Biosynthesis of reovirus-specified polypeptides: Initiation of reovirus messenger RNA translation in vitro and identification of methionyl-x initiation peptides

The initiation of reovirus messenger RNA-directed protein synthesis in vitro was investigated in a cell-free protein synthesizing system prepared from Krebs ascites tumor cells. The principal translation products of the mixture of 10 reovirus mRNA species transcribed in vitro by reovirus cores were polypeptides μ₀, μ₁, σ_2a, and σ₃. Translation could be initiated with formylmethionine transferred from rat liver methionyl-tRNA formylated by Escherichia coli formyltransferase with 10-formyltetrahydrafolate as the formyl donor. Formylmethionine incorporation was complete within 10 to 15 min and was inhibited by aurin tricarboxylic acid and pactamycin; by contrast, incorporation of methionine and leucine continued for 30 to 60 min.The identification of the amino acids at the amino termini of polypeptides μ₀, μ₁, σ_2a, and σ₃ synthesized in vitro was elucidated. Protein synthesis was carried out in the presence of rat liver formylmethionyl-tRNA and various groups of radioactively labeled amino acids. The viral polypeptides that were synthesized were isolated by urea-SDS-polyacrylamide gel electrophoresis and digested with pronase. N-Formylmethionylcontaining peptides were then separated from other peptides by fractionation on Dowex-50 and hydrolyzed with acid. The radioactive amino acids that were liberated were then identified by two-dimensional thin-layer chromatography. The following amino terminal assignments were elucidated: μ₀, (N-formyl)methionyl-valyl-(proline); μ1, (N-formyl)methionyl-leucyl-valine; σ_2a, (N-formyl)methionyl-threonyl-valine; and σ₃, (N-formyl)methionyl-valyl-tyrosyl-(proline).No evidence was obtained for amino terminal acetylation or formylation of reovirus-specific protein synthesized in vitro in the absence of exogenously added formylmethionyl-transfer RNA.     

Cell-free translation of soil-borne wheat mosaic virus RNAs

The genome of soil-borne wheat mosaic (SBWMV) virus appears to be composed of two RNAs. Three strains have the same large RNA, designated 1.0L RNA, but differ in the size of the smaller RNA, designated 0.5L RNA for wild type (WT), 0.4L RNA for mutant Lab 2, and 0.35L RNA for mutant Lab 1, where L corresponds to approximately 6700 nucleotide residues. The major translation products of 1.0L RNA in rabbit reticulocyte lysates had apparent molecular weights of 180,000 (180K), 152K, 135K, 80K, and 45K. None of these were precipitated with antiserum against virions. The 0.5L RNA stimulated the synthesis of products of 90K, 28K, and 19.7K, the 0.4L RNA of 66K, 28K, and 19.7K, the 0.35L RNA of 55K, 28K, and 19.7K. Protein of 19.7K comigrated with viral coat protein and was the predominant product in all cases. Immunoprecipitation, peptide mapping, and the time course of appearance of products suggest that the larger products of RNA Its (0.5L, 0.4L, and 0.35L RNA) arise from readthrough. The pattern of products is consistent with formation of 0.4L and 0.35L by internal deletions in the 3' region of 0.5L RNA. Extracts of SBWMV-WT-infected wheat contained polypeptides that corresponded to the translation products of 0.5L RNA in electrophoretic mobility and immunological reactivity.     

Biosynthesis of reovirus-specified polypeptides: the reovirus s1 mRNA encodes two primary translation products

Reovirus serotypes 1 (Lang strain) and 3 (Dearing strain) code for a hitherto unrecognized low-molecular-weight polypeptide of Mr approximately 12,000. This polypeptide (p12) was synthesized in vitro in L-cell-free protein synthesizing systems programmed with either reovirus serotype 1 mRNA, reovirus serotype 3 mRNA, or with denatured reovirus genome double-stranded RNA, and in vivo in L-cell cultures infected with either reovirus serotype. The synthesis of p12 in vivo was insensitive to actinomycin D, and occurred at similar times after infection as the previously identified reovirus encoded lambda, mu, and sigma polypeptides. Pulse-chase experiments in vivo, and the relative kinetics of synthesis of p12 in vitro, indicate that it is a primary translation product. Fractionation of reovirus mRNAs by velocity sedimentation and translation of separated mRNAs in vitro suggests that p12 is coded for by the s1 mRNA, which also codes for the previously recognized sigma 1 polypeptide. Synthesis of both p12 and sigma 1 in vitro in L-cell-free protein synthesizing systems programmed with denatured reovirus genome double-stranded RNA also suggests that these two polypeptides can be coded by the same mRNA species. The Mr approximately 12,000 polypeptide was not a detectable structural component of purified virions, and antiserum prepared against purified reovirions did not immunoprecipitate p12. It is proposed that the Mr approximately 12,000 polypeptide encoded by the S1 genome segment be designated sigma 1bNS, and that the polypeptide previously designated sigma 1 be renamed sigma 1a.     

