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.     

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.     

Mapping RNase T1-resistant oligonucleotides of avian tumor virus RNAs: sarcoma-specific oligonucleotides are near the poly(A) end and oligonucleotides common to sarcoma and transformation-defective viruses are at the poly(A) end.

The large RNase T1-resistant oligonucleotides of the nondefective (nd) Rous sarcoma virus (RSV): Prague RSV of subgroup B (PR-B), PR-C and B77 of subgroup C; of their transformation-defective (td0 deletion mutants: td PR-B, td PR-C, and td B77; and of replication-defective (rd) RSV(-) were completely or partially mapped on the 30 to 40S viral RNAs. The location of a given oligonucleotide relative to the poly(A) terminus of the viral RNAs was directly deduced from the smallest size of the poly(A)-tagged RNA fragment from which it could be isolated. Identification of distinct oligonucleotides was based on their location in the electrophoretic/chromatographic fingerprint pattern and on analysis of their RNase A-resistant fragments. The following results were obtained. (i) The number of large oligonucleotides per poly(A)-tagged ffagment increased with increasing size of the fragment. This implies that the genetic map is linear and that a given RNase T1-resistant oligonucleotides has, relative to the poly(A) end, the same location on all 30 to 40S RNA subunits of a given 60 to 70S viral RNA complex, (ii) Three sarcoma-specific oligonucleotides were identified in the RNAs of Pr-B, PR-C and B77 by comparison with the RNAs of the corresponding td viruses...     

Polymorphism of avian sarcoma virus src proteins.

The src gene products of seven different avian sarcoma viruses were compared. In vitro translation of virion RNA yielded products identified unambiguously as p60src in the case of two stocks of the Schmidt-Ruppin strain, three stocks of the Prague strain, the Bryan strain, and the Bratislava 77 strain of avian sarcoma virus. Differences in the electrophoretic mobility of these seven p60src proteins in sodium dodecyl sulfate-polyacrylamide gels, corresponding to variation in the apparent molecular weights ranging from 56,000 to 60,500, were observed. Antigenic variability was also found; only three of the seven viruses tested encoded a p60src, which was precipitated by antisera derived from rabbits bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Examination of the methionine-containing tryptic peptides of the seven ;60src proteins by two-dimensional mapping revealed four common peptides but marked variability in the five to eight other peptides in each protein. Clear differences in the peptide maps of p60src were observed, both between different strains of virus and within strains. In the three cases examined, p60src synthesized in transformed cells was found to be essentially identical to that synthesized in vitro. We conclude that there is significant polymorphism in the p60src proteins of the avian sarcoma viruses.     

Identification of a Packaged Cellular mRNA in Virions of Rous Sarcoma Virus

A novel messenger activity has been identified by in vitro translation of the 70S virion RNAs of a variety of avian leukosis and avian sarcoma viruses. When the 70S virion RNA complex was heat dissociated and the polyadenylated RNA was fractionated on neutral sucrose gradients, a polypeptide of 34,000 daltons (34K) was observed in the translation products of 18S polyadenylic acid-containing virion RNA. Aside from the p60(src)-related subgenomic messenger activities, this was the only prominent messenger activity that sedimented at <20S. It was determined that the 34K protein was not virally coded because (i) messenger activity for the 34K protein was not generated by mild alkaline hydrolysis of 35S genomic RNA, (ii) the 34K proteins synthesized in response to different virion RNAs had identical tryptic peptide maps, and (iii) the tryptic peptide map of the 34K protein coded for by virion RNA was identical to that of a major in vitro translation product of 34,000 daltons made from 18S uninfected chick cell polyadenylated RNA. The 18S RNA was shown to be contained within virion particles, rather than part of a cellular structure copurifying with virus preparations, by demonstrating the presence of 34K messenger activity in virion cores made from detergent-disrupted virus. This cellular mRNA, however, was not observed in the virion RNAs of Rous-associated virus types 0 and 2 avian leukosis viruses and therefore is not packaged by all avian retroviruses. Since no other cellular message has been detected by this assay, it seems likely that the 34K mRNA found in 70S virion RNA is the result of selective packaging of an abundant host cell mRNA by certain avian retroviruses.     

Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses.

Thirteen clones of hybrid cells which synthesize antibodies directed against the Rous sarcoma virus (RSV) transforming protein, pp60src, were isolated. Mouse myeloma cells were fused with spleen cells from mice that had been immunized with purified pp60src from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were screened by immunoprecipitation of pp60src from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp60src encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellular homolog of pp60src. Antibody from clone 261 had a high affinity for the viral yes gene product, and antibodies from clones 443 and 463 recognized the transforming proteins encoded by viruses containing the related transforming genes fps and ros. Several other clones had a low affinity for the viral yes, fps, and ros gene products which could be detected by in vitro phosphorylation of the transforming proteins after immunoprecipitation with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60src and casein in an immune complex-bound reaction.     

Protein-coding potential of mouse mammary tumor virus genome RNA as examined by in vitro translation.

The protein-coding capacity of the mouse mammary tumor virus genome has been examined by in vitro translation of genome length and polyadenylated subgenomic fragments of viral RNA. Intact genome RNA of about 35S programmed synthesis of the Pr77gag, Pr110gag and Pr160gag/pol precursors seen in infected cells in vivo. Polyadenylated RNA fragments of 18 to 28S encoded products whose tryptic peptide maps resembled those of the nonglycosylated precursor to the envelope glycoproteins, confirming the gene order 5'-gag-pol-env-3'. Translation of polyadenylated RNA fragments smaller than 18S yielded a series of related proteins whose peptide maps bore no resemblance to any of the virion structural proteins. Thus, a region of the mouse mammary tumor virus genome distal to the env gene appears to have an open reading frame sufficient to encode at least 36,000 daltons of protein as of yet unknown function.     

Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

Sac(-) cells infected with murine coronavirus strain JHM shut off host cell protein synthesis and synthesized polypeptides with molecular weights of 150,000, 60,000, and 23,000. The 60,000- and 23,000-molecular-weight polypeptides comigrated with virion structural proteins p60 and p23, and the 60,000-molecular-weight protein was identified as p60 by tryptic peptide fingerprinting. Polyadenylate-containing RNA [poly(A) RNA] extracted from the cytoplasm of infected cells directed the synthesis of both 60,000- and 23,000-molecular-weight polypeptides in messenger-dependent cell-free systems derived from mouse L-cells and rabbit reticulocytes. The reticulocyte system also synthesized a 120,000-molecular-weight polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity of the 60,000- and 23,000-molecular-weight in vitro products was established by comigration with virion proteins, immunoprecipitation, and in the case of p60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNAs which encoded p60 and p23 sedimented in sucroseformamide gradients at 17S and 19S, respectively, and were clearly separable. These RNAs were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that they represent physiological mRNA's. These results suggest that the expression of the coronavirus JHM genome involves more than one subgenomic mRNA.     

Metabolism of viral RNA in murine leukemia virus-infected cells; evidence for differential stability of viral message and virion precursor RNA

