Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase

Lee, M. T. Michael; Bishop, Konrad; Medcalf, Liz; Elton, Debra; Digard, Paul; Tiley, Laurence

doi:10.1093/nar/30.2.429

Cited by 68 publications

(69 citation statements)

References 57 publications

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“…Although PB1 alone can bind the 5Ј-terminal vRNA sequence (10), its binding is more efficient upon PB1-PA complex formation (20), suggesting a role for PA in the ability of the polymerase to interact with the vRNA 5Ј terminus. A polymerase complex with alterations in the RNA binding properties could be unsuitable for cRNA synthesis.…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

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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“…95 Although it has been reported that alternative binary complexes of the polymerase subunits might be responsible for transcription or replication, 120,121 a number of other reports indicate that the polymerase heterotrimer is the functional unit in both processes. 8,48,49,63,66,122 Altogether, the genetic approaches indicated above enabled the proposal of a new model for influenza RNA replication, which is depicted in the diagram presented in Figure 5. Activation of vRNA synthesis would result from the interaction of a soluble polymerase complex (Non-resident polymerase-NRP1-) with the cRNP (Fig.…”

Section: Virus Genome Amplificationmentioning

confidence: 99%

The influenza virus RNA synthesis machine

et al. 2011

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“…It has been reported that the PB1-PA dimeric complex can bind to the 5Ј terminus of vRNA in a gel shift experiment (23).…”

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confidence: 99%

In Vitro Assembly of PB2 with a PB1-PA Dimer Supports a New Model of Assembly of Influenza A Virus Polymerase Subunits into a Functional Trimeric Complex

Deng

Sharps

Fodor

et al. 2005

J Virol

137

171

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Influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex of PB1, PB2, and PA. We show that the individually expressed PB2 subunit can be assembled with the coexpressed PB1-PA dimer in vitro into a transcriptionally active complex. Furthermore, we demonstrate that a model viral RNA promoter can bind to the PB1-PA dimer prior to assembly with PB2. Our results are consistent with a recently proposed model for the sequential assembly of viral RNA polymerase complex in which the PB1-PA dimeric complex and the PB2 monomer are transported into the nucleus separately and then assembled in the nucleus.

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Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase

Cited by 68 publications

References 57 publications

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

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

The influenza virus RNA synthesis machine

In Vitro Assembly of PB2 with a PB1-PA Dimer Supports a New Model of Assembly of Influenza A Virus Polymerase Subunits into a Functional Trimeric Complex

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