In vitro translation of avian erythroblastosis virus RNA: Identification of two major polypeptides

RNA of avian erythroblastosis virus (AEV) pseudotyped with RAV-1, AEV(RAV-1), and its fragments containing poly(A) were translated in an mRNA-dependent reticulocyte lysate. p74 and 46 were found to be AEV RNA specific out of several main products, pr76, p74, p46, and p37. p74 was translated from 30 S AEV RNA and contained some peptides homologous to pr76, but most peptides as specific. On the other hand p46 was translated from 20 S AEV RNA containing poly(A). However, no relation to known viral proteins was established. This preliminary observation suggests that the 5′-half of AEV genome encodes p74 and the 3′-half p46.     

RNA specific for the transforming component of avian erythroblastosis virus strain R.

Avian erythroblastosis virus strain R (AEV) contains two species of RNA, 35 S and 30 S, when rescued from nonproducing AEV-transformed chicken cells as pseudotypes of Rous-associated virus-1 (RAV-1) or of AEV-associated virus (REAV). Biological and electrophoretic data suggest that the 30 S RNA is specific for the transforming component of AEV. In fingerprint analysis of RNase T₁-resistant oligonucleotides the 30 S RNA of AEV(RAV-1) yields 13 distinct oligonucleotide spots, 7 of which are unique to the 30 S RNA of AEV, the others are common to RAV-1. The fingerprint of 30 S RNA obtained from AEV(REAV) revealed a similar pattern of unique and shared oligonucleotides. These data suggest that the genome of AEV strain R contains specific genetic sequences which may code for a product that causes transformation of the host cell.     

Cell-free translation of purified avian sarcoma virus src mRNA

Avian sarcoma virus 21 S RNA, purified by hybridization from virus-infected cells, was translated in a cell-free system. The major product of translation was a protein of 60,000 daltons. This protein was the same as authentic pp60^src, the product of the ASV src gene, when compared by electrophoretic mobility in polyacrylamide gels, immunological reactivity and partial protease digestion. These findings confirm that the 21 S ASV RNA serves as mRNA for pp60^src. Furthermore, pp60^src is the only major product of translation of the src gene and is apparently synthesized without a cleavable signal sequence.     

Site-specific mutagenesis of avian erythroblastosis virus: erb-B is required for oncogenicity

Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds. Two domains within its replication-defective genome, erb-A and erb-B, have been implicated in AEV-mediated oncogenesis. An efficient transfection system for generating infectious, transforming virus from molecular clones of AEV and RAV-1 (helper virus) was combined with the techniques of site-specific mutagenesis to investigate the contribution of erb-B to the two forms of oncogenesis induced by AEV. Deletion and frameshift mutations were constructed in the erb-B locus of cloned AEV DNA in vitro. Infectious retroviruses harboring these mutations were recovered and their ability to transform fibroblasts in vitro or induce erythroleukemia in vivo was assessed. The presence of mutant viral genomes in chick embryo fibroblasts or erythroblasts of infected birds was confirmed by suitable biochemical analyses. Expression of viral genes in cells infected with AEV mutants was examined by immunoprecipitation with antisera to erb-A and erb-B proteins. It was found that the product of erb-B is necessary for transformation of fibroblasts and induction of erythroblastosis by AEV, although a small portion of this protein at the carboxy terminus is dispensable.     

Transformation parameters of chicken embryo fibroblasts infected with the ts34 mutant of avian erythroblastosis virus

A mutant of avian erythroblastosis virus, ts34 AEV, previously shown to be temperature sensitive for the maintenance of erythroblast transformation, was examined for its effect on the expression of transformation parameters in chicken embryo fibroblasts. Focus formation and growth of colonies in semisolid agar as well as the disarrangement of actin cables and of LETS protein fibrils were not affected by the incubation of infected cells at 41°. In contrast, plasminogen activator protease, rate of hexose uptake, and sarcomagenicity were found to be expressed in a temperature-dependent manner. Our results suggest that the same gene required for the transformation of erythroblasts by AEV is also required for the transformation of fibroblasts.     

