Initiation of translation directed by 42S and 26S RNAs from Semliki Forest virus in vitro.

Glanville, N. Theresa; Ranki, Marjut; Morser, John; Kääriäinen, Leevi; Smith, Alan E.

doi:10.1073/pnas.73.9.3059

Cited by 66 publications

(39 citation statements)

References 27 publications

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“…Since the polymerase components are synthesized only in small amounts early in infection (Lachmi & K~i~iri~iinen, 1977), the isolation and purification of sufficient amounts of the enzyme for analysis has been a very difficult task (Clewley & Kennedy, 1976;Ranki & K~i~iri~tinen, 1979;Gomatos et al, 1980). The putative RNA polymerase components of SFV, non-structural proteins nsP1, nsP2, nsP3 and nsP4 (previously designated ns72), are synthesized as a large polyprotein (2431 amino acids) from the 5' region of the 42S genomic RNA (Lachmi & Kfifiri~iinen, 1976;Glanville et al, 1976;Kalkkinen et al, 1981;Kerfinen & Ruohonen, 1983;Takkinen, 1986). The nsP1 is the major virus-specific protein which together with smaller amounts of nsP2 and nsP4, copurifies with template-associated RNA polymerase preparations (Ranki & K~i~irifiinen, 1979;Gomatos et al, 1980;Ker~inen & Ruohonen, 1983).…”

Section: Introductionmentioning

confidence: 99%

Semliki Forest Virus-specific Non-structural Protein nsP3 Is a Phosphoprotein

Peränen

Takkinen

Kalkkinen

et al. 1988

Journal of General Virology

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SUMMARYAntisera were raised in rabbits against fusion proteins consisting of fl-galactosidase and partial amino acid sequences of Semliki Forest virus (SFV)-specific non-structural proteins nsP1, nsP2, nsP3 and nsP4. The antisera were specific since each of them precipitated only one labelled protein of a size expected for nsP 1, nsP2, nsP3 or nsP4 from lysates of [35S]methionine-labelled SFV-infected BHK-21 cells. The specific antisera also precipitated p220 (with sequences of nsP1, nsP2 and nsP3), p155 (nsP1 and nsP2) and p135 (nsP3 and nsP4) which have been previously shown to be cleavage products of the polyprotein precursor of the non-structural proteins, nsP1, nsP4 and most of nsP3, together with the virus-specific RNA polymerase activity, were in the mitochondrial pellet (P 15) fraction of infected BHK-21 cells whereas nsP2 was evenly distributed between P15 and the supernatant fraction (S15). Only antisera directed against nsP3 sequences precipitated a labelled protein from cells incubated with [32p]orthophosphate during SFV infection. Treatment of the immunoprecipitate with calf alkaline intestinal phosphatase reduced the amount of labelled nsP3 considerably. Immunoprecipitated 32p-labelled nsP3, isolated by SDS-PAGE, was subjected to acid hydrolysis. Both phosphoserine and phosphothreonine but not phosphotyrosine could be identified in the hydrolysate. Approximately twice as much [32p]serine as [32p]threonine was detected in nsP3. PI5 and S15 fractions were prepared from [35S]methionine-and 32p-labelled SFV-infected cells and the 3sS/32p ratio of nsP3 was determined after immunoprecipitation and SDS-PAGE. The nsP3 in S15 was less heavily phosphorylated (about 50%) than P15-associated nsP3. Anti-nsP3 serum revealed large cytoplasmic vesicles in SFV-infected cells in indirect immunofluorescence microscopy.

