Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants.

The src genes of four Rous sarcoma virus (RSV) mutants temperature-sensitive (ts) for cell transformation were analyzed. The mutant src genes were cloned into a replication-competent RSV expression vector, and the contribution of individual mutations to the ts phenotype was assessed by in vitro recombination with wild-type src sequences. Three of the mutants, which were derived from the Schmidt-Ruppin strain of RSV, each encoded two mutations within the conserved kinase domain. In all three cases, one of the two mutations was an identical valine to methionine change at amino acid position 461. Virus encoding recombinant src genes containing each of these mutations alone were not ts for transformation, demonstrating that two mutations are required for temperature sensitivity. The sequence of the src gene of the Bryan high-titer strain of RSV was determined and compared with that of the fourth ts mutant which was derived from it, again revealing two lesions in the kinase domain of the mutant.     

env Gene of Rous sarcoma virus: identification of the gene product by cell-free translation.

Cell-free translation of polyadenylic acid-selected, denatured virion 70S RNA of the Schmidt-Ruppin strain of Rous sarcoma virus (subgroup A) yields a 64,000-Mr polypeptide which is specifically immunoprecipitated by a group-specific serum raised against envelope glycoprotein gp85. This polypeptide is not synthesized from the virion RNA of the replication-defective mutant rdNY8SR-A, which contains an extensive deletion within the envelope (env) gene. From this genetic evidence we conclude that the 64,000-Mr polypeptide represents the nonglycosylated product of the env gene and propose the designation of P64env. The 64,000-Mr polypeptide is translated from a 26S to 28S polyadenylated RNA species, whereas the p60src product is synthesized from a 20S to 22S RNA, and both Pr76gag and P180gag-pol are synthesized predominately from 34S RNA. The product of the env gene of Rous-associated virus-2 was also identified by cell-free translation.     

Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein that block processing to gp85 and gp37.

We have investigated the specificity of the proteolytic cleavage of the Rous sarcoma virus glycoprotein precursor by introducing two mutations into the putative cleavage region (Arg-Arg-Lys-Arg). We show that neither a deletion of the cleavage sequence nor a glutamic acid for lysine substitution altered intracellular transport or surface expression of the env gene products. However, both the four-amino-acid deletion and the glutamic acid substitution block processing of the env precursor. Susceptibility of the glutamic acid-substituted env precursor to proteases indicated that tertiary protein structure was unaffected. While inhibitor experiments suggested that more than one endopeptidase might be capable of mediating the proteolytic cleavage, the results presented here point to the presence in the Golgi apparatus of a novel endopeptidase, required for retroviral glycoprotein cleavage, that has a high specificity for lysine-containing peptides.     

Complete sequence of the Rous sarcoma virus env gene: identification of structural and functional regions of its product.

The amino-terminal amino acid sequences of gp85 and gp37, the envelope glycoproteins of Rous sarcoma virus (RSV), were determined. Alignment of these sequences with the amino acid sequence predicted from the complete nucleotide sequence of the Prague strain of RSV, subgroup C (PR-C), has allowed us to delineate the env gene-coding region of this virus. The coding sequences for gp85 and gp37 have been placed in an open reading frame that extends from nucleotide 5045 to nucleotide 6862 and predict sizes of 341 amino acids (36,962 molecular weight) for gp85 and 198 amino acids (21,566 molecular weight) for gp37. Carbohydrate makes a significant contribution to the observed molecular weights of these polypeptides--the amino acid sequence contains 14 potential glycosylation sites (Asn-X-Ser/Thr) in gp85 and two in gp37. Experiments aimed at estimating the number of carbohydrate side chains yielded results consistent with most or all of these sites being occupied. Although an initiation codon is located early (codon 4) in the open reading frame, it is likely that splicing yields an mRNA on which translation initiates at the same AUG as that of the gag gene to produce a nascent polypeptide in which gp85 is preceded by a 62-amino-acid-long leader peptide. This leader contains the hydrophobic sequence (signal sequence) necessary for translocation across the endoplasmic reticulum and is completely removed from the env gene product during translation. The polyprotein precursor, Pr95env, is cleaved to gp85 and gp37 at the carboxyl side of the basic sequence:-Arg-Arg-Lys-Arg-. gp85 is attached through a disulphide linkage to gp37, and although the positions of the cysteines involved in this linkage are not known, the presence of a 27-amino-acid-long hydrophobic region at the carboxy-terminus of gp37 is consistent with its role as a membrane anchor for the viral glycoprotein complex. The location of host range variable regions with respect to the possible tertiary structure of the complex is discussed.     

Genetic analysis of interactions between Gag proteins of Rous sarcoma virus.

