Characterization of ribosome binding on Rous sarcoma virus RNA in vitro.

We determined the sites at which ribosomes form initiation complexes on Rous sarcoma virus RNA in order to determine how initiation of Pr76gag synthesis at the fourth AUG codon from the 5' end of Rous sarcoma virus strain SR-A RNA occurs. Ribosomes bind almost exclusively at the 5'-proximal AUG codon when chloride is present as the major anion added to the translational system. However, when chloride is replaced with acetate, ribosomes bind at the two 5'-proximal AUG codons, as well as at the initiation site for Pr76gag. We confirmed that the 5'-proximal AUG codon is part of a functional initiation site by identifying the seven-amino acid peptide encoded there. Our results suggest that (i) translation in vitro of Rous sarcoma virus virion RNA results in the synthesis of at least two polypeptides; (ii) the pattern of ribosome binding observed for Rous sarcoma virus RNA can be accounted for by the modified scanning hypothesis; and (iii) the interaction between 40S ribosomal subunits or 80S ribosomal complexes is stronger at the 5'-proximal AUG codon than at sites farther downstream, including the initiation site for the major viral proteins.     

Specific nuclear proteins interact with the Rous sarcoma virus internal enhancer and share a common element with the enhancer located in the long terminal repeat of the virus.

We have documented that the Rous sarcoma virus (RSV) internal enhancer functions in the nontransformed Baby Hamster Kidney (BHK) cell line. The sequences within this region were assayed for their ability to bind to specific factors present in BHK nuclear extracts using the gel retardation assay and DNAse I footprinting. At least two sequences within the internal enhancer which can specifically bind nuclear factors in vitro have been identified. These regions are located between nucleotides 813-850 and 856-877. These sites map within the overall region of the internal enhancer which has been shown to be essential for enhancer activity and within the specific region which can function as an orientation independent enhancer. Using the DNase I footprinting and binding data to design an oligonucleotide, we have demonstrated that an oligonucleotide extending from nucleotides 804-877 will substitute efficiently as an enhancer. We also demonstrate that the SV40 enhancer does not compete for the factors which bind to the RSV internal enhancer, whereas an oligonucleotide to the binding site for EFII in the LTR can compete for factor binding to the internal enhancer.     

Feline sarcoma virus-coded polyprotein: enzymatic cleavage by a type C virus-coded structural protein.

A purified 15,000-molecular-weight (Mr) Prague strain Rous sarcoma virus gag gene-coded structural protein, p15, was shown to enzymatically cleave the previously described 130,000 Mr feline sarcoma virus-coded polyprotein, Pr130. Cleavage products included proteins ranging in molecular weight from 12,000 to 110,000. The specificity of this cleavage reactivity was indicated by the fact that, under similar conditions, neither purified type C viral structural proteins nor nonviral proteins such as bovine serum albumin were cleaved to significant extents. Moreover, feline leukemia virus Pr65gag was efficiently cleaved, resulting in the generations of proteins of 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10) Mr. Using enzymatically (p15) treated feline sarcoma virus Pr130 as starting material, we were able to purify a major 72,000 Mr cleavage product and to show it to contain the previously described feline sarcoma virus-coded nonstructural component.     

Identification of a transformation-specific protein induced by a Rous sarcoma virus.

Using antisera obtained from rats bearing Schmidt-Ruppin strain Rous sarcoma virus-induced tumors, we have idnetified a protein with an apparent molecular weight of 56,000 daltons and an isoelectric point of 6.3 in extracts of chick embryo fibroblasts transformed by a wild-type nondefective Rous sarcoma virus (Schmidt-Ruppin strain). This protein was not found in cells infected by trnasformation-defective mutants with either a partial or complete deletion of the src gene, nor in cells infected by a nontransforming avian leukosis virus. The 56,000 dalton molecular weight protein was found to be synthesized at both the permissive and nonpermissive temperatures in cells infected by either of two conditionallethal mutants that are temperature-sensitive in cell transformation. The amount of this protein, however, accumulated in cells infected by these temperature-sensitive mutants, relative to the structural polypeptides, differed significnatly from that seen with the nondefective virus. Pulsechase experiments indicate that the protein is extremely unstable, with a half-life of about 20 min, and does not serve as a precursor to any of the detectable virion polypeptides. Furthermore, incubation of the rat antiserum with purified, disrupted virus did not affect its immunoreactivity to this particular protein. We conclude that this 56,000 dalton molecular weight protein is a nonstructural protein specific to cells transformed by Rous sarcoma virus.     

Effect of p15-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors.