A comparative study on the translation of cardiovirus RNAs in rabbit reticulocyte lysates

In rabbit reticulocyte lysates the RNAs of encephalomyocarditis (EMC) virus, mengovirus, and Mous-Elberfeld (ME) virus directed the synthesis of similar sets of products. Moreover, the viral protease synthesized from any one of the three viral RNAs could cause cleavage of the viral capsid precursor proteins synthesized from any of the three RNAs. However, the three RNAs differed in their dependence on tRNA supplementation (to the lysates) for effective translation. In the absence of tRNA supplementation, synthesis of 5'-derived proteins of EMC viral RNA proceeded normally, but little synthesis of the proteins coded by the remaining portion of the viral genome occurred. In the case of mengoviral RNA, omission of tRNA supplementation caused mostly a generalized reduction of the synthesis of all viral proteins. In contrast, synthesis of ME viral proteins stopped almost completely in the absence of tRNA supplementation.     

Characterization of the cell-free translation products of carnation mottle virus genomic and subgenomic RNAs

The in vitro translation products of carnation mottle virus (CarMV) genomic and subgenomic RNAs were analysed using a rabbit reticulocyte lysate system. Viral RNAs directed synthesis of three main polypeptides, p80, p40, and p34. p40, which was the predominant product using unfractionated virion RNA as template, was identified as coat protein based on electrophoretic mobility through SDS-polyacrylamide gels and immunoprecipitation with anti-CarMV serum. Upon size-fractionation of virion RNAs by sucrose gradient centrifugation and subsequent translational analysis, p40 was found to be encoded by subgenomic RNA, whereas p80 and p34 were synthesized from templates of genome length. p80 and p34 were found to contain common or overlapping amino acid sequences by analysis of cleavage peptides formed during proteolysis with alpha-chymotrypsin. These data support CarMV translational mechanisms other than those proposed in a previous study (R. Saloman, M. Bar-Joseph, H. Soreq, I. Gozes, and U. Z. Littauer, 1978,Virology 90, 288-298).     

Comparison of messenger RNAs induced in cells infected with each member of the morbillivirus group

Virus-specific mRNAs radiolabelled with [32P]orthophosphate in the presence of actinomycin D were extracted from the cytoplasm of Vero cells infected with each of the known morbilliviruses: measles virus, canine distemper virus, rinderpest virus, and peste des petits ruminants virus. When analysed on denaturing agarose-formaldehyde gels the major RNA species from all viruses in the group were identical, except for canine distemper virus where one of the virus-specific mRNAs (mRNA 5), which probably codes for the virus haemagglutinin (S.E.H. Russell, D. K. Clarke, E. M. Hoey, B. K. Rima, S. J. Martin, J. Gen. Virol. 66, 433-441 (1985], was significantly smaller than the corresponding mRNA induced by the other viruses. Plasmid DNA containing a virus-specific insert, representing greater than 98% of the gene derived from the P-protein mRNA of canine distemper virus, showed significant cross-hybridisation with all the other members of the morbillivirus group.     

Presence in infected cells of nonvirion proteins encoded by adenovirus messenger RNAs of the major late transcription regions L0 and L1