The genomic RNA of avian reticuloendotheliosis virus REV

Purified virus obtained from a subline of chicken bone marrow cells transformed by avian reticuloendotheliosis virus (REV) was found to contain the RNA of REV in excess over the RNA of its associated helper virus REV-A. Electrophoretic and sedimentation analyses resolved these RNAs into a 28 S and a 34 S component, respectively. Comparison of these RNA species with the RNA obtained from plaque-purified preparations of REV-A confirmed that the 28 S RNA represents the genome of transforming REV. The small size of 28 S REV RNA suggests that the defectiveness of REV is due to a deletion of replicative sequences. Hybridization experiments indicated that about 25–30% of REV RNA sequences are unrelated to REV-A. These may include the putative transforming sequences of REV. REV shared 12–15 of 42 identifiable large RNase T₁-resistant oligonucleotides with REV-A. The 28 S REV RNA did not contain the transformation-specific oligonucleotides which are largely conserved among avian acute leukemia viruses MC29, MH2, and CMII or the src-specific oligonucleotides of avian sarcoma viruses. It is concluded that the sequences which are unique for REV contain a new class of avian tumor virus transforming genes.     

In vitro translation products of turnip crinkle virus RNA

Turnip leaves infected with turnip crinkle virus accumulate a 35-kd polypeptide which comigrates with the major protein from isolated virions. RNA from TCV virions directs the synthesis of a number of polypeptides in vitro including a 35-kd protein which is immunoprecipitable with antiserum to virus particles. Translation of viral RNA size-fractionated on sucrose gradients or methyl mercurichydroxide-containing gels shows that the TCV coat protein is synthesized primarily from RNA fragments which are smaller than the genomic RNA. A satellite RNA species found in virions does not direct the synthesis in vitro of any identifiable protein.     

Cell-free translation of tobacco vein mottling virus RNA

Tobacco vein mottling virus (TVMV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system. The RNA was approximately 5% as active as rabbit globin mRNA in directing protein synthesis. Translation was stimulated approximately 15% by the cap analog pppm7G, a phenomenon which has also been observed with uncapped viral RNAs. Treatment with NaIO4 and NaB(3H]4 under conditions which label the caps of globin and ovalbumin mRNA failed to produce labeled cap structures in TVMV RNA. Approximately 20 polypeptides, ranging from 20,000 to 100,000 daltons, were produced in the reticulocyte system programmed with TVMV RNA. The predominant species (P75) had a molecular weight of 75,000. The same major polypeptides were produced in the wheat germ system, but there was relatively greater synthesis of the smaller polypeptides. Incubation of the reticulocyte system in the presence of cycloheximide following an initial period of synthesis failed to cause breakdown of larger polypeptides into smaller polypeptides. Preincubation of TVMV RNA with the reticulocyte system before addition of labeled amino acids caused greater synthesis of the smaller polypeptides, suggesting that they are derived from fragments of the RNA produced during the incubation. Translation of TVMV RNA which had been bound to oligo(dT)-cellulose yielded nearly the same spectrum of polypeptides, but synthesis of polypeptides smaller than 52,000 daltons was reduced. At low ionic strength, only polypeptides of 52,000 daltons and smaller were synthesized. At high ionic strength, P75 was essentially the only product synthesized. Antibody to whole virus precipitated many of the polypeptides, including P75, suggesting that they contain the coat protein sequence. No polypeptide with the electrophoretic mobility of authentic coat protein, however, was precipitated. Comparison of partial proteolytic digests of P75 and authentic coat protein provided further evidence for sequence homology.     

Translation of potyvirus RNA in a rabbit reticulocyte lysate: Cell-free translation strategy and a genetic map of the potyviral genome

