Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site

Hara, Koyu; Shiota, Mayumi; Kido, Hiroshi; Ohtsu, Yasushi; Kashiwagi, Takanari; Iwahashi, Jun; Hamada, Nobuyuki; Mizoue, Kazutoshi; Tsumura, Naoki; Kato, Hirohisa; Toyoda, Tetsuya

doi:10.1046/j.1365-2443.2001.00399.x

Cited by 67 publications

(51 citation statements)

References 73 publications

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“…1A). This inhibitory effect of PA has been described before and is probably due to the expression of the PA-X protein (16,22,43,44). Coexpression of increasing amounts of PB2 and, to some extent, of NP counteracted the inhibition of viral polymerase activity by Mx1 (Fig.…”

Section: Pb2supporting

confidence: 66%

Interferon-Inducible Protein Mx1 Inhibits Influenza Virus by Interfering with Functional Viral Ribonucleoprotein Complex Assembly

Verhelst

Parthoens

Schepens

et al. 2012

J Virol

200

168

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bMx1 is a GTPase that is part of the antiviral response induced by type I and type III interferons in the infected host. It inhibits influenza virus infection by blocking viral transcription and replication, but the molecular mechanism is not known. Polymerase basic protein 2 (PB2) and nucleoprotein (NP) were suggested to be the possible target of Mx1, but a direct interaction between Mx1 and any of the viral proteins has not been reported. We investigated the interplay between Mx1, NP, and PB2 to identify the mechanism of Mx1's antiviral activity. We found that Mx1 inhibits the PB2-NP interaction, and the strength of this inhibition correlated with a decrease in viral polymerase activity. Inhibition of the PB2-NP interaction is an active process requiring enzymatically active Mx1. We also demonstrate that Mx1 interacts with the viral proteins NP and PB2, which indicates that Mx1 protein has a direct effect on the viral ribonucleoprotein complex. In a minireplicon system, avian-like NP from swine virus isolates was more sensitive to inhibition by murine Mx1 than NP from human influenza A virus isolates. Likewise, murine Mx1 displaced avian NP from the viral ribonucleoprotein complex more easily than human NP. The stronger resistance of the A/H1N1 pandemic 2009 virus against Mx1 also correlated with reduced inhibition of the PB2-NP interaction. Our findings support a model in which Mx1 interacts with the influenza ribonucleoprotein complex and interferes with its assembly by disturbing the PB2-NP interaction.

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Section: Pb2supporting

confidence: 66%

Interferon-Inducible Protein Mx1 Inhibits Influenza Virus by Interfering with Functional Viral Ribonucleoprotein Complex Assembly

Verhelst

Parthoens

Schepens

et al. 2012

J Virol

200

168

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“…Hara et al reported that the PA subunit is in fact a chymotrypsin-like protease having its catalytic site at serine 624 (14). However, by mutation analysis, we observed that the ability of PA to induce in vivo protein degradation lay within the 247 N-terminal amino acids of the protein, threonine 157 being involved in this activity (32,39).…”

mentioning

confidence: 60%

Threonine 157 of Influenza Virus PA Polymerase Subunit Modulates RNA Replication in Infectious Viruses

Huarte

Falcón

Nakaya

et al. 2003

J Virol

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Previous results have shown a correlation between the decrease in protease activity of several influenza A virus PA protein mutants and the capacity to replicate of the corresponding mutant ribonucleoproteins (RNPs) reconstituted in vivo. In this work we studied the phenotype of mutant viruses containing these mutations. Viruses with a T162A mutation, which showed a very moderate decrease both in protease and replication activities of reconstituted RNPs, showed a wild-type phenotype. Viruses with a T157A mutation, which presented a severe decrease in protease activity and replication of RNPs, showed a complex phenotype: (i) transport to the nucleus of PAT157A protein was delayed, (ii) virus multiplication was reduced at both low and high multiplicities, (iii) transcriptive synthesis was unaltered while replicative synthesis, especially cRNA, was diminished, and (iv) viral pathogenesis in mice was reduced, as measured by loss of body weight and virus titers in lungs. Finally, recombinant viruses with a T157E mutation in PA protein, which resulted in a drastic reduction of protease and replication activities of RNPs, were not viable. These results indicate that residue T157 in PA protein is important for the capacity of viral polymerase to synthesize cRNA.The influenza virus RNA polymerase is a complex composed of three subunits, PA, PB1, and PB2. The polymerase, together with the nucleoprotein (NP) and the viral RNA template, form viral ribonucleoproteins (RNPs), which carry out viral transcription and replication. The polymerase complex synthesizes three different RNA classes: (i) mRNAs that contain a cap structure and 10 to 15 nucleotides derived from host cell mRNAs at their 5Ј end and a poly(A) tail at their 3Ј end; (ii) virion RNAs found in the viral particle (vRNAs); and (iii) cRNAs that are full-length complements of vRNA molecules and act as replicative intermediates (19,24).The PB1 subunit contains the polymerase active site, with sequence motifs characteristic of viral RNA-dependent RNA polymerases (34) which are essential for its activity (5). It also contains sites for sequence-specific binding to the conserved 5Ј-and 3Ј-terminal sequences of the vRNA and cRNA molecules (10,11,22). The PB2 protein binds cap1 structures (6, 15, 43). Although previous evidence indicated that the endonucleolytic activity responsible for the cleavage of host mRNA precursors could be contained on this subunit (7, 23), it has been proposed that the endonucleolytic activity lies on the PB1 subunit (21). Because it is responsible for cap-binding activity, PB2 is involved in transcription, but recently, we identified mutations in the PB2 subunit that specifically altered viral RNA replication (9a). The phenotype of viral temperaturesensitive mutants suggests that the PA subunit may be involved in the transition from mRNA transcription to cRNA and vRNA synthesis (reviewed in reference 24), but its precise role in this process is unknown at present. A recent report identified a mutation in the PA protein that shows a phenotype i...

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“…47,68 The fold of the endonuclease domain and its biochemical properties are similar to those of type II restriction endonucleases. 63,66 In addition of the endonuclease active site, protease activities have been associated to the PA subunit of the polymerase [69][70][71] and a correlation has been reported between such an activity and the capacity of the polymerase to replicate. 72,73 The mechanism by which the polymerase subunits interact, the site where the trimeric complex is formed and its transport to the nucleus have been analysed in detail.…”

Section: To Switch or Not To Switchmentioning

confidence: 99%

The influenza virus RNA synthesis machine

et al. 2011

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Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site

Cited by 67 publications

References 73 publications

Interferon-Inducible Protein Mx1 Inhibits Influenza Virus by Interfering with Functional Viral Ribonucleoprotein Complex Assembly

Interferon-Inducible Protein Mx1 Inhibits Influenza Virus by Interfering with Functional Viral Ribonucleoprotein Complex Assembly

Threonine 157 of Influenza Virus PA Polymerase Subunit Modulates RNA Replication in Infectious Viruses

The influenza virus RNA synthesis machine

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