Segment NS of influenza a virus contains an additional gene NSP in positive-sense orientation

Жирнов, О. П.; Poyarkov, Stanislav; Воробьева, И. В.; Сафонова, О. А.; Malyshev, N A; Klenk, Hans‐Dieter

doi:10.1134/s1607672907030106

Cited by 28 publications

(31 citation statements)

References 11 publications

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“…For WSN, the database of proteins searched included all known viral proteins, as well as the translations of hypothetical open reading frames. No peptides were found from PB1-F2, or the putative ambisense gene product NSP/NEG8 [34], [35], and no peptides were found matching the unique sequences of the PA-X [36] or PB1-N40 proteins [37]. Somewhat surprisingly, the non-structural protein NS1 was readily detected in all preparations of influenza A and B viruses.…”

Section: Resultsmentioning

confidence: 87%

Mapping the Phosphoproteome of Influenza A and B Viruses by Mass Spectrometry

Hutchinson¹,

Denham²,

Thomas³

et al. 2012

PLoS Pathog

125

160

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Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses – to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins (PB2, PB1 and PA), glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation (PB2, M1, NP, NS1 and, through NP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets.

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Section: Resultsmentioning

confidence: 87%

Mapping the Phosphoproteome of Influenza A and B Viruses by Mass Spectrometry

Hutchinson¹,

Denham²,

Thomas³

et al. 2012

PLoS Pathog

125

160

View full text Add to dashboard Cite

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“…Additional inspection of other viral proteins revealed numerous caspase cleavage motifs in the proteins PB1, PA, PB2, NS1, NA and HA (summarized in Table 1). The proteins NEP, M1, and the hypothetical NSP (Zhirnov et al, 2007) appeared to lack any caspase cleavage motifs. Wide distribution of caspase motifs in different influenza virus proteins implied that these sites could play an important role in functioning of the viral proteins and virus biology.…”

Section: Resultsmentioning

confidence: 99%

Influenza virus pathogenicity is determined by caspase cleavage motifs located in the viral proteins

Жирнов

Syrtzev²

2009

J Mol Genet Med

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Almost all influenza virus proteins are found to contain caspase cleavage motifs. Two caspase cleavage consensus sequences, EXD↓Y and D/EXXD↓Y (caspase motifs) were identified in N- and C-terminal regions of influenza virus proteins nucleocapsid NP (positions D16 and D497) and ionic channel M2 (positions D23 and D87). Using reverse genetics with the highly-virulent avian influenza virus A/FPV/Rostock (H7N1), as a vector precursor, these NP and M2 caspase motifs were artificially altered by site-directed mutagenesis and pathogenicity of the generated caspase mutant viruses was tested in chickens. Three main groups of virus mutants were identified. The first group of mutants was characterized by high replication in cells and low virulence in chickens. These virus mutants possessed the altered N-terminal NP and C-terminal M2 caspase motifs. The second group of virus mutants, possessing the altered N-terminal caspase motif of M2, was characterized by attenuated replication in cultured cells and reduced pathogenic properties in chickens. Third, mutations generated in the C-terminus of NP were lethal and restricted virus rescue by reverse genetics, implying a critical role of this caspase site in virus replication. Thus, these data suggested that, (i) caspase motifs in virus proteins play a significant role in virus pathogenicity; (ii) the lack of direct correlation between replication potential and pathogenicity, observed in caspase mutants of the first virus group, implied that virus caspase motifs could affect immunopathogenesis during the infection process, rather than simply controlling virus production in target cells in the chicken host.

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“…Therefore it was not surprising that our PSI-BLAST searches and analysis with HHSearch failed to find any distantly related protein sequences. We believe that the previously noted [30] similarity of the predicted NEG8 protein and a Tetrahymena protein (Accession no. Q950Z5) is spurious and the result of matching of the hydrophobic signal sequence of NEG8 protein and the highly skewed amino acid composition of the Tetrahymena protein.…”

Section: Resultsmentioning

confidence: 88%

“…Although we are not the first to observe this ORF, it has been rarely been commented upon by others. It was observed when segment 8 was first sequenced [29] and more recently, the ubiquity of this ORF was briefly noted after we began this work [30]. …”

Section: Introductionmentioning

confidence: 99%

Evidence for a novel gene associated with human influenza A viruses

Clifford

Twigg

Upton

2009

Virol J

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BackgroundInfluenza A virus genomes are comprised of 8 negative strand single-stranded RNA segments and are thought to encode 11 proteins, which are all translated from mRNAs complementary to the genomic strands. Although human, swine and avian influenza A viruses are very similar, cross-species infections are usually limited. However, antigenic differences are considerable and when viruses become established in a different host or if novel viruses are created by re-assortment devastating pandemics may arise.ResultsExamination of influenza A virus genomes from the early 20th Century revealed the association of a 167 codon ORF encoded by the genomic strand of segment 8 with human isolates. Close to the timing of the 1948 pseudopandemic, a mutation occurred that resulted in the extension of this ORF to 216 codons. Since 1948, this ORF has been almost totally maintained in human influenza A viruses suggesting a selectable biological function. The discovery of cytotoxic T cells responding to an epitope encoded by this ORF suggests that it is translated into protein. Evidence of several other non-traditionally translated polypeptides in influenza A virus support the translation of this genomic strand ORF. The gene product is predicted to have a signal sequence and two transmembrane domains.ConclusionWe hypothesize that the genomic strand of segment 8 of encodes a novel influenza A virus protein. The persistence and conservation of this genomic strand ORF for almost a century in human influenza A viruses provides strong evidence that it is translated into a polypeptide that enhances viral fitness in the human host. This has important consequences for the interpretation of experiments that utilize mutations in the NS1 and NEP genes of segment 8 and also for the consideration of events that may alter the spread and/or pathogenesis of swine and avian influenza A viruses in the human population.

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Segment NS of influenza a virus contains an additional gene NSP in positive-sense orientation

Cited by 28 publications

References 11 publications

Mapping the Phosphoproteome of Influenza A and B Viruses by Mass Spectrometry

Mapping the Phosphoproteome of Influenza A and B Viruses by Mass Spectrometry

Influenza virus pathogenicity is determined by caspase cleavage motifs located in the viral proteins

Evidence for a novel gene associated with human influenza A viruses

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