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 Role of Ran Binding Protein 5 in Nuclear Import and Assembly of the Influenza Virus RNA Polymerase Complex

2006 ◽  
Vol 80 (24) ◽  
pp. 11911-11919 ◽  
Author(s):  
Tao Deng ◽  
Othmar G. Engelhardt ◽  
Benjamin Thomas ◽  
Alexandre V. Akoulitchev ◽  
George G. Brownlee ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Nuclear Import ◽  
Influenza A ◽  
Binding Protein ◽  
Nuclear Accumulation ◽  
Polymerase Complex ◽  
Knock Down ◽  
Virus Rna ◽  
Infected Cells

ABSTRACT The influenza A virus RNA-dependent RNA polymerase is a heterotrimeric complex of polymerase basic protein 1 (PB1), PB2, and polymerase acidic protein (PA) subunits. It performs transcription and replication of the viral RNA genome in the nucleus of infected cells. We have identified a nuclear import factor, Ran binding protein 5 (RanBP5), also known as karyopherin β3, importin β3, or importin 5, as an interactor of the PB1 subunit. RanBP5 interacted with either PB1 alone or with a PB1-PA dimer but not with a PB1-PB2 dimer or the trimeric complex. The interaction between RanBP5 and PB1-PA was disrupted by RanGTP in vitro, allowing PB2 to bind to the PB1-PA dimer to form a functional trimeric RNA polymerase complex. We propose a model in which RanBP5 acts as an import factor for the newly synthesized polymerase by targeting the PB1-PA dimer to the nucleus. In agreement with this model, small interfering RNA (siRNA)-mediated knock-down of RanBP5 inhibited the nuclear accumulation of the PB1-PA dimer. Moreover, siRNA knock-down of RanBP5 resulted in the delayed accumulation of viral RNAs in infected cells, confirming that RanBP5 plays a biological role during the influenza virus life cycle.

scholarly journals Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses

2016 ◽  
Vol 90 (7) ◽  
pp. 3684-3693 ◽  
Author(s):  
Léa Meyer ◽  
Alix Sausset ◽  
Laura Sedano ◽  
Bruno Da Costa ◽  
Ronan Le Goffic ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Wild Type Virus ◽  
Deletion Mutants ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type

ABSTRACTThe influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA–PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for furtherin vitroandin vivocharacterization. Four viable mutants with single-codon deletions were generated; all of them exhibited atsphenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serialin vitropassages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stabletsmutants that could be used to design novel attenuated vaccines.IMPORTANCEIn order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA–PB1 dimer to the nucleus, where viral replication occurs. Thesetsdeletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.

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 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 A Single Amino Acid Mutation in the PA Subunit of the Influenza Virus RNA Polymerase Promotes the Generation of Defective Interfering RNAs

2003 ◽  
Vol 77 (8) ◽  
pp. 5017-5020 ◽  
Author(s):  
Ervin Fodor ◽  
Louise J. Mingay ◽  
Mandy Crow ◽  
Tao Deng ◽  
George G. Brownlee
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A ◽  
Single Amino Acid ◽  
Amino Acid Mutation ◽  
Virus Rna ◽  
Form Part ◽  
Single Amino Acid Mutation

ABSTRACT An R638A mutation of the polymerase acidic protein (PA) subunit of the RNA polymerase of influenza A/WSN/33 virus results in severe attenuation of viral growth in cell culture by promoting the synthesis of defective interfering RNAs. We propose that R638A is an “elongation” mutant that destabilizes PA-RNA template interactions during elongation. A C453R mutation in PA can compensate for this defect, suggesting that amino acids C453 and R638 form part of the same domain.

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 The PB2 Subunit of the Influenza A Virus RNA Polymerase Is Imported into the Mitochondrial Matrix

2016 ◽  
Vol 90 (19) ◽  
pp. 8729-8738 ◽  
Author(s):  
Joshua C. D. Long ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Rna Polymerase ◽  
Influenza A ◽  
Influenza Viruses ◽  
Mitochondrial Matrix ◽  
Innate Immune Responses ◽  
Mitochondrial Import ◽  
Virus Rna ◽  
Mitochondrial Antiviral Signaling

ABSTRACTThe polymerase basic 2 (PB2) subunit of the RNA polymerase complex of seasonal human influenza A viruses has been shown to localize to the mitochondria. Various roles, including the regulation of apoptosis and innate immune responses to viral infection, have been proposed for mitochondrial PB2. In particular, PB2 has been shown to inhibit interferon expression by associating with the mitochondrial antiviral signaling (MAVS) protein, which acts downstream of RIG-I and MDA-5 in the interferon induction pathway. However, in spite of a growing body of literature on the potential roles of mitochondrial PB2, the exact location of PB2 in mitochondria has not been determined. Here, we used enhancedascorbateperoxidase (APEX)-tagged PB2 proteins and electron microscopy to study the localization of PB2 in mitochondria. We found that PB2 is imported into mitochondria, where it localizes to the mitochondrial matrix. We also demonstrated that MAVS is not required for the import of PB2 into mitochondria by showing that PB2 associates with mitochondria in MAVS knockout mouse embryo fibroblasts. Instead, we found that amino acid residue 9 in the N-terminal mitochondrial targeting sequence is a determinant of the mitochondrial import of PB2, differentiating the localization of PB2 of human from that of avian influenza A virus strains. We also showed that a virus encoding nonmitochondrial PB2 is attenuated in mouse embryonic fibroblasts (MEFs) compared with an isogenic virus encoding mitochondrial PB2, in a MAVS-independent manner, suggesting a role for PB2 within the mitochondrial matrix. This work extends our understanding of the interplay between influenza virus and mitochondria.IMPORTANCEThe PB2 subunit of the influenza virus RNA polymerase is a major determinant of viral pathogenicity. However, the molecular mechanisms of how PB2 determines pathogenicity remain poorly understood. PB2 associates with mitochondria and inhibits the function of the mitochondrial antiviral signaling protein MAVS, implicating PB2 in the regulation of innate immune responses. We found that PB2 is imported into the mitochondrial matrix and showed that amino acid residue 9 is a determinant of mitochondrial import. The presence of asparagine or threonine in over 99% of all human seasonal influenza virus pre-2009 H1N1, H2N2, and H3N2 strains is compatible with mitochondrial import, whereas the presence of an aspartic acid in over 95% of all avian influenza viruses is not, resulting in a clear distinction between human-adapted and avian influenza viruses. These findings provide insights into the interplay between influenza virus and mitochondria and suggest mechanisms by which PB2 could affect pathogenicity.

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.

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