Molecular hybridization techniques were used to examine the stability of viral message and virion precursor RNA in murine leukemia virus-infected cells treated with actinomycin D. Under the conditions used, viral RNA synthesis was inhibited, but viral protein synthesis continued, and the cells produced noninfectious particles (actinomycin D virions) lacking genomic RNA (J. G. Levin and M. J. Rosenak, Proc. Natl. Acad. Sci. U.S.A. 73:1154-1158, 1976). Analysis of total RNA in virions revealed that the amount of hybridizable viral RNA decreased steadily after the addition of actinomycin D and by 8 h was 10% of the control value. Studies on fractionated viral RNA showed that this low level of hybridization is due to residual 70S RNA in the virion population. The results indicated that viral RNA which is destined to be encapsidated into virions has a half-life of approximately 3 to 4 h. In contrast, other intracellular virus-specific RNA molecules appeared to be quite stable and persisted for a long period of time, with a half-life of at least 12 h. These observations support the idea that two independent functional pools of 35S viral RNA exist within the infected cell: one serving as message and the other as precursor to virion RNA. The existence of two viral RNA pools was further documented by the finding that 12 h after the addition of actinomycin D, when virion precursor RNA was depleted, 35S and 21S viral nRNA species could be identified in polyribosomal RNA as well as in total polyadenylated cell RNA. Surprisingly, 35S and mRNA declined more rapidly than did 21S mRNA, which appeared to be increased in amount.     

Defective interfering passages of Sindbis virus: nature of the defective virion RNA.

Defective interfering particles of Sindbis virus contain 20S RNA identical to that found in BHK cells co-infected with standard and defective virions. We have characterized these RNAs by their oligonucleotide fingerprints. Most of the oligonucleotides were identical to those found in the mRNA (26S RNA) that codes for the virion structural proteins. Three oligonucleotides found in 20S RNA were absent from the 26S RNA pattern and may represent sequences from the 5' end of the virion RNA. Previous difficulties in describing the nature of the defective virion RNA were due to the aggregated state of the RNA. Nucleocapsids obtained from standard and defective virions were essentially the same size and had about the same density, suggesting that defective particles contain more than a single molecule of 20S RNA.     

Nucleic acids of respiratory syncytial virus.

Analysis of purified respiratory syncytial virus revealed that the virion RNA was composed of 50S, 28S, 18S, and 4S species. The 18S and 28S species were presumed to represent host rRNA since virus grown in actinomycin D-treated cells contained only 50S and 4S RNAs. Actinomycin D treatment stimulated production of infectious respiratory syncytial virus 5- to 10-fold. The 50S virion RNA was shown to hybridize with polyadenylated mRNA's isolated from infected cells, indicating that respiratory syncytial virus RNA is of negative-strand sense. Six mRNA's were identified by polyacrylamide gel electrophoresis.     

Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85.

Subgroup D avian sarcoma and leukosis viruses can penetrate a variety of mammalian cells in addition to cells from their natural host, chickens. Sequences derived from the gp85-coding domain within the env gene of a mammal-tropic subgroup D virus (Schmidt-Ruppin D strain of Rous sarcoma virus [SR-D RSV]) and a non-mammal-tropic subgroup B virus (Rous-associated virus type 2) were recombined to map genetic determinants that allow penetration of mammalian cells. The following conclusions were based on host range analysis of the recombinant viruses. (i) The determinants of gp85 that result in the mammal tropism phenotype of SR-D RSV are encoded within the 160 codons that lie 3' of codon 121 from the corresponding amino terminus of the gp85 protein. (ii) Small linear domains of the SR-D RSV gp85-coding domain placed in the subgroup B background did not yield viruses with titers equal to that of the subgroup D virus in a human cell line. (iii) Recombinant viruses that contained subgroup D sequences within the hr1 variable domain of gp85 showed modest-to-significant increases in infectivity on human cells relative to chicken cells. A recombinant virus that contained three fortuitous amino acid substitutions in the gp85-coding domain was found to penetrate the human cell line and give a titer similar to that of the subgroup D virus. In addition, we found that the subgroup D virus, the mutant virus, and recombinant viruses with an increased mammal tropism phenotype were unstable at 42 degrees C. These results suggest that the mammal tropism of the SR-D strain is not related to altered receptor specificity but rather to an unstable and fusogenic viral glycoprotein. A temperature sensitivity phenotype for infectivity of mammalian cells was also observed for another mammal-tropic avian retrovirus, the Bratislava 77 strain of RSV, a subgroup C virus, but was not seen for any other avian retrovirus tested, strengthening the correlation between mammal tropism and temperature sensitivity.     

