologically differentiated, or later after they became pigmented. Cells infected and maintained at the temperature permissive for transformation (37 degrees C) proliferated rapidly compared to uninfected cells are produced extensive cytoplasmic vacuoles in a fashion similar to other types of cells transformed with RSV-BH-Ta at 37 degrees C. Cells infected and maintained at the non-permissive temperature for transformation (41 degrees C) also proliferated rapidly but did not become morphologically transformed. Transformation occurred reversibly following a shift of temperature. Infection of morphologically undifferentiated neural crest cells at either temperature prevented their differentiation into pigment cells, and infection of pigmented neural crest cells at either temperature led to a gradual loss of pigmentation. These results suggest that even at the non-permissive temperature the virus may regulate the state of differentiation of certain types of cells.     

Nucleotide sequence of Rous sarcoma virus RNA at the initiation site of DNA synthesis: The 102nd nucleotide is U.

The T₁ oligonucleotide in the genome Rous sarcoma virus (RSV) that corresponds to the initiation site of DNA synthesis in vitro was identified by hybridization of genome RNA with RSV strong stop DNA (the initial 101-nucleotide long fragment synthesized in endogenous reactions) and partially sequenced. The sequence of (C₂, U₂) A-U-U-U-G found corresponds to the d(A-A-T-G-A-A-G) sequence at the 5′ end of the DNA product plus the CA-OH sequence at the 3′ end of the tRNA^Trp primer. Therefore the nucleotide opposite the terminal A of the primer is the complementary U. Furthermore, no internal repetition of more than 30 nucleotides of the 5′ sequence could be detected.     

Effects of alterations in the leader sequence of Rous sarcoma virus RNA on initiation of translation.

The 372-nucleotide leader sequence of Rous sarcoma virus RNA contains three conserved short open reading frames and other sequences responsible for a variety of life cycle functions. We have investigated several aspects of the leader RNA which may influence the translation of the major coding regions to which the leader is juxtaposed. We found that small perturbations of the leader length do not affect the binding and scanning of ribosomal subunits by more than about 10%, that the length and/or structure of the RSV RNA leader is near optimal for translation of the major coding regions of the viral RNA, that inclusion or deletion of open reading frames influences downstream initiation in a manner that is not strictly additive, and that reinitiation of translation at the gag gene is very efficient.