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

2009 ◽  
Vol 90 (6) ◽  
pp. 1398-1407 ◽  
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.

scholarly journals Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Benjamin E. Nilsson ◽  
Aartjan J. W. te Velthuis ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Influenza A Viruses ◽  
Viral Polymerase ◽  
Virus Rna ◽  
Rna Genome ◽  
Transcription And Replication

ABSTRACT The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome. IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

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

2005 ◽  
Vol 79 (9) ◽  
pp. 5812-5818 ◽  
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.

scholarly journals Polyuridylated mRNA Synthesized by a Recombinant Influenza Virus Is Defective in Nuclear Export

2000 ◽  
Vol 74 (1) ◽  
pp. 418-427 ◽  
Author(s):  
Leo L. M. Poon ◽  
Ervin Fodor ◽  
George G. Brownlee
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Nuclear Export ◽  
Influenza A ◽  
Viral Rna ◽  
Attenuated Virus ◽  
Infected Cells ◽  
Novel Virus ◽  
Virus Mutant ◽  
Novel Influenza A

ABSTRACT The poly(A) tail of influenza virus mRNA is synthesized by reiterative copying of a U track near the 5′ end of the virion RNA (vRNA) template by the viral RNA polymerase. We have engineered a novel influenza A/WSN/33 virus which contains a neuraminidase (NA) vRNA with its U track mutated into an A track. Instead of synthesizing poly(A)-tailed NA mRNA, this novel virus synthesizes poly(U)-tailed NA mRNA. In infected cells, most poly(U)-tailed NA mRNA was retained in the nucleus, while most control polyadenylated NA mRNA was transported to the cytoplasm. These results suggest that the poly(A) tail is important for efficient nuclear export of NA mRNA. The mutant virus produced a reduced amount of NA and showed an attenuated phenotype, suggesting that poly(A) signal mutants of this type might be useful as potential live attenuated virus vaccines. In addition, this virus mutant might provide a useful model to further elucidate the basic mechanisms of mRNA nuclear export.

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

2001 ◽  
Vol 276 (33) ◽  
pp. 31179-31185 ◽  
Author(s):  
Ayae Honda ◽  
Atsushi Endo ◽  
Kiyohisa Mizumoto ◽  
Akira Ishihama
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Viral Rna ◽  
Virus Rna ◽  
Functional Modulation

scholarly journals 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

2015 ◽  
Vol 89 (12) ◽  
pp. 6376-6390 ◽  
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.

scholarly journals Involvement of Hsp90 in Assembly and Nuclear Import of Influenza Virus RNA Polymerase Subunits

2006 ◽  
Vol 81 (3) ◽  
pp. 1339-1349 ◽  
Author(s):  
Tadasuke Naito ◽  
Fumitaka Momose ◽  
Atsushi Kawaguchi ◽  
Kyosuke Nagata
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Nuclear Transport ◽  
Intracellular Localization ◽  
Viral Rna ◽  
Polymerase Complex ◽  
Virus Rna ◽  
Binary Complexes ◽  
Infected Cells ◽  
Rna Polymerase Subunits

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.

scholarly journals Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT

2010 ◽  
Vol 84 (17) ◽  
pp. 8691-8699 ◽  
Author(s):  
Tatiana Fislová ◽  
Benjamin Thomas ◽  
Katy M. Graef ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Viral Replication ◽  
Mammalian Cells ◽  
Influenza A ◽  
Mitochondrial Protein ◽  
Host Factors ◽  
Fk506 Binding Protein ◽  
Virus Rna ◽  
Rna Accumulation

ABSTRACT The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), α- and β-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.

scholarly journals Role of the influenza virus heterotrimeric RNA polymerase complex in the initiation of replication

2006 ◽  
Vol 87 (11) ◽  
pp. 3373-3377 ◽  
Author(s):  
Tao Deng ◽  
Jane L. Sharps ◽  
George G. Brownlee
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Replication Initiation ◽  
Polymerase Complex ◽  
Virus Rna ◽  
Initiation Of Replication ◽  
Rna Genome ◽  
Transcription And Replication

Both transcription and replication of the influenza virus RNA genome are catalysed by a virus-specific RNA polymerase. Recently, an in vitro assay, based on the synthesis of pppApG, for the initiation of replication by recombinant RNA polymerase in the absence of added primer was described. Here, these findings are extended to show that adenosine, AMP and ADP can each substitute for ATP in reactions catalysed by either recombinant ribonucleoprotein or RNA polymerase complexes with either model virion RNA (vRNA) or cRNA promoters. The use of either adenosine or AMP, rather than ATP, provides a convenient, sensitive and easy assay of replication initiation. Moreover, no pppApG was detected when a PB1–PA dimer, rather than the trimeric polymerase, was used to catalyse synthesis, contrasting with a previous report using baculovirus-expressed influenza RNA polymerase. Overall, it is suggested that the heterotrimeric polymerase is essential for the initiation of replication.

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