The adenovirus major late promoter functions at early and intermediate times to produce a limited set of mRNAs that appear in the cytoplasm of productively infected HeLa cells. These mRNAs may be translated in cell-free systems to produce two unrelated polypeptides of approximately 13,500 Mr (L0-13.5K and L0-13.6K) and a pair of related polypeptides of approximately 55,000 Mr (the L1-52K/55K proteins). Radiochemical protein sequence analysis of in vitro synthesized proteins has identified the N-terminal sequences of the L0-13.5K and L0-13.6K proteins (J. B. Lewis and C. W. Anderson (1983), Virology 127, 112-123). Additional sequence analyses confirmed the identification of the open reading frame for the L0-13.5K protein, and identified the ATG encoded by nucleotides 11,040 to 11,042 from the left end of the adenovirus genome as the initial codon of the L1-52K/55K protein. Antisera raised against synthetic peptides homologous to these three amino termini were used to demonstrate the presence of the L0-13.5K protein, the L0-13.6K protein, and the L1-52K/55K proteins in extracts of HeLa cells infected by adenovirus 2. The L0-13.5K protein was detected at early, intermediate, and late times after infection. The L0-13.6K and L1-52K/55K proteins were detected only at late times. Immunofluorescence microscopy indicated that the L0-13.6K protein is distributed around the periphery of the nucleus and along fibers running the length of the cell. Nonpermeabilized infected cells were stained by anti-L0-13.6K peptide serum at a single spot on the cell surface. Neither the L0-13.6K nor the L1-52K/55K proteins were detected in purified virus.     

The in vitro translation of Pea enation mosaic virus RNA

Pea enation mosaic virus (PEMV) RNA was isolated from virions and then translated in rabbit reticulocyte lysates and in wheat germ extracts. RNA 1 (apparent molecular weight, Mr 1.77 x 106) was shown to code for two major translation products, vp2 (Mr 88,000) and vp4 (Mr 36,000) plus a high-molecular-weight minor product vp1 (Mr 147,000). Tryptic peptide comparisons of vp2 and vp4 revealed unique amino acid sequences for each product indicating that vp2 was not a read through protein of vp4. The in vitro translation products were synthesized in the order of their molecular weight, with vp4 appearing first. Therefore, vp4 and vp2 were not produced from a slowly processed precursor. RNA 2 (Mr 1.2 x 106) was shown to code for one product, vp3 (Mr 45,000). Northern analysis of total virion RNA, which was hybridized to cDNA transcribed from total virion RNA, did not detect additional RNA species. Therefore, gene products, vp2, vp3, and vp4, appear to be translated from the two genomic RNAs rather than from encapsidated subgenomic RNAs. PEMV antiserum specifically immunoprecipitated vp2, indicating that vp2 has amino acid sequences that are related to those of capsid protein.     

Molecular cloning of the complete genome of reovirus serotype 3

All 10 genes of the Dearing strain of reovirus serotype 3 have been cloned in their entirety into pBR322. This has been proved by sequencing the terminal regions (50-100 residues long) of all 10 cloned genes; all such regions were found to be identical to the corresponding terminal regions of the double-stranded RNA genes that are present in reovirus particles. The lengths of the cloned reovirus genes were estimated by comparing their electrophoretic mobilities (and those of their PstI cleavage fragments) with those of accurately known marker DNA molecules. The genes range in size from about 3825 bp for the L genes to about 1200 bp for the S4 gene.     

Comparative studies of the 3''-terminal sequences of several alpha virus RNAs

We have investigated the mechanism by which ribavirin (Rbv), mycophenolic acid, and 2-amino-1,3,4-thiadiazole inhibit the replication of Sindbis virus in Aedes albopictus cells. In each case there was a good correlation between inhibition of virus replication and a reduction (80–90%) in the level of cellular GTP. The antiviral effects of all three compounds could be reversed by (1) equimolar amounts of xanthosine but not by guanosine, (2) actinomycin D (0.2 μg/ml), and (3) α-amanitin (10 μg/ml) in α-amanitin-sensitive cells but not in α-amanitin-resistant cells. In the case of actinomycin D the reversal of the antiviral effects was correlated with a restoration of the GTP pool to near normal levels and a decrease in the amount of phosphorylated Rbv in acid-soluble cell extracts. The role of the phosphorylated forms of Rbv in inhibiting virus replication and the relationship to cellular RNA synthesis are discussed.     

Genetic variation during persistent reovirus infection: isolation of cold-sensitive and temperature-sensitive mutants from persistently infected L cells

We have examined the evolution of reovirus in two independently established persistently infected (p.i.) cell lines. We found that reovirus undergoes extensive mutation during persistent infection in L cells. However, there was no consistent pattern of virus evolution; in one p.i. cell line temperature-sensitive (ts) mutants were selected, whereas cold-sensitive (cs) mutants were isolated from the second p.i. culture. Neither the cs nor the ts mutants isolated from the carrier cultures expressed their defect at 37 degrees, the temperature at which the p.i. cells were maintained, indicating that the cs and ts phenotypes were nonselected markers. These results emphasize the point that emergence of the ts or cs mutants during persistent infection only signifies that the virus has changed; it does not necessarily imply that the particular mutant is essential for the maintenance of the persistent infection. Given the high mutation rate of viruses, and the wide spectrum of viral mutants present in carrier cultures, it is essential to distinguish the relevant changes from those which may simply represent an epiphenomenon. In the accompanying paper (R. S. Kauffman, R. Ahmed, and B. N. Fields Virology, 130, 79-87, 1983), we show that by using a genetic approach, it is possible to identify the viral gene(s) which are critical for the maintenance of persistent reovirus infection.     

