Nucleotide sequence of the influenza A/duck/Alberta/60/76 virus NS RNA: Conservation of the NS1/NS2 overlapping gene structure in a divergent influenza virus RNA segment

Baez, Melvyn; Zazra, James J.; Elliott, Richard M.; Young, James F.; Palese, Peter

doi:10.1016/0042-6822(81)90166-5

Cited by 53 publications

(25 citation statements)

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“…For example, the nucleotide sequence of A/Duck/ Alberta/60/76 shows only 73% homology between it and A/PR/8/34 (Baez et al, 1981) which is in agreement with the proposed groups. However, the sequences of A/PR/8/34 (Baez et al, 1980;Winter et al, 198 lb), A/Udorn/72 ) and A/FPV/Rostock/34 (Porter et al, 1980) were all closely related, showing about 90% nucleotide sequence homology, a result which was unexpected from the hybridization data.…”

Section: The Virionsupporting

confidence: 91%

See 1 more Smart Citation

Structure and function of the influenza virus genome

McCauley¹,

Mahy²

1983

Biochemical Journal

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Section: The Virionsupporting

confidence: 91%

“…Allen et al (1980) ; McCauley et al (1982) Baez et al (1980) Winter etal. (1981b) Baez et al (1981) Porter et al (1980) Krystal et al (1983) Krystal et al (1983) Krystal et al (1983) Briedis & Lamb (1982) * Non-coding or signal sequence not reported.…”

Section: The Virionmentioning

confidence: 94%

Structure and function of the influenza virus genome

McCauley¹,

Mahy²

1983

Biochemical Journal

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“…NS segments of influenza A viruses are phylogenetically separated into two groups, alleles A and B (1,8,25). Alignment of the nucleotide sequences of several influenza A viruses revealed that the sequences important for efficient incorporation of NS segment (nucleotides 16 to 56 or 841 to 870 in the vRNA sense orientation) are relatively conserved (Fig.…”

Section: Discussionmentioning

confidence: 99%

Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions

Fujii

Noda

et al. 2005

J Virol

165

164

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The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3 coding region are critical for efficient NS vRNA incorporation and that deletion of the 3 segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3 NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3 end) form a structure that is required for efficient influenza A virus vRNA packaging.The genetic information that characterizes all life systems must be transmitted to each generation. For viruses, this is achieved by amplification of the genome within cells and its subsequent packaging into progeny virions. The influenza A virus genome is fragmented into eight single-strand negativesense RNA segments that encode at least 11 proteins (3, 10). These viral RNAs (vRNAs) form the ribonucleoprotein (RNP) complex with heterotrimeric viral polymerase subunit proteins (PB2, PB1, and PA) and nucleoprotein (NP) (10). After introduction into cells via receptor-mediated endocytosis, the viral RNPs are transported into the nucleolus, where viral mRNAs are transcribed and translated into viral proteins (10). The vRNAs are replicated and, together with the viral proteins, form the RNP complexes (10), which are then exported to the cytoplasm in a CRM-1-dependent manner (4,14,15,18,21,26). The final step is packaging of the RNP complexes into progeny virions at the plasma membrane (10).Influenza A virus RNA contains noncoding regions at both ends of its coding region (10). These noncoding regions are composed of sequences that are conserved among eight vRNAs (U13 and U12) as well as sequences specific for each segment that are located inside the terminal noncoding region (10).These noncoding regions regulate replication of the viral genome and transcription (22). Luytjes et al. (13) generated an artificial viral RNA comprising the chloramphenicol acetyltransferase (CAT) gene flanked by the noncoding regions of an NS segment. They showed that this RNA was incorporated into influenza virus virions, using the CAT activity of virusinfected cells as an indicator of segment incorporation. However, the CAT activity gradually decreased upon passaging. Recently, we showed that both ends of th...

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“…4 shows the autoradiograms of one such experiment. The sequences of the NS genes of the strains used have been analyzed (5,6,24,25) proteins were labeled with ['Slmethionine as described (16)(17). Cell extracts were prepared and polypeptides were separated on a 7-14% polyacrylamide gradient.…”

Section: Methodsmentioning

confidence: 99%

Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli.

Young¹,

Desselberger²,

Palese³

et al. 1983

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

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RNA segment 8 of the influenza A virus genome codes for two nonstructural proteins, NS1 and NS2, for which the functions are unknown. Cloned cDNA copies of this gene from three different influenza A virus strains were inserted into an Escherichia coli plasmid expression vector, pASI, carrying the strong regulatable A phage promoter, PL. After induction, the NS1 proteins were overproduced to levels of 20-25% of total cellular protein. This was surprising in that the codon composition for these eukaryotic genes is similar to that for weakly expressed proteins in E. coli. Thus, under the appropriate conditions, it appears that high level expression of genes containing a relatively large proportion of minor codons can be obtained. The NS1 protein produced in bacteria from a cloned cDNA copy of the A/PR/8/34 virus NS gene was purified to apparent homogeneity and used to generate a high-titer monospecific rabbit antiserum. Immunoprecipitation studies showed this antibody to be crossreactive against the NS1 proteins produced by several different influenza A virus strains. Immunofluorescence experiments in Madin-Darby canine kidney cells showed the NS1 proteins to be located in the nucleoplasm early in infection for all strains examined. With some of the strains, NS1-specific immunofluorescence was observed predominantly in the nucleoli later in infection. This technology can be used to obtain other viral proteins in pure form for structural, functional, and immunological studies.Influenza A viruses contain a genome of eight segmented single-stranded RNAs of negative polarity that, when transcribed into mRNA, code for at least 10 polypeptides (1,2). Three of these gene products (NS1, NS2, and M2) are found only in virus-infected cells. The other seven proteins are present in the virion, associated with the viral envelope (HA, NA, and Ml), or as part of the core complex (PB1, PB2, PA, and NP). The ability to obtain large quantities of these viral proteins in pure form will greatly enhance studies of the structure of these polypeptides and help to elucidate the function(s) of these gene products during the virus replication cycle.With the emergence of recombinant DNA technology, it has been possible to clone cDNA copies of the eight influenza virus genomic RNAs into Escherichia coli plasmid vectors (3). In this paper, we describe the bacterial production of the influenza virus NS1 nonstructural proteins from three different viral strains. The viral cDNA coding for the NS1 protein was inserted into a plasmid vehicle, pASl, that contains transcriptional and translational signals derived from bacteriophage A (4). Under the appropriate induction conditions, large quantities of NSL protein were produced in an E. coli host. The protein was then extracted and purified for use as an immunogen to generate a hightiter monospecific antiserum in rabbits for in vitro and in vivo studies.Immunoprecipitation studies using this antiserum showed it to be crossreactive with all strains tested, and immunofluorescence experiments re...

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Nucleotide sequence of the influenza A/duck/Alberta/60/76 virus NS RNA: Conservation of the NS1/NS2 overlapping gene structure in a divergent influenza virus RNA segment

Cited by 53 publications

References 9 publications

Structure and function of the influenza virus genome

Structure and function of the influenza virus genome

Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions

Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli.

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