A proteolytic activity is associated with structural protein p15 in avian RNA tumor viruses. Its effect on the known intracellular viral polyprotein precursors obtained by immunoprecipitation was investigated. Cleavage of Pr76gag resulted in the sequential appearance of p15, p27, and p19. The intracellular precursor Pr180gag-pol was also cleaved by p15, whereas the intracellular glycoprotein precursors of avian RNA tumor viruses, Pr92env, remained unaffected by p15 under all conditions tested. The specificities of the antibodies used to precipitate the precursors influenced the pattern of intermediates and cleavage products obtained by p15 treatment. If virus harvested from the the Prague strain of Rous sarcoma virus, subgroup C-transformed cells at 15-min intervals was incubated at 37 degrees C for further maturation, RNA-dependent DNA polymerase activity showed an optimum of DNA synthesis with 70S viral RNA or synthetic template-primers after short incubation periods. The presence of additional p15 during incubation resulted in a shift of the enzyme activity peak toward earlier time points. Virus harvested at 3-h intervals contained significant amounts of Pr180gag-pol and Pr76gag. The addition of p15 resulted in the cleavage of Pr180gag-pol and Pr76gag, but only a few distinct low-molecular-weight polypeptides appeared. Treatment of purified RNA-dependent DNA polymerase with p15 in vitro resulted in a disappearance of the beta subunit and an enrichment of the alpha subunit. In addition, a polypeptide of 32 x 10(3) molecular weight was generated. The cleavage pattern observed differed from the one obtained by trypsin treatment.     

The same normal cell protein is phosphorylated after transformation by avian sarcoma viruses with unrelated transforming genes.

The phosphorylation of a normal cellular protein of molecular weight 34,000 (34K) is enhanced in Rous sarcoma virus-transformed chicken embryo fibroblasts apparently as a direct consequence of the phosphotransferase activity of the Rous sarcoma virus-transforming protein pp60src. We have prepared anti-34K serum by using 34K purified from normal fibroblasts to confirm that the transformation-specific phosphorylation described previously occurs on a normal cellular protein and to further characterize the nature of the protein. In this communication, we also show that the phosphorylation of 34K is also increased in cells transformed by either Fujinami or PRCII sarcoma virus, two recently characterized avian sarcoma viruses whose transforming proteins, although distinct from pp60src, are also associated with phosphotransferase activity. Moreover, comparative fingerprinting of tryptic phosphopeptides shows that the major site of phosphorylation of 34K is the same in all three cases.     

Determination of the proteolytic processing sites in the polyprotein encoded by the bottom-component RNA of cowpea mosaic virus

We have purified two low-molecular-weight polypeptides from the Prague C strain of Rous sarcoma virus and have identified these as products of the gag precursor Pr76 by protein sequencing and by amino acid analysis. Both polypeptides are derived from a stretch of 22 amino acids within Pr76 that separates p19 and p10. We refer to this region as p2. Together the two cleavage products form the entire p2 region. The junctions of p19 with the amino-terminal fragment of p2 and of p10 with the carboxy-terminal fragment of p2 define two new processing sites within the gag precursor, Tyr-155-His-156 and Gly-177-Ser-178. Both polypeptides are major cleavage products of Pr76 that occur in Prague C Rous sarcoma virus at an estimated 1,000 copies per virion. They also are prominent components of avian myeloblastosis virus. The combination of gel filtration and reverse-phase high-pressure liquid chromatography, which was used for the isolation of the two fragments of p2, resolved over a dozen other low-molecular-weight polypeptides from avian sarcoma and leukemia viruses that previously were undetected. This technique thus should serve as a useful procedure for further characterization of viral components.     

Identification of a ribosome-binding site for a leader peptide encoded by Rous sarcoma virus RNA.

A new method for identifying ribosome-binding sites was developed to determine whether AUG codons in the 5'-terminal RNA sequence of Rous sarcoma virus were used to initiate protein synthesis. We found that when translation is inhibited, the major ribosome-binding site on Rous sarcoma virus RNA is at the 5'-proximal AUG codon, even though the primary translational product from this RNA, Pr76gag, is encoded behind the fourth AUG codon 331 bases downstream from the observed initiation site. These results suggest that ribosomes can initiate translation on Rous sarcoma virus RNA at more than one site, thereby producing a seven-amino-acid peptide, as well as the gag gene polyprotein precursor of Mr 76,000.     

Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.     

At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.