The translations of tobacco etch virus (TEV) RNA and pepper mottle virus (PeMV) in a rabbit reticulocyte lysate were analyzed to determine the effect of RNA quality on template activity and to identify all the products of the potyviral genome. The size distribution of the in vitro translation products, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was dependent on RNA quality. Full-length RNA stimulated the cell-free synthesis primarily of a 87,000 (87K) molecular weight product for TEV and a 78K product for PeMV. RNA with limited fragmentation stimulated a number of discrete products, while highly fragmented RNA did not act as a template. RNA monocistronic for capsid protein was not detected. Four of the six discrete translation products were identified by reactivity with antisera to four virus-specific proteins. A number of other products, which were consistently immunoprecipitated with more than one antiserum, were detected. Products were presumed to be premature terminations and/or gene readthroughs on the basis of serological reactions and of molecular weight estimates. These readthroughs were useful in linking genes and in constructing a genetic map of the potyviral genome. The proposed genetic map for TEV is as follows: 5' end-87K protein gene-49K nuclear inclusion protein gene-50K protein gene-70K cylindrical inclusion protein gene-54K nuclear inclusion protein gene-30K capsid protein gene-3' end. The proposed genetic map for PeMV is as follows: 5' end-78K protein gene-49K protein gene-41K protein gene-68K cylindrical inclusion protein gene-56K protein gene-33K capsid protein gene-3' end. The proposed genetic map accounts for 95% of the estimated coding capacity of the TEV RNA and 93% of the PeMV RNA.     

The protease inhibitor TLCK alters the apparent molecular weights of some measles virus proteins by a mechanism unrelated to inhibition of proteolytic cleavage

Limited DNase I digestion and subsequent restriction enzyme analysis were used to compare the chromatin conformation of different proviral genome copies in M-MuLV-induced tumors of Mov-3 mice. The results show that the endogenous, genetically transmitted viral genome copy is highly resistant, whereas the somatically acquired genome copies are sensitive to digestion with DNase I.     

Turnip crinkle virus RNA and its translation in rabbit reticulocyte and wheat embryo extracts

Turnip crinkle virions contain genomic RNA, Mr approximately 1.4 x 10(6), small RNA, Mr approximately 1.5 x 10(5) and a heterogeneous collection of intermediate size. Turnip crinkle virion RNA was translated in rabbit reticulocyte lysates into three major unrelated products of molecular weight 38,000 (p38, the virion coat protein), 36,000 (p36), and 25,000 (p25). The same three products, but in different proportion, were synthesized in wheat embryo extracts. Certain intermediate size RNA fractions translated into p38 and p36 products much more efficiently than did unfractionated RNA. The 1.5 x 10(5) molecular weight RNA did not induce amino acid incorporation. We propose a translation map of the TCV genome, based on tryptic analyses of the major products and of putative read-through proteins.     

Antigenic and structural studies on the transforming proteins of Rous sarcoma virus and Yamaguchi 73 avian sarcoma virus

A widely cross reactive antiserum raised against denatured pp60v-src, the transforming protein encoded by Rous sarcoma virus, was used to test antigenic relationships with the transforming gene products encoded by other avian sarcoma viruses. The results showed that P90gag-yes, the transforming protein of a representative of Class III avian sarcoma viruses, is antigenically related to pp60v-src. Tryptic phosphopeptide analysis of P90gag-yes revealed two phosphotyrosine-containing peptides and one phosphoserine-containing peptide. One of the phosphotyrosine-containing peptides comigrated with the phosphotyrosine-containing tryptic peptide from pp60v-src.     

Cell-free synthesis of enzymatically active vaccinia virus thymidine kinase

Mouse L cells that lacked thymidine kinase (L_tk^? cells) were infected with wild-type vaccinia virus or with a mutant that was deficient in the viral thymidine kinase. Cytoplasmic messenger RNA preparations were isolated 2 hr after infection and translated in a messenger RNA-dependent reticulocyte lysate. Both messenger RNA preparations directed the efficient synthesis of early vaccinia virus polypeptides. Furthermore, translation of the messenger RNA from cells infected with wild-type virus resulted in a substantial increase in the thymidine kinase activity of the lysate which was prevented by inhibitors of cell-free protein synthesis. Translation of the messenger RNA from cells infected with the viral tk^? mutant, or from uninfected L_tk^? cells, did not increase the thymidine kinase activity of the extract. The enzyme synthesized in vitro was compared with that isolated from vaccinia virus-infected cells. The two enzymes were indistinguishable with respect to their rates of thermal inactivation, their sedimentation coefficients in glycerol gradients, and their relative electrophoretic mobilities in nondenaturing polyacrylamide gels. We conclude that active viral thymidine kinase can be synthesized de novo in a cell-free system directed by early messenger RNA from vaccinia virus-infected cells.     