Studies on the mechanism of interferon action: Characterization of polysome-associated cellular RNAs modified by interferon

A recent publication indicated that certain polysome-associated RNA species are altered in interferon-treated cells. The present data show that these RNA species are poly(A)-containing mRNAs, RNAs without a poly(A)-rich region and tRNAs. In addition, we show that in polyacrylamide gels in aqueous medium as well as in nonaqueous medium (formamide) the mRNAs from interferon-treated cells migrate more slowly than do control cell mRNAs, suggesting that the interferon mRNAs are slightly larger than normal. Transfer RNAs from interferon-treated cells, on the other hand, move more slowly than control tRNAs in aqueous medium, but not in formamide, suggesting that the difference in mobility in tRNAs is associated with factors other than size.     

Proteins encoded near the adenovirus late messenger RNA leader segments

Small fragments of adenovirus 2 DNA cloned into the single-strand phage M13 were used to select adenoviral messenger RNAs transcribed from the R-strand between map positions 16 and 30. Cell-free translation of these mRNAs produced proteins of 13.5K, 13.6K, and 11.5K, respectively encoded between the first and second segments of the tripartite major late leader, within the “i”-leader segment, and immediately preceding the third leader segment. Partial sequence analysis of the 13.6K protein is consistent with the hypothesis that it is encoded within the Header segment.     

Independent replication of cowpea mosaic virus bottom component RNA: in vivo instability of the viral RNAs

We compared the accumulation of cowpea mosaic virus (CPMV) RNAs after inoculation of cowpea and Chenopodium quinoa protoplasts with bottom (B) or middle (M) component virions alone, or with a mixture of the M and B components. RNA extracted from infected protoplasts was subjected to quantitative spot hybridization after electrophoresis and blotting. Compared to the complete M plus B mixture, the B inoculum induced (1) a reduced rate and extent of accumulation of B-RNA of both the (+) and (-) polarities concomitant with a balance between synthesis and degradation and (2) B-RNA that decreased in amount after further RNA synthesis was prevented by administration of cordycepin. In contrast, the (-) B-RNA apparently was stable, possibly sequestered in double-stranded form. M-component-inoculated cowpea protoplasts were subsequently inoculated with B component at intervals, and, after further incubation, RNA was analyzed by electrophoresis and hybridization. Rescue of the ability to form M-RNAs was lost when the interval between inoculations was greater than 17 hr, although the protoplasts remained sensitive to the complete inoculum. The data suggest that both of the genomic RNAs are unstable in protoplasts when inoculated alone, i.e., under conditions that leave viral RNA unprotected by coat protein.     

Sequence comparison of the 3' ends of a subgenomic RNA and the genomic RNAs of barley stripe mosaic virus

All strains of barley stripe mosaic virus examined encapsidate small amounts of an 800-nucleotide (NT) gamma-subgenomic (sg) RNA. This sgRNA has been isolated from genomic (g) RNAs of the Type and North Dakota 18 (ND18) strains and the sequence of these RNAs has been compared near the 3' end. The immediate 3' termini of the gRNAs terminate in the icosomer-GGUCCCCCAAGGGAAGACCAOH-3' and differ from the sgRNAs, which are polyadenylated. The poly(A) tracts of the sgRNAs are heterogeneous with lengths ranging from 10 to greater than 150 NT. Polyacrylamide gel electrophoresis of complementary (c) DNAs transcribed in the presence of dideoxynucleotides reveals that the sgRNAs from Type and ND18 have almost identical sequences for at least 160 NT adjacent to the 5' side of the poly(A) region. This region of the sgRNA from the ND18 strain is nearly identical to a 95-NT sequence adjacent to a poly(A) tract located at the 3' end of a 2050-base pair cDNA cloned from the gamma-genomic RNA of ND18. These results suggest that the sequences encoding the sgRNA are located upstream of an internal poly(A) region situated more than 200 NT from the 3' end of the gamma-genomic RNA.     