Translation of HTLV (human T-cell leukemia virus) RNA in a nuclease-treated rabbit reticulocyte system

A human type-C retrovirus, designated HTLV (human T-cell leukemia virus), was isolated from the HTLV producer cell line MT-2. Agarose gel electrophoresis analysis 32P-labeled HTLVMT-2 virion RNA revealed that HTLVMT-2 virion RNA consists mainly of 24S and small amounts of 35S and 32S RNAs. The 24S HTLVMT-2 virion RNA and unfractionated HTLVMT-2 virion RNA were translated in a rabbit reticulocyte lysate system in vitro. The predominant polypeptide synthesized from 24S RNA had an apparent mol. wt. of 28 000 (28 K); unfractionated HTLVMT-2 virion RNA directed the synthesis of 53 000 (53 K), 33 000 (33 K) and 28 000 (28 K) polypeptides as main components. Most of the polypeptides synthesised in vitro by translation of HTLVMT-2 virion RNAs possess the same sizes as the proteins formerly designated as ATLA (ATL-associated antigen) in SDS-polyacrylamide gel electrophoresis and immunologically precipitated with sera of ATL patients. Therefore, the antigens termed ATLA, found by the serological study of ATL, are HTLVMT-2 encoded polypeptides.     

Messenger Activity of Virion RNA for Avian Leukosis Viral Envelope Glycoprotein

An intracellular assay for viral envelope glycoprotein (env) messenger was employed to analyze the RNA from virus particles of Rous-associated virus type 2. For this assay RNA was microinjected into cells infected by the env-deficient Bryan strain of Rous sarcoma virus [RSV(-) cells]. Only when the injected RNA could be translated by the recipient cells to produce viral envelope glycoprotein was the env deficiency of the RSV(-) cells complemented, enabling them to release focus-forming virus. RNA in a 21S size fraction from the Rous-associated virus particle promoted the release of numerous focus-forming virus from RSV(-) cells, whereas the major 35S virion RNA species was inactive. The env messenger activity sedimented as a sharp peak with high specific activity. RNase T1-generated fragments of virion 35S RNA were unable to promote the release of infectious virus from RSV(-) cells. Consequently, the active molecule was most likely to be env messenger which had been encapsulated by the virus particle from the cytoplasm of infected cells. Approximately 95% of the env messenger within the virion was associated with the virion high-molecular-weight RNA complex. The temperature required to dissociate env messenger from the high-molecular-weight complex was indistinguishable from the temperature required to disrupt the complex itself. Virion high-molecular-weight RNA that was associated with env messenger sedimented slightly more rapidly than the bulk virion RNA; this was the strongest evidence that the 21S messenger had been encapsulated directly from the infected cells. These data are considered along with a related observation [concerning the prolonged expression of env messenger after injection into RSV(-) cells] to raise the possibility that virus-encapsulated env messenger can become expressed within subsequently infected cells.     

Immune response to the src gene product in mice bearing tumors induced by injection of avian sarcoma virus-transformed mouse cells.

A single subcutaneous injection of 10(7) live cells of the highly tumorigenic avian sarcoma virus (Schmidt-Ruppin strain, subgroup D)-transformed BALB/c line into BALB/c mice resulted in the production of an antiserum specific for the avian sarcoma virus gene product pp60src. All sera taken from mice 3 weeks after injection of tumor cells contained antibodies to pp60src. Immunoprecipitation experiments showed that all sera precipitated pp60src from Schmidt-Ruppin-infected chicken cells, but only a portion of these sera precipitated pp60src from chicken cells infected with other strains of avian sarcoma virus, i.e., Prague and Bratislava-77. Analysis of the cross-reactivity patterns of these antisera demonstrated a minimum of three to four antigenic determinants on pp60src. The findings reported here should facilitate the production of monoclonal antibodies to pp60src, which in turn will provide highly specific probes for further investigations into the structure and function of this protein.