Effect of double-stranded RNA associated with viral messenger RNA on in vitro protein synthesis (original) (raw)
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On the biosynthesis and structure of double-stranded RNA in vaccinia virus-infected cells
Proceedings of the National Academy of Sciences of the United States of America, 1969
The virus-specific, RNase-resistant RNA appearing in vaccinia virus-infected cells was directly shown to be an RNA duplex. After its melting and subsequent banding on Cs(2)SO(4) or incubation with DNase, the RNA could be reannealed and then hybridized with vaccinia virus DNA.The double-stranded virus-specific RNA appears to exist in the cell in the form of heterogeneous partially double-stranded RNA's sedimenting between 9 and 22S. The kinetics of the appearance of the double-stranded RNA in the cell show a dependence on the multiplicity of infection and suggest that the double-stranded RNA is a "late" product of viral intracellular biosynthesis. The synthesis of the double-stranded RNA is inhibited by actinomycin D.
Journal of virology, 1991
We have recently demonstrated that the poly(A) moieties of short RNAs obtained from both in vitro transcription and from vaccinia virus (VV)-infected cells exhibit dissimilar effects on the in vitro translation of cellular and VV mRNAs (R. Bablanian, G. Coppola, P. Masters, and A. K. Banerjee, Virology 148:375-380, 1986; M. J. Su and R. Bablanian, Virology 179:679-693, 1990). In the present study, we have investigated the roles of poly(A), m7GTP, and initiation factors in the mechanism of selective translation of VV mRNAs. The effects of unfractionated poly(A) [termed poly(A)un, with various chain lengths up to 3,000 nucleotides] and a 150- to 300-nucleotide fraction of synthetic poly(A) [termed poly(A)150-300] on the translation of HeLa cell mRNAs and early and late VV mRNAs were studied. Both the poly(A)un and the poly(A)150-300 completely inhibited the translation of HeLa cell mRNAs obtained from total cytoplasmic RNA in the nuclease-treated reticulocyte lysates. Viral mRNAs from...
Journal of General Virology, 1979
Cytoplasmic RNA synthesis can be detected in vaccinia virus-infected HeLa cells in the presence of 2/zg/ml but not 2o/*g/ml of actinomycin D. When RNA synthesis is observed protein synthesis is inhibited in infected, treated cells. We had previously noted that such a correlation may also be observed in infected, cycloheximide-treated cells. If actinomycin D (20/zg/ml) is added to these cells at various times after infection and treatment, the inhibition of protein synthesis seen upon removal of cycloheximide does not continue beyond the point to which it had developed before the actinomycin D was added. These results indicate that the inhibition of protein synthesis can be correlated with the amount of cytoplasmic RNA synthesized in infected cells and that this RNA synthesis and the subsequent inhibition of protein synthesis can be prevented by sufficiently high concentrations of actinomycin D. The cytoplasmic RNA which is synthesized does not appear to consist of double-stranded RNA nor of extensive self complementary regions. The cytoplasmic RNA synthesized in infected, cycloheximide treated cells appears to consist of early virus mRNA which can function as mRNA in vitro in a cell-free system derived from normal cells. An examination of the phosphorylation of ribosomal proteins shows six additional phosphoproteins in infected cells, two of which may be observed in infected cycloheximide-treated ceils, suggesting that phosphorylation of ribosomal proteins cannot be directly correlated with the inhibition of overall protein synthesis seen in infected cycloheximide-treated cells.