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Section: Introductionmentioning

confidence: 99%

Semliki Forest Virus-specific Non-structural Protein nsP3 Is a Phosphoprotein

Peränen

Takkinen

Kalkkinen

et al. 1988

Journal of General Virology

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“…In standard translation assays in vitro both RNAs have only one functional ribosome binding site. These are unique since in the case of SFV the initiation dipeptides are Met-Asn and Met-Ala for 26-S and 42-S RNA, respectively [12]. Furthermore, the oligonucleotide maps of the riboAbbreviations.…”

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confidence: 99%

The Nucleotide Sequences of the 5′‐Terminal T1 Oligonucleotides of Semliki‐Forest‐Virus 42‐S and 26‐S RNAs are Different

Pettersson¹,

Söderlund²,

Kääriäinen³

1980

European Journal of Biochemistry

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To study the mechanism of the synthesis of Semliki Forest virus (SFV) 26-S RNA, we have isolated the 5'-terminal 'cap'-containing RNase-T1-resistant oligonucleotide (T1 cap) from the genomic 42-S RNA and from the subgenomic 26-S RNA and determined their nucleotide sequences. The T1 caps were purified from 32P-labelled RNAs on two successive two-dimensional fractionation systems : (a) electrophoresis on cellulose acetate paper followed by homochromatography and (b) two-dimensional polyacrylamide gel electrophoresis. The T1 caps derived from the two RNAs had different mobilities in both systems. Their nucleotide sequence was found to be : m7G(5')ppp-(5')ApUpGp-for the 42-S RNA and m7G(5')ppp(5')ApUpUpGp-for the 26-S RNA, respectively. Thus, it appears that the 26-S RNA is not formed by initiation of the RNA polymerase at the 3' end of the negative-strand template followed by 'cleavage and splicing' or as the result of a 'polymerase jump'. Our results, instead, favour the model of internal initiation of the polymerase on the 42-S negative-strand RNA template.

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“…This pellet was dissolved in pyridine (50% in water) and chromatographed on a Sephadex G-25 column with pyridine (20 "/, in water) as elution buffer. Column fractions 210-230 containedpurepeptide P5 (10)(11)(12)(13)(14)(15)(16)(17)(18)(19) as was shown by manual sequencing. However, the amino acid composition was obscure, since many amino acids present in the analysis in high yields do not belong to peptide P5.…”

Section: Peptides Derivedfsom Pepsin Digestion Of the Proteinmentioning

confidence: 99%

“…After pepsin digestion of the protein and centrifugation of the lyophilized and redissolved peptides prior to column chromatography, a residue insoluble in dilute formic acid was found, which contained mainly the sequence of peptide P5 (10)(11)(12)(13)(14)(15)(16)(17)(18)(19). This pellet was dissolved in pyridine (50% in water) and chromatographed on a Sephadex G-25 column with pyridine (20 "/, in water) as elution buffer.…”

Section: Peptides Derivedfsom Pepsin Digestion Of the Proteinmentioning

confidence: 99%

The Core Protein of Alphaviruses

Boege

Wengler

Wittmann‐Liebold

1983

European Journal of Biochemistry

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The primary structure of the core protein of Semliki Forest virus has been established by protein chemical characterization of 102 peptides, generated by digestion with trypsin, pepsin, thermolysin, and by partial acid cleavage of the protein. Besides a difference in one position, the sequence as established by these experiments is in agreement with the sequence predicted from the nucleotide sequence of the mRNA Pvoc. Nut1 Acad. Sci. U S A , 77,6376 -63801. The core protein has a blocked N terminus, consists of 267 amino acid residues, and has the following amino acid composition:

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Initiation of translation directed by 42S and 26S RNAs from Semliki Forest virus in vitro.

Abstract: The proteins synthesized in vitro in response to 42S and 26S RNAs

Cited by 66 publications

References 27 publications

Semliki Forest Virus-specific Non-structural Protein nsP3 Is a Phosphoprotein

Semliki Forest Virus-specific Non-structural Protein nsP3 Is a Phosphoprotein

The Nucleotide Sequences of the 5′‐Terminal T1 Oligonucleotides of Semliki‐Forest‐Virus 42‐S and 26‐S RNAs are Different

The Core Protein of Alphaviruses

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