The yeast two-hybrid system was used to characterize homomeric interactions between the Gag proteins of Rous sarcoma virus (RSV). The RSV Gag precursor was found to interact strongly with itself and not with various control proteins. The RSV Gag did not interact significantly with Gag proteins of a variety of other retroviruses, including murine leukemia viruses and primate lentiviruses. Deletion analysis suggested that two nonoverlapping regions are independently sufficient to mediate RSV Gag-Gag dimerization. One such region lies near the N terminus and contains p2, p10, and a large N-terminal part of the capsid (CA) domain; the other is localized in the C terminus and includes a small C-terminal portion of CA and the nucleocapsid protein. These interaction domains may play roles in viral assembly.     

Infection of resistant avian cells by subgroup B Rous sarcoma virus.

Chicken fibroblasts derived from the H & N flock, which have been characterized as resistant to subgroup B avian oncornaviruses in focus assays, can be infected in suspension shortly after trypsinization by subgroup B sarcoma and leukosis viruses. Once cells are plated, resistance to infection reappears rapidly. C/BE cell suspensions obtained by treatment with EDTA instead of trypsin are not as sensitive to infection. Late interference established by preinfection with subgroup B leukosis viruses is not overcome by trypsinization. In addition to C/BE H & N chicken cells, C/ABE RPRL line 7 cells can also be infected by subgroup B viruses shortly after trypsinization; however, none of the cell types can be made sensitive to subgroup E infection. These results are discussed in relation to current information on the genetic control of resistance to avian oncornaviruses.     

Inhibition of Rous sarcoma virus replication by 2-deoxyglucose and tunicamycin: identification of an unglycosylated env gene product.

Two inhibitors of glycosylation, 2-deoxyglucose and tunicamycin, depressed the synthesis of infectious Rous sarcoma virus greater than 100-fold. Under the same conditions only a two- to threefold decrease in the production of virus particles was observed. The noninfectious particles had a lower density (1.145 g/ml) in isopycnic sucrose gradients and lacked the two virion glycoproteins, gp85 and gp37, found on infectious virions. The four internal structural proteins of the virus, p27, p19, p15, and p12, appeared to be assembled normally into the noninfectious virus. Polypeptides related to the Rous sarcoma virus glycoproteins were immunoprecipitated from pulse-labeled Rous sarcoma virus (Prague strain, subgroup B)-transformed cells. pr95gp, the polyprotein precursor to gp85 and gp37, was the major protein precipitated from untreated cells. PR95GP, THE POLYPROTEIN PRECURSOR TO GP85 AND GP37, WAS THE MAJOR PROTEIN PRECIPITATED FROM UNTREATED CELLS. This was absent in both tunicamycin- and 2-deoxyglucose-treatec ells, and a new polypeptide of molecular weight 57,000 to 58,000 was the major species precipitated. In tunicamycin-treated cells this product was unstable and was degraded during a 2-h chase; in 2-deoxyglucose-treated cells, on the other hand, the polypeptide appeared to be more stable and underwent partial glycosylation. The synthesis and processing of pr76, the polyprotein precursor to the internal structural proteins of the virion, occurred normally in both treated and untreated cells. It is concluded that the unglycosylated env gene product is a polypeptide of molecular weight 57,000 to 58,000.     

Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.     

Evolution of gp85 gene of subgroup J avian leukosis virus under the selective pressure of antibodies

Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without anti-ALV-J serum in the medium) and B (with anti-ALV-J serum in the medium), then viruses from each well of both groups were separately passed in CEF every 6 d and formed their independent passage lineages. For each lineage of both groups, gp85 genes of the viruses in the 10th, 20th and 30th passages were amplified, cloned and sequenced. The sequence data indicated that the homologies of gp85 at aa level between the primary virus and the passed viruses of different passages of 3 lineages in group A were 97.7%-99.7%; and the homologies of gp85 between the primary virus and the passed viruses of different passages of 3 lineages in group B were 93.8%-96.1%. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S in 3 highly variable (hr-) regions at aa#110-120, aa#141-151 and aa#189-194 of gp85 in 3 lineages of group A were 2 (8/4), 1(3/3) and 1.3 (4/3), however, NS/S in the same 3 hr-regions of group B were 4.1 (13/3), 4.7 (14/3) and 3.3 (11/3). This study is the first demonstration of influence of immune selective pressure on evolution of ALV-J gp85 by specific antibodies under the controlled in vitro experiments.     

