Fine mapping of the subunit binding sites of influenza virus RNA polymerase

Ohtsu, Yasushi; Honda, Yoshikazu; Toyoda, Tetsuya

doi:10.1016/s0531-5131(01)00395-8

Cited by 14 publications

(25 citation statements)

References 31 publications

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“…2). These results are in good agreement with a previous study that inferred a core binding site between residues 718-732 from the behaviour of small deletions within larger polypeptides [23]. However, our results suggest a binding site extending slightly further towards the C-terminus and we suspect this explains the relatively poor inhibitory activity exhibited by a peptide including residues 715-740 noted in a recent study [15].…”

Section: Discussionsupporting

confidence: 93%

“…Contradicting this, a subsequent study showed direct binding by the C-terminal 57 amino-acids (residues 700-757; [23]) and in agreement with this we found that a GST fusion protein bearing the last 75 amino-acids of PB1 (G683) bound PB2 as efficiently as cognate polyclonal antiserum [14]. Based on the behaviour of overlapping C-terminal deletions, Ohtsu and colleagues suggested that amino-acids 718-732 were the most important for PB2-binding [23]. However, a polypeptide containing amino-acids 715-740 showed only weak trans-dominant inhibitory activity against the wild-type polymerase [15], perhaps indicating that additional sequences are required for efficient binding.…”

Section: Delineation Of C-terminal Pb1 Residues Involved Inmentioning

confidence: 97%

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Evidence that the C‐terminal PB2‐binding region of the influenza A virus PB1 protein is a discrete α‐helical domain

et al. 2007

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The influenza A virus RNA-dependent RNA polymerase is a heterotrimer composed of PB1, PB2 and PA subunits and essential for viral replication. However, little detailed structural information is available for this important enzyme. We show by circular dichroism spectroscopy that polypeptides from the C-terminus of PB1 that are capable of binding efficiently to PB2 fold into stable a-helical structures. Structure prediction analysis of this region of PB1 indicates that it likely consists of a three-helical bundle. Deletion of any of the helices abrogated transcriptional function. Thus, PB1 contains a C-terminal a-helical PB2-binding domain that is essential for nucleotide polymerization activity.

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Section: Discussionsupporting

confidence: 93%

Section: Delineation Of C-terminal Pb1 Residues Involved Inmentioning

confidence: 97%

Evidence that the C‐terminal PB2‐binding region of the influenza A virus PB1 protein is a discrete α‐helical domain

et al. 2007

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“…Toyoda et al (1996) used an immunoprecipitation assay and deletion mutants to show that the N-terminal 249 amino acid residues of PB2 can bind to PB1. However, a subsequent study from the same laboratory detected PB1 by co-precipitation with PB2 minus the N-terminus, suggesting the possibility of another region of interaction with PB1 (Ohtsu et al, 2002). This was supported by Poole et al (2004), who also identified a second PB1-binding site in the C-terminal half of PB2.…”

Section: Introductionmentioning

confidence: 84%

Structural insight into the essential PB1–PB2 subunit contact of the influenza virus RNA polymerase

Sugiyama

Obayashi

Kawaguchi

et al. 2009

EMBO J

172

202

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Influenza virus RNA-dependent RNA polymerase is a multi-functional heterotrimer, which uses a 'cap-snatching' mechanism to produce viral mRNA. Host cell mRNA is cleaved to yield a cap-bearing oligonucleotide, which can be extended using viral genomic RNA as a template. The cap-binding and endonuclease activities are only activated once viral genomic RNA is bound. This requires signalling from the RNA-binding PB1 subunit to the cap-binding PB2 subunit, and the interface between these two subunits is essential for the polymerase activity. We have defined this interaction surface by protein crystallography and tested the effects of mutating contact residues on the function of the holo-enzyme. This novel interface is surprisingly small, yet, it has a crucial function in regulating the 250 kDa polymerase complex and is completely conserved among avian and human influenza viruses.

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“…PA N has proteolytic activity and is important for RNA synthesis activity of the polymerase complex (43,46,47). PA C binds to PB1 for complex formation and nuclear transport (48)(49)(50). Recently, it has been shown that the PA gene contains a second ORF (X-ORF) whose N-terminal 191 amino acids are derived from the PA ORF, while the C-terminal sequence (positions 190 to 253 of the PA gene) is derived from the ϩ1-frameshifted X-ORF (11).…”

Section: Discussionmentioning

confidence: 99%

“…cells, and 293T cells, were inoculated with virus at a multiplicity of infection (MOI) of 0.01 and incubated in the appropriate medium containing 1% fetal bovine serum (FBS) at 37°C. Supernatants were collected at 12,24,48,72, and 96 h p.i., and virus titers were determined as the number of 50% tissue culture infectious doses (TCID 50 ) per ml in MDCK cells using the method of Reed and Muench (27).…”

mentioning

confidence: 99%

The PA-Gene-Mediated Lethal Dissemination and Excessive Innate Immune Response Contribute to the High Virulence of H5N1 Avian Influenza Virus in Mice

Song

et al. 2013

J Virol

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Highly pathogenic H5N1 influenza A virus remains a substantial threat to public health. To understand the molecular basis and host mechanism for the high virulence of H5N1 viruses in mammals, we compared two H5N1 isolates which have similar genetic backgrounds but greatly differ in their virulence in mice. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is nonpathogenic. We first showed that CK10 elicited a more potent innate immune response than did GS10 in mouse lungs by increasing the number and expression levels of activated genes. We then generated a series of reassortants between the two viruses and evaluated their virulence in mice. Inclusion of the CK10 PA gene in the GS10 background resulted in a dramatic increase in virulence. Conversely, expression of the GS10 PA gene in the CK10 background significantly attenuated the virulence. These results demonstrated that the PA gene mainly determines the pathogenicity discrepancy between CK10 and GS10 in mice. We further determined that arginine (R) at position 353 of the PA gene contributes to the high virulence of CK10 in mice. The reciprocal substitution at position 353 in PA or the exchange of the entire PA gene largely caused the transfer of viral phenotypes, including virus replication, polymerase activity, and manipulation of the innate response, between CK10 and GS10. We therefore defined a novel molecular marker associated with the high virulence of H5N1 influenza viruses, providing further insights into the pathogenesis of H5N1 viruses in mammals.

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Fine mapping of the subunit binding sites of influenza virus RNA polymerase

Cited by 14 publications

References 31 publications

Evidence that the C‐terminal PB2‐binding region of the influenza A virus PB1 protein is a discrete α‐helical domain

Evidence that the C‐terminal PB2‐binding region of the influenza A virus PB1 protein is a discrete α‐helical domain

Structural insight into the essential PB1–PB2 subunit contact of the influenza virus RNA polymerase

The PA-Gene-Mediated Lethal Dissemination and Excessive Innate Immune Response Contribute to the High Virulence of H5N1 Avian Influenza Virus in Mice

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