We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at least two protein factors (EFI and EFII) that bound specifically to the Rous sarcoma virus (RSV) enhancer in vitro. These factors were differentially extracted from quail cell nuclei, recognized different nucleotide sequences in the U3 region of the RSV long terminal repeat, and appeared to bind preferentially to opposite DNA strands as monitored by the DNase I protection assay. The EFI- and EFII-protected regions within U3 corresponded closely to sequences previously demonstrated by deletion mutagenesis to be required for enhancer activity, strongly suggesting a functional significance for these proteins. Only weak homologies between other enhancer consensus sequence motifs and the EFI and EFII recognition sites were observed, and other viral enhancers from simian virus 40 and Moloney murine sarcoma virus did not compete effectively with the RSV enhancer for binding either factor.     

Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer.

We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to the sequence motif, PEA2, located within the polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having a considerably reduced molecular size. In this study, the altered form, PEBP3, was purified to near homogeneity. The purified PEBP3 comprised two sets of families of polypeptides, alpha-1 to alpha-4 and beta-1 to beta-2, which were 30 to 35 kilodaltons and 20 to 25 kilodaltons in size, respectively. Both kinds of polypeptides possessed DNA-binding activities with exactly the same sequence specificity. Individual alpha or beta polypeptides complexed with DNA showed faster gel mobilities than did PEBP3. However, the original gel retardation pattern was restored when alpha and beta polypeptides were mixed together in any arbitrary pair. These observation along with the results of UV- and chemical-cross-linking studies led us to conclude that PEBP3 is a heterodimer of alpha and beta subunits, potentially having a divalent DNA-binding activity. Furthermore, PEBP3 was found to bind a second, hitherto-unnoticed site of the polyomavirus enhancer that is located within the B element and coincides with the sequence previously known as the simian virus 40 enhancer core homology. From comparison of this and the original binding sites, the consensus sequence for PEBP3 was defined to be PuACCPuCA. These findings provided new insights into the biological significance of PEBP3 and PEBP2.     

Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo.

We have compared the polypeptide products of the src gene of several strains of Rous sarcoma virus produced by in vitro translation of heat-denatured 70S virion RNA in the nuclease-treated reticulocyte lysate with those present in chick cells transformed by these viruses. We have done this by immunoprecipitation, using sera from rabbits injected at birth with Schmidt-Ruppin Rous sarcoma virus. In vitro translation results in the synthesis of at least nine polypeptides which appear to be encoded by the src gene. These range in size from 17,000 to 60,000 daltons. The sera from tumor-bearing rabbits precipitated these polypeptides arising from the in vitro translation of RNA from Schmidt-Ruppin Rous sarcoma virus of both subgroup A and subgroup D and from one stock of Prague Rous sarcoma virus of subgroup C. In each case, all of this family of related polypeptides could be precipitated except the smallest, the 17,000-dalton polypeptide. No precipitation of analogous polypeptides resulting from the translation of RNA from other strains of Rous sarcoma virus was observed. Cells transformed by these three strains of Rous sarcoma virus contain easily detectable amounts of a polypeptide, p60src, essentially identical to the 60,000-dalton in vitro product. With one exception, they do not contain significant amounts of polypeptides analogous to the smaller in vitro products which can be precipitated by these sera. Cells transformed by one stock of Schmidt-Ruppin Rous sarcoma virus of subgroup A did contain a 39,000-dalton polypeptide, which was related, by peptide mapping, to the 60,000-dalton polypeptide and was similar in size to a precipitable in vitro product. The 60,000-dalton polypeptide present in transformed cells appeared to be phosphorylated 10 to 25 min after its synthesis, metabolically very stable, and not derived from a precursor polypeptide. All immunoprecipitates from transformed cells which contained p60src also contained an 80,000-dalton phosphoprotein. This polypeptide is unrelated to p60src, as determined by peptide mapping, and may well be a host cell polypeptide which is specifically associated with p60src.     

Mutation of a termination codon affects src initiation.

The four Rous sarcoma virus messages gag, gag-pol, env, and src all derive from a full-length RNA precursor. All four messages contain the same 5' leader segment. Three of the messages, gag, gag-pol, and env, use an AUG present in this leader to initiate translation. The src AUG initiation codon lies 3' of the leader segment, 90 bases downstream of the gag initiation codon in the spliced src message. However, in the spliced src message a UGA termination codon lies between the gag AUG and the src AUG. All three codons are in the same reading frame. By using oligonucleotide-directed mutagenesis, the UGA termination codon has been converted to CGA. Cells infected with the mutant (called 1057 CGA) were spindle shaped, distinct from the rounded shape of cells infected with the parental Rous sarcoma virus. The mutant virus initiates src translation at the gag AUG, producing a 63,000-dalton src protein. We suggest that the wild-type src message produces two polypeptides, a very small (nine-amino acid) peptide that is initiated at the gag AUG and the 60,000-dalton src protein that is initiated at the src AUG.