Biochemical and Biophysical Research Communications, 1975
Wheat germ cell-free extracts translate reovirus ssRNA as efficiently as brome mosaic virus ssRNA. The magnesium optima, kinetics, dependence on messenger RNA concentration, and high level amino acid incorporation are similar for the animal virus and plant virus messenger RNAs. With unfractionated mRNAs from either virus, the largest messengers are not translated, or are very poorly translated. The smaller mRNAs, including the reovirus messengers for polypeptides a I, a3, a2 and ~I, are translated with fidelity. Evidence suggests that after several translation cycles, the ribosomes are not efficiently released, perhaps creating a "ribosome-jam" resulting in the synthesis of incomplete polypeptides. Brome mosaic virus and reovirus are representatives of different plant and animal virus groups whose genetic information is distributed among several RNAs. The reovirus genome consists of ten segments of double-stranded RNA; a virion core-associated RNA-dependent RNA polymerase catalyzes the synthesis in vitro of I0 single-stranded RNA species of three size classes: small (s), molecular weight 0.3 to 0.4 x 106; medium (m), 0.6 to 0.7 x 106; and large (£), 1.3 to 1.4 x 106 (1,2). The reovirion consists of seven polypeptides (3), whereas reovirus-infected cells contain nine detectable virus-specific polypeptides of three size classes: X (molecular weight 140 to 155,000); ~ (72 to 88,000); and a (34 to 42,000) (4). The brome mosaic virus genome consists of four species of single-stranded RNA (5) which are of the same polarity as mRNA. The two smallest BMV ssRNAs, RNA3 and RNA4, code for the coat protein; RNAs I, 2 and 3 also code for other polypeptides (6). The regulation of the translation
On the Regulation of Protein Synthesis in Vaccinia Virus Infected Cells
Journal of General Virology, 1976
All eukaryotic mRNA species show a characteristic individual translational efficiency under conditions of restricted polypeptide chain initiation caused by an increase in the osmolarity of the growth medium. In vaccinia virus infected L cells or HeLa cells virus mRNAs can be grouped into classes on the basis of their relative labelling under standard and hypertonic conditions. Under the latter conditions, most of the 'early' mRNAs possess very high translational efficiencies, most of the 'intermediate' mRNAs show an intermediate efficiency and the most prominent 'late' mRNAs show a translational efficiency which is lower than that of other virus mRNAs but still higher than the average cellular mRNA. Late in the infection cycle virus mRNAs with a relative low translational efficiency are preferentially translated under standard growth conditions whereas 'early' virus mRNAs which are still present and which show a higher translational resistance to hypertonic conditions are not translated. These results indicate a unique translational control operating late in the growth cycle of vaccinia virus.
Cell-free translation of carnation latent virus RNA and analysis of virus-specific dsRNA
Virus Genes, 1991
Carnation latent virus was shown to direct the synthesis of virus-specific polypeptides in both reticulocyte lysate and wheat germ in vitro translation systems. The L-(4,S3H)-leucine-labeled products ranged in molecular mass from M, 190 to 33 kD. The 33 kD product, synthesized after only 15 min incubation, was the only major polypeptide that immunoprecipitated with antiserum to CarLV. Coatprotein synthesis does not occur as a result of proteolytic processing, but may arise as a result of translation of a subgenomic RNA species. Subgenomic RNA species were not detected by Northern hybridization of CarLV cDNA to either viral RNA or total nucleic acid from systemically infected plants, although CarLV-specific dsRNA species equivalent to 1.6 and 2.1 kb were detected.
Characterization and temporal regulation of mRNAs encoded by vaccinia virus intermediate-stage genes
Journal of virology, 1993
The steady-state levels of mRNAs encoded by three intermediate-stage genes of vaccinia virus, A1L, A2L, and G8R, were compared with those encoded by well-characterized early- and late-stage genes. After synchronous infection of HeLa cells, the early mRNA was detected within 20 min and peaked at about 100 min; all three intermediate mRNAs were detected at 100 min and peaked at about 120 min; and the late mRNA was detected at 140 min and increased thereafter. Upon reaching maximum levels, the early and intermediate mRNAs declined at rates consistent with half-lives of about 30 min, providing the basis for rapid changes in gene expression. Intermediate mRNA was not detected when viral DNA synthesis was prevented, whereas its accumulation was enhanced by blocking translation after removal of the replication inhibitor. The 5' ends of the mRNAs initiated within a TAAAT or TAAAAT sequence in the coding DNA strand but contained a poly(A) leader of up to 30 additional bases. Diffuse band...