Three low molecular weight RNA species detected in tobacco ringspot virus

A culture line of tobacco ringspot virus (TRSV) has changed in sedimentation properties and does not resolve into components normally found in this virus. Virus preparations contain a large proportion of a new type of particle whose protein shell is structurally and immunologically the same as normal TRSV capsids but encapsidates three species of RNA with molecular weights of approximately 114,000, 245,000, and 367,000 daltons.     

The 28 S genomic RNA of avian sarcoma virus PRCII codes for the transformation-specific polyprotein P105

We have characterized the genomic RNA of the defective avian sarcoma virus PRCII. Replicating virus which consists of transforming PRCII and a nontransforming but replication-competent helper, PRCII-AV, contains two RNA species. One is identical in size to the 35 S genome of avian leukosis helper viruses. The second component migrates slightly faster than 28 S ribosomal RNA in polyacrylamide gel electrophoresis but co-sediments with this RNA in sucrose gradients. In vitro translation across a gradient of velocity-sedimented poly(A)-containing PRCII virion RNA yielded three major proteins. The virion protein precursors Pr76^gag and Pr180^gag-pol were translated from 35 S RNA, while the transformation-specific polyprotein P105 was translated from 28 S RNA. P105 may be the only protein coded for by the PRCII genome, although this product would not exhaust the coding capacity of 28 S RNA. Whether translated in vitro or immunoprecipitated from transformed cells, P105 was essentially identical as demonstrated by comparative peptide maps.     

A temperature-sensitive lesion affecting levels of transformation-specific viral RNA in a mutant of avian sarcoma virus PRCII

LA46 is a mutant of avian sarcoma virus PRCII that is temperature sensitive (ts) in the maintenance of oncogenic transformation and in the production of infectious virus. These ts properties are independent of the helper virus. At the nonpermissive temperature LA46-infected cells also fail to produce significant quantities of the transformation-specific protein, and the amount of intracellular transformation-specific viral RNA is reduced at least 100-fold. The probable primary defect of LA46, a temperature sensitivity in transformation-specific viral RNA, can account for the phenotype of the mutant. The molecular mechanism of this defect is currently investigated.     

Characteristics of avian sarcoma virus strain PRCIV and comparison with strain PRCII-p

Avian sarcoma virus strain Poultry Research Centre IV (PRCIV) (J. G. Carr and J. G. Campbell (1958)Brit. J. Cancer12, 631–635) is highly oncogenic in chickens and transforms chicken embryo fibroblasts in culture. It is defective in replication, unable to code for complete functional products of the three essential viral genes gag, pol, or env. Nonproducing cells transformed by PRCIV lack the transforming protein of Rous sarcoma virus, pp6^src but synthesize a new transformation-specific protein of 170,000 MW. This P170 contains partial gag sequences, probably at its N-terminus and transformation-specific, cell-derived sequences, probably in the C-terminal portion of the molecule. In two-dimensional tryptic peptide maps P170 of PRCIV is indistinguishable from the transformation-specific protein P170 of the previously described PRCII-p (M. L. Breitman, J. C. Neil, C. Moscovici, and P. K. Vogt (1981)Virology108, 1–12). Both P170 proteins also contain all but one tryptic peptide of the transformation-specific gag-linked protein P105 found in cells transformed by avian sarcoma virus PRCII. The P170 proteins of PRCIV and PRCII-p have an associated tyrosine-specific kinase activity similar to P105 of PRCII. All three agents, PRCII, PRCII-p, and PRCIV, belong to class II of avian sarcoma viruses. In cells transformed by PRCIV or PRCII-p secondary gag-linked transformation-specific proteins of less than 170,000 MW are sometimes seen, but limited in vivo and in vitro passage of the viruses shows the P170 to be a stable characteristic of PRCIV and PRCII-p transformation. The genomic RNA of PRCIV and PRCII-p sediments at 30 S corresponding to a size of about 6.1 kilobases.