Differential Roles of Viral RNA and cRNA in Functional Modulation of the Influenza Virus RNA Polymerase

ABSTRACT Transcription and replication of the influenza virus RNA genome occur in the nuclei of infected cells through the viral RNA-dependent RNA polymerase consisting of PB1, PB2, and PA. We previously identified a host factor designated RAF-1 (RNA polymerase activating factor 1) that stimulates viral RNA synthesis. RAF-1 is found to be identical to Hsp90. Here, we examined the intracellular localization of Hsp90 and viral RNA polymerase subunits and their molecular interaction. Hsp90 was found to interact with PB2 and PB1, and it was relocalized to the nucleus upon viral infection. We found that the nuclear transport of Hsp90 occurs in cells expressing PB2 alone. The nuclear transport of Hsp90 was in parallel with that of the viral RNA polymerase binary complexes, either PB1 and PB2 or PB1 and PA, as well as with that of PB2 alone. Hsp90 also interacted with the binary RNA polymerase complex PB1-PB2, and it was dissociated from the PB1-PB2 complex upon its association with PA. Furthermore, Hsp90 could form a stable PB1-PB2-Hsp90 complex prior to the formation of a ternary polymerase complex by the assembly of PA in the infected cells. These results suggest that Hsp90 is involved in the assembly and nuclear transport of viral RNA polymerase subunits, possibly as a molecular chaperone for the polymerase subunits prior to the formation of a mature ternary polymerase complex.

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NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome

Journal of General Virology ◽

10.1099/vir.0.009639-0 ◽

2009 ◽

Vol 90 (6) ◽

pp. 1398-1407 ◽

Cited By ~ 137

Author(s):

Nicole C. Robb ◽

Matt Smith ◽

Frank T. Vreede ◽

Ervin Fodor

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Nuclear Export ◽

Influenza A ◽

Viral Rna ◽

Viral Transcription ◽

Virus Rna ◽

Specific Alteration ◽

Transcription And Replication ◽

Rna Levels

The influenza virus RNA polymerase transcribes the negative-sense viral RNA segments (vRNA) into mRNA and replicates them via complementary RNA (cRNA) intermediates into more copies of vRNA. It is not clear how the relative amounts of the three RNA products, mRNA, cRNA and vRNA, are regulated during the viral life cycle. We found that in viral ribonucleoprotein (vRNP) reconstitution assays involving only the minimal components required for viral transcription and replication (the RNA polymerase, the nucleoprotein and a vRNA template), the relative levels of accumulation of RNA products differed from those observed in infected cells, suggesting a regulatory role for additional viral proteins. Expression of the viral NS2/NEP protein in RNP reconstitution assays affected viral RNA levels by reducing the accumulation of transcription products and increasing the accumulation of replication products to more closely resemble those found during viral infection. This effect was functionally conserved in influenza A and B viruses and was influenza-virus-type-specific, demonstrating that the NS2/NEP protein changes RNA levels by specific alteration of the viral transcription and replication machinery, rather than through an indirect effect on the host cell. Although NS2/NEP has been shown previously to play a role in the nucleocytoplasmic export of viral RNPs, deletion of the nuclear export sequence region that is required for its transport function did not affect the ability of the protein to regulate RNA levels. A role for the NS2/NEP protein in the regulation of influenza virus transcription and replication that is independent of its viral RNP export function is proposed.

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Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis

Nature Reviews Microbiology ◽

10.1038/nrmicro.2016.87 ◽

2016 ◽

Vol 14 (8) ◽

pp. 479-493 ◽

Cited By ~ 159

Author(s):

Aartjan J. W. te Velthuis ◽

Ervin Fodor

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Rna Synthesis ◽

Viral Rna ◽

Virus Rna

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Different De Novo Initiation Strategies Are Used by Influenza Virus RNA Polymerase on Its cRNA and Viral RNA Promoters during Viral RNA Replication

Journal of Virology ◽

10.1128/jvi.80.5.2337-2348.2006 ◽

2006 ◽

Vol 80 (5) ◽

pp. 2337-2348 ◽

Cited By ~ 79

Author(s):

Tao Deng ◽

Frank T. Vreede ◽

George G. Brownlee

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

De Novo ◽

Viral Rna ◽

Virus Rna ◽

Initiation Of Replication ◽

Replicative Intermediates ◽

Internal Initiation

ABSTRACT Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of cRNA and viral RNA (vRNA) differed. To do this, two in vitro replication assays, which generated transcripts that had 5′ triphosphate end groups characteristic of authentic replication products, were developed. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppApG synthesis internally on the model cRNA promoter, whereas it initiated pppApG synthesis terminally on the model vRNA promoter. The internally synthesized pppApG could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model cRNA and vRNA promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a cRNA chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its cRNA and vRNA promoters. To our knowledge, this is novel and has not previously been described for any viral RNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells.