Evolution of gp85 gene of subgroup J avian leukosis virus under the selective pressure of antibodies

Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without anti-ALV-J serum in the medium) and B (with anti-ALV-J serum in the medium), then viruses from each well of both groups were separately passed in CEF every 6 d and formed their independent passage lineages. For each lineage of both groups, gp85 genes of the viruses in the 10th, 20th and 30th passages were amplified, cloned and sequenced. The sequence data indicated that the homologies of gp85 at aa level between the primary virus and the passed viruses of different passages of 3 lineages in group A were 97.7%–99.7%; and the homologies of gp85 between the primary virus and the passed viruses of different passages of 3 lineages in group B were 93.8%–96.1%. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S in 3 highly variable (hr-) regions at aa#110–120, aa#141–151 and aa#189–194 of gp85 in 3 lineages of group A were 2 (8/4), 1(3/3) and 1.3 (4/3), however, NS/S in the same 3 hr-regions of group B were 4.1 (13/3), 4.7 (14/3) and 3.3 (11/3). This study is the first demonstration of influence of immune selective pressure on evolution of ALV-J gp85 by specific antibodies under the controlled in vitro experiments.     

High-frequency recombination within the gag gene of Rous sarcoma virus.

We isolated 28 recombinants of Rous sarcoma virus at early (24 h) and late (7 days) times after infection. These recombinants were selected for wild type in the pol and src genes and analyzed for their env and gag phenotypes. We were unable to show strong linkage between any two markers, including two markers within a single gene (gag).     

The family of env genes of avian retroviruses: molecular analysis of Rous sarcoma virus adapted to duck cells

For the elucidation of the molecular basis of RSV adaptation to conditionally permissive host from the genome library of duck embryo fibroblasts, transformed by Rous sarcoma virus in 30 passages on these cells, recombinant bacteriophages that include provirus sequences, were obtained. Complete and transformation-defective proviruses were characterized, nucleotide sequences of their env-genes were compared with their counterparts the original RSV (Pr-RSV-C) and with viruses of other subgroups (A, B, D and E). The possible relation of the revealed changes in domains coding gp85 and gp37, with the changes of chicken RSV characteristics during adaptation to duck cells is discussed.     

Regulation of cellular morphology by the Rous sarcoma virus src gene: analysis of fusiform mutants.

We have been interested in how Rous sarcoma virus (RSV) influences transformed cell morphology and compared the molecular properties of chicken embryo cells (CEC) infected with mutants of RSV that induce the fusiform transformed cell morphology with those of CEC infected by wild-type RSV, which induces the more normal round transformed cell morphology. We looked for properties shared by all fusiform mutant-infected cells, because these may be responsible for maintaining the fusiform morphology. Five different fusiform mutants, two wild-type RSVs, and one wild-type back revertant of a fusiform mutant were studied. In the fusiform mutant-infected cells, the localization and myristylation of pp60src were determined and the extent of expression of the extracellular matrix protein fibronectin was examined at both the mRNA and protein levels. The phosphorylation of vinculin on tyrosine also was examined in the same CEC. Within all fusiform mutant-transformed CEC, pp60src was dramatically absent from the adhesion plaque sites normally seen in cells transformed with wild-type RSV, and these transformed CEC all expressed more fibronectin mRNA and protein in the extracellular matrix than did the wild-type RSV-transformed CEC. The absence of pp60src from the adhesion plaques was not due to lack of myristylation of the src protein, and tyrosine phosphorylation of vinculin was not related to fibronectin expression. These results suggest that the inverse relationship between pp60src in the adhesion plaques and fibronectin expression in the extracellular matrix may be interconnected phenomena and could be related to the maintenance of the fusiform transformed morphology.     

Characterization of the Rous sarcoma virus transforming gene product

This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus transforming gene product, pp60src, using ATP or GTP as phosphate donors. The Km values for the phosphorylation of pp60src by ATP and GTP were similar (10-36 and 25-36 microM, respectively) and the Vmax values were different (5-7 and 1.5-1.7 nmol min-1 mg-1 of pp60src, respectively). The radiolabeling of pp60src by [gamma-32P] ATP was inhibited by ADP and dATP at 20-fold higher concentrations by 75 and 83%, respectively. Other nucleotides served as weaker inhibitors under the same conditions. The radiolabeling of pp60src by [gamma-32P]GTP had lower specificity for this nucleotide, since ATP, dATP, ADP, dGTP, GDP, and TTP had at least a 50% inhibitory effect. The phosphorylated products of approximate Mr = 60,000 that were produced with ATP or GTP were shown to be the same protein molecule since they both could be immunoprecipitated with antibody raised against p60src produced by bacterial recombinants. Structural analysis revealed that the use of GTP resulted in phosphorylation of a tyrosine residue on the COOH-terminal region of pp60src, apparently the same site which contains the tyrosine phosphorylated in infected cells. In contrast, the use of ATP resulted in phosphorylation of several additional tyrosine residues on the NH2-terminal region of the molecule. In thermolability studies, the t1/2 values for the phosphorylation of pp66src in preparations from wild type virus-infected chicken cells were 5.1 min for both ATP and GTP, whereas the t1/2 values for the phosphorylation of pp60src in preparations from temperature-sensitive transformation mutant-infected cells were 1.1 min for both phosphate donors. Similar observations were found with alpha-casein as substrate.