A reexamination of influenza single- and double-stranded RNAs by gel electrophoresis

Pons, Marcel W.

doi:10.1016/0042-6822(76)90508-0

Cited by 95 publications

(33 citation statements)

References 9 publications

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“…Each segment is transcribed ipto a single mRNA which is subsequently polyadenylated and translated to produce a unique viral polypeptide (reviewed in reference 1). Various methods have been used to estimate the molecular weights of the genome segments, including sucrose density gradient centrifugation (2,3) and polyacrylamide gel electrophoresis under neutral (2)(3)(4)(5) or partially denaturing conditions (6)(7)(8). More recently, Desselberger and Palese (9), using glyoxylation to ensure RNA denaturation, obtained much lower molecular weight estimates than those reported previously, with segment sizes ranging from 8.9 x 105 to 2.1 x 10 daltons for the genome of influenza A/PR/8/34.…”

Section: Introductionmentioning

confidence: 99%

A new method for the size estimation of the RNA genome segments of influenza virus

1979

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Previous estimates of the size of the RNA genome segments of influenza virus have been unreliable because of a lack of suitable RNA species as size markers. We have attempted to overcome this problem by utilising the ability of AMV reverse transcriptase to synthesise full length DNA copies of RNA molecules in the presence of a suitable primer. By comparing such DNA copies of the RNA segments of the influenza virus genome with sequenced restriction fragments from the E. coli plasmid pBR322, we have made more reliable estimates of the sizes of the eight genome segments from influenza virus A/NT/60/68.

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Section: Introductionmentioning

confidence: 99%

A new method for the size estimation of the RNA genome segments of influenza virus

1979

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“…The genome of influenza virus consists of eight single-stranded RNA segments (1)(2)(3), which have been shown to contain the genetic information for the following eight virus-specific polypeptides: three polypeptides associated with RNA polymerase activity (P1, P2, P3), the hemagglutinin (HA), the nucleocapsid protein (NP), the neuraminidase (NA), the membrane protein (M), and a nonstructural protein (NS) (4)(5)(6)(7)(8). Assignments of these eight polypeptides to the eight genome segments have been made by several methods (8)(9)(10)(11).…”

mentioning

confidence: 99%

Segment 8 of the influenza virus genome is unique in coding for two polypeptides.

Lamb¹,

Choppin²

1979

Proc. Natl. Acad. Sci. U.S.A.

138

111

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In previous studies we showed that a ninth polypeptide with a molecular weight of t11,000 (NS2) found in influenza virus-infected cells was unique, that it could be synthesized in vitro, and that its expression in vivo required early protein synthesis. On the basis of these results we suggested that one of the eight genome RNA segments of influenza virus codes for two polypeptides [Lamb, R. A., Etkind, P. R. & Choppin, P.W. (1978) Virology 91, 60-78]. We describe here differences in the electrophoretic mobility of the NS2 polypeptides of different strains of influenza A virus. These results provided further evidence that NS2 is virus coded and also made possible genetic studies using recombinants between two virus strains (HK and PR8) whose NS2 polypeptides differ. These studies showed that the gene for NS2 reassorts with that of the nonstructural polypeptide NS,, which is coded by genome segment 8. A mRNA for NS2 has been separated from that of NS1 and the other viral polypeptides by centrifugation and has been translated in vitro.Hybridization of genome segment 8 to the total mRNAs from infected cells specifically prevented the synthesis of NS2 and NS1. These results indicate that influenza virus genome segment 8 is transcribed into two separate mRNAs that code for two polypeptides, NS1 and NS2. Possible mechanisms for the transcription of the two mRNAs from either contiguous or overlapping genes are discussed.

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“…The eight single-stranded RNA segments of the influenza virus genome (1)(2)(3), which are of opposite polarity to mRNA, have been shown (4)(5)(6) by various methods to contain the genetic information for the following eight virus-specific polypeptides: three polypeptides associated with RNA polymerase activity (P1, P2, P3), the hemagglutinin (HA), the nucleocapsid protein (NP), the neuraminidase (NA), the membrane protein M, and a nonstructural protein (NS1) (7)(8)(9)(10). In addition to these polypeptides, we and others (8,(11)(12)(13)(14)(15)(16) have observed a ninth polypeptide (Mr, 11,000) now designated NS2.…”

mentioning

confidence: 99%

Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome.

Lamb

Choppin

Chanock

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

102

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RNA segment 8 of the influenza virus genome is unique in coding for two polypeptides, NS, (Mr, ut25,000) The eight single-stranded RNA segments of the influenza virus genome (1-3), which are of opposite polarity to mRNA, have been shown (4-6) by various methods to contain the genetic information for the following eight virus-specific polypeptides: three polypeptides associated with RNA polymerase activity (P1, P2, P3), the hemagglutinin (HA), the nucleocapsid protein (NP), the neuraminidase (NA), the membrane protein M, and a nonstructural protein (NS1) (7-10). In addition to these polypeptides, we and others (8,(11)(12)(13)(14)(15)(16) have observed a ninth polypeptide (Mr, 11,000) now designated NS2. This polypeptide is not synthesized from primary transcripts of viral genome RNA, and early viral protein synthesis is required for its synthesis (13,14). NS2 was shown to be a unique ninth influenza virus polypeptide on the basis of its peptide composition, its synthesis in vitro using mRNAs from infected cells, the isolation of a separate mRNA for it, and strain-specific differences in its migration in polyacrylamide gels (13)(14)(15). Because of the evidence for nine virus-coded polypeptides and the existence of only eight influenza virus RNA segments, we postulated that one RNA segment must code for two polypeptides (14). It was subsequently shown that virus RNA segment 8 coded for both NS1 and NS2 in studies with recombinant viruses in which NS1 and NS2 reassorted together, and, in addition, hybridization of segment 8 to total viral mRNAs specifically prevented the synthesis of both NS1 and NS2 in vitro (15,17).We describe here results obtained by using cloned DNA HeLa (human) cell lines were grown as described (14).mRNA Extraction and Protein Synthesis In Vitro. These were as described (15).Isotopic Labeling of Polypeptides in Infected Cells, Polyacrylamide Gel Electrophoresis, and Peptide Maps. These were all as described (14).Preparation of Cloned NS DNA. Clone PFV 26/NS was constructed previously (18) by using pBR322 (19) and was produced by transformation of Escherichia coil K-12 strain HB101. The bacteria were grown in L broth with tetracycline (20 ,ig/ml), and the plasmid was amplified by using chloramphenicol. The plasmid DNA was isolated by the cleared lysate technique (20) followed by centrifugation in isopycnic CsCl/ethidium bromide gradients. The NS gene was released from the plasmid DNA by digestion with Pst I and purification on 4% polyacrylamide gels (acrylamide/bisacrylamide, 20:1) (18). These experiments were conducted under P1 containment as prescribed in the National Institutes of Health guidelines.Restriction Enzyme Analysis. Uniformly 32P-labeled NS gene DNA, electrophoretically eluted from polyacrylamide gels, was digested with restriction enzymes and the products were analyzed on 4% polyacrylamide gels. 32P-Labeled simian virus 40 DNA segments obtained by digestion with HindII and HindIII were used as markers.Analysis of mRNA by Nuclease SI Digestion. The technique of Berk an...

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A reexamination of influenza single- and double-stranded RNAs by gel electrophoresis

Cited by 95 publications

References 9 publications

A new method for the size estimation of the RNA genome segments of influenza virus

A new method for the size estimation of the RNA genome segments of influenza virus

Segment 8 of the influenza virus genome is unique in coding for two polypeptides.

Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome.

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