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Association of the Influenza A Virus RNA-Dependent RNA Polymerase with Cellular RNA Polymerase II

Journal of Virology ◽

10.1128/jvi.79.9.5812-5818.2005 ◽

2005 ◽

Vol 79 (9) ◽

pp. 5812-5818 ◽

Cited By ~ 148

Author(s):

Othmar G. Engelhardt ◽

Matt Smith ◽

Ervin Fodor

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Influenza A ◽

Large Subunit ◽

Viral Rna ◽

Rna Dependent Rna Polymerase ◽

Pol Ii ◽

Virus Rna ◽

Transcription Sites

ABSTRACT Transcription by the influenza virus RNA-dependent RNA polymerase is dependent on cellular RNA processing activities that are known to be associated with cellular RNA polymerase II (Pol II) transcription, namely, capping and splicing. Therefore, it had been hypothesized that transcription by the viral RNA polymerase and Pol II might be functionally linked. Here, we demonstrate for the first time that the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain. The viral polymerase binds hyperphosphorylated forms of Pol II, indicating that it targets actively transcribing Pol II. In addition, immunofluorescence analysis is consistent with a new model showing that influenza virus polymerase accumulates at Pol II transcription sites. The present findings provide a framework for further studies to elucidate the mechanistic principles of transcription by a viral RNA polymerase and have implications for the regulation of Pol II activities in infected cells.

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The role of the pseudoknot at the 3' end of turnip yellow mosaic virus RNA in minus-strand synthesis by the viral RNA-dependent RNA polymerase.

Journal of Virology ◽

10.1128/jvi.71.8.5990-5996.1997 ◽

1997 ◽

Vol 71 (8) ◽

pp. 5990-5996 ◽

Cited By ~ 27

Author(s):

B A Deiman ◽

R M Kortlever ◽

C W Pleij

Keyword(s):

Rna Polymerase ◽

Mosaic Virus ◽

Viral Rna ◽

Yellow Mosaic Virus ◽

Turnip Yellow Mosaic Virus ◽

Minus Strand ◽

Rna Dependent Rna Polymerase ◽

Virus Rna

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Temperature-Sensitive Mutants in the Influenza A Virus RNA Polymerase: Alterations in the PA Linker Reduce Nuclear Targeting of the PB1-PA Dimer and Result in Viral Attenuation

Journal of Virology ◽

10.1128/jvi.00589-15 ◽

2015 ◽

Vol 89 (12) ◽

pp. 6376-6390 ◽

Cited By ~ 13

Author(s):

Bruno Da Costa ◽

Alix Sausset ◽

Sandie Munier ◽

Alexandre Ghounaris ◽

Nadia Naffakh ◽

...

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Nuclear Import ◽

Influenza A ◽

Rational Design ◽

Hot Spot ◽

Temperature Sensitive ◽

Nuclear Targeting ◽

Virus Rna ◽

Endonuclease Domain

ABSTRACTThe influenza virus RNA-dependent RNA polymerase catalyzes genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits PB1, PB2, and PA are critical steps in the virus life cycle. We investigated the structure and function of the PA linker (residues 197 to 256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroism experiments revealed that the PA linker by itself is structurally disordered. A large series of PA linker mutants exhibited a temperature-sensitive (ts) phenotype (reduced viral growth at 39.5°C versus 37°C/33°C), suggesting an alteration of folding kinetic parameters. Thetsphenotype was associated with a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed thattsand lethal PA mutants did not efficiently recruit PB1 to reach the nucleus at 39.5°C. A protein complementation assay using PA mutants, PB1, and β-importin IPO5 tagged with fragments of theGaussia princepsluciferase showed that increasing the temperature negatively modulated the PA-PB1 and the PA-PB1-IPO5 interactions or complex stability. The selection of revertant viruses allowed the identification of different types of compensatory mutations located in one or the other of the three polymerase subunits. Twotsmutants were shown to be attenuated and able to induce antibodies in mice. Taken together, our results identify a PA domain critical for PB1-PA nuclear import and that is a “hot spot” to engineertsmutants that could be used to design novel attenuated vaccines.IMPORTANCEBy targeting a discrete domain of the PA polymerase subunit of influenza virus, we were able to identify a series of 9 amino acid positions that are appropriate to engineer temperature-sensitive (ts) mutants. This is the first time that a large number oftsmutations were engineered in such a short domain, demonstrating that rational design oftsmutants can be achieved. We were able to associate this phenotype with a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restored the ability of the complex to move to the nucleus. Two of thesetsmutants were shown to be attenuated and able to produce antibodies in mice. These results are of high interest for the design of novel attenuated vaccines and to develop new antiviral drugs.

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