Alfalfa mosaic virus temperature-sensitive mutants. II. Early functions encoded by RNAs 1 and 2

Mutants Bts 03 and Mts 04 of alfalfa mosaic virus (AIMV) have temperature-sensitive mutations in genomic RNAs 1 and 2, respectively. These mutants are defective in the production of viral minus-strand RNA, coat protein, and infectious virus when assayed in cowpea protoplasts at the nonpermissive temperature (30 degrees). To determine the temperature-sensitive step in the replication cycle, mutant-infected protoplasts were shifted from an incubation temperature of 25 degrees (permissive temperature) to 30 degrees at different times during a 24-hr incubation period. For both mutants an initial incubation of infected protoplasts for 6 hr at 25 degrees was sufficient to permit a normal minus-strand RNA synthesis, translation of RNA 4 into coat protein, and assembly of infectious virus during the subsequent incubation at the nonpermissive temperature. Probably, AIMV RNAs 1 and 2 encoded proteins are produced early in infection and the mutant proteins are protected from inactivation at 30 degrees once they are incorporated in a functional structure.     

Coding assignments of Drosophila X virus genome segments: in vitro translation of native and denatured virion dsRNA

Both dsRNA genome segments of Drosophila X virus (DXV) were denatured and translated in vitro using nuclease-treated rabbit reticulocyte lysates. The synthesis of all four primary gene products was detected by polyacrylamide gel electrophoresis and autoradiography. Genome segment A (mol wt 2.3 X 10(6)) encoded polypeptides with molecular weights of 67,000 (67K), 34K, and 27K, whereas segment B (mol wt 22 X 10(6)) encoded the 110K polypeptide. The proteolytic processing of 67K which generates a 49K polypeptide in infected cells was also observed in vitro. Pulse-chase experiments indicated that synthesis of the three polypeptides encoded by genome segment A initiated independently and simultaneously, suggesting that segment A is polycistronic. Native (undenatured) DXV dsRNA could also be translated with high fidelity (vitro). The messenger activity of native dsRNA was abolished by S1 nuclease treatment but completely restored on subsequent denaturation. In vitro "pulse-chase" experiments using native dsRNA as messenger, indicated that the order of translation of polypeptides on genome segment A was 5'-67K-27K-34K-3'.     

Synthesis and processing of the translation products of tobacco ringspot virus in rabbit reticulocyte lysates

In reticulocyte lysates a predominant protein of apparent molecular weight 116,000 (116K) was synthesized from tobacco ringspot virus RNA-2 and proteins of 225K and 205K were synthesized from RNA-1. In time-course experiments using unfractionated RNA the levels of nine additional proteins of 195K, 135K, 92K, 77K, 65K, 53K, 40K, 30K, and 23K increased with time. The levels of these nine proteins relative to the 225K, 205K, and 116K proteins decreased when unfractionated RNA was translated in reticulocyte lysates in the presence of the amino acid analogs canavanine, S-aminoethylcysteine, and p-fluorophenylalanine. A protease induced in the reticulocyte lysates by RNA-1 catalyzed cleavage of the 116K protein into proteins of 53K, 40K, and 23K. Proteins of 116K, 77K, and 53K were immunoprecipitated with antiserum to tobacco ringspot virus particles.     

Interactions of plus and minus strand leader RNAs of the New Jersey serotype of vesicular stomatitis virus with the cellular La protein

The New Jersey serotype of vesicular stomatitis virus (VSV-NJ) was found to synthesize a minus strand leader RNA of 44-46 bases long and a plus strand leader RNA of 47-50 bases long in infected cells. The minus strand leader RNA of VSV-NJ was found associated with the host cell La protein in infected cells by immunoprecipitation with antisera from patients with systemic lupus erythematosus. These results differ from those reported previously (J. Wilusz , M. G. Kurilla , and J. D. Keene (1983). Proc. Natl. Acad. Sci. USA 80, 5827-5831) for the similarly sized species of minus strand leader RNA made by the Indiana serotype of VSV (VSV-IND). Despite sequence differences between the 3' ends of the plus strand leader RNAs of the two serotypes, the plus strand leader RNA of VSV-NJ was found to have a pattern of La protein accumulation similar to that reported previously for the plus strand leader RNA of VSV-IND. These results provide additional support for a role for La protein in VSV replication and help further delineate the sequence requirements for La protein binding to VSV leader RNAs.