Influenza A Virus RNA Polymerase Has the Ability To Stutter at the Polyadenylation Site of a Viral RNA Template during RNA Replication

ABSTRACT The viral polymerase of influenza virus, a negative-strand RNA virus, is believed to polyadenylate the mRNAs by stuttering at a stretch of five to seven uridine residues which are located close to the 5′ ends of the viral RNA templates. However, a mechanism of polyadenylation based on a template-independent synthesis of the poly(A) tail has not been excluded. In this report, we present new evidence showing the inherent ability of the viral polymerase to stutter at the poly(U) stretch of a viral RNA template during RNA replication. Variants which possess 1- to 13-nucleotide-long insertions at the poly(U) stretch have been identified. These results support a stuttering mechanism for the polyadenylation of influenza virus mRNAs.

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Fundamental contribution and host range determination of ANP32 protein family in influenza A virus polymerase activity

10.1101/529412 ◽

2019 ◽

Cited By ~ 2

Author(s):

Haili Zhang ◽

Zhenyu Zhang ◽

Yujie Wang ◽

Meiyue Wang ◽

Xuefeng Wang ◽

...

Keyword(s):

Influenza Virus ◽

Viral Replication ◽

Influenza A Virus ◽

Mammalian Cells ◽

Influenza A ◽

Rna Replication ◽

Polymerase Activity ◽

Viral Polymerase ◽

Human Influenza Virus ◽

Virus Rna

ABSTRACTThe polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase, and to both influence polymerase activity and interspecies restriction. Here we report that either ANP32A or ANP32B is indispensable for influenza A virus RNA replication. The contribution of ANP32B is equal to that of ANP32A, and together they play a fundamental role in the activity of mammalian influenza A virus polymerase, while neither human ANP32A nor ANP32B support the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving ANP32A alone to support viral replication. Two amino acid mutations at sites 129-130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A&B as key factors for both virus replication and adaption.IMPORTANCEThe key host factors involved in the influenza A viral the polymerase activity and RNA replication remain largely unknown. Here we provide evidence that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza virus RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily non-functional in supporting viral replication because of a 129-130 site mutation. The 129-130 site plays an important role in ANP32A/B and viral polymerase interaction, therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

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Mutations in the N-Terminal Region of Influenza Virus PB2 Protein Affect Virus RNA Replication but Not Transcription

Journal of Virology ◽

10.1128/jvi.77.9.5098-5108.2003 ◽

2003 ◽

Vol 77 (9) ◽

pp. 5098-5108 ◽

Cited By ~ 33

Author(s):

Pablo Gastaminza ◽

Beatriz Perales ◽

Ana M. Falcón ◽

Juan Ortín

Keyword(s):

Influenza Virus ◽

Rna Replication ◽

Terminal Region ◽

Viral Rna ◽

Mrna Accumulation ◽

Virus Rna ◽

Transcription In Vitro ◽

Type Mutant

ABSTRACT PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.

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Fundamental Contribution and Host Range Determination of ANP32A and ANP32B in Influenza A Virus Polymerase Activity

Journal of Virology ◽

10.1128/jvi.00174-19 ◽

2019 ◽

Vol 93 (13) ◽

Cited By ~ 16

Author(s):

Haili Zhang ◽

Zhenyu Zhang ◽

Yujie Wang ◽

Meiyue Wang ◽

Xuefeng Wang ◽

...

Keyword(s):

Influenza Virus ◽

Viral Replication ◽

Influenza A Virus ◽

Mammalian Cells ◽

Influenza A ◽

Rna Replication ◽

Polymerase Activity ◽

Cellular Protein ◽

Human Influenza ◽

Viral Polymerase

ABSTRACTThe polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase and to influence both polymerase activity and interspecies restriction. We report here that either human ANP32A or ANP32B is indispensable for human influenza A virus RNA replication. The contribution of huANP32B is equal to that of huANP32A, and together they play a fundamental role in the activity of human influenza A virus polymerase, while neither human ANP32A nor ANP32B supports the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving avian ANP32A alone to support viral replication. Two amino acid mutations at sites 129 to 130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A and ANP32B as key factors for both virus replication and adaptation.IMPORTANCEThe key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. We provide evidence here that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza viral RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily nonfunctional in supporting viral replication because of a mutation at sites 129 and 130. These sites play an important role in ANP32A/ANP32B and viral polymerase interaction and therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

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

Journal of Virology ◽

10.1128/jvi.02467-16 ◽

2017 ◽

Vol 91 (7) ◽

Cited By ~ 13

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.

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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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CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

mBio ◽

10.1128/mbio.00597-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 19

Author(s):

Yu-Chen Lin ◽

King-Song Jeng ◽

Michael M. C. Lai

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Ubiquitin Ligase ◽

Influenza A ◽

Rna Binding ◽

Rna Replication ◽

Viral Rna ◽

Mass Spectrometry Analysis ◽

Positive Role

ABSTRACT Influenza A virus (IAV) RNA segments are individually packaged with viral nucleoprotein (NP) and RNA polymerases to form a viral ribonucleoprotein (vRNP) complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4), which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro -translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication. IMPORTANCE Influenza virus, particularly influenza A virus, causes severe and frequent outbreaks among human and avian species. Finding potential target sites for antiviral agents is of utmost importance from the public health point of view. We previously found that viral nucleoprotein (NP) is ubiquitinated, and ubiquitination enhances viral RNA replication. In this study, we found a cellular ubiquitin ligase, CNOT4, capable of ubiquitinating NP. The ubiquitination sites are scattered on the surface of the NP molecule, which is critical for RNA replication. CNOT4 and a ubiquitin protease, USP11, together regulate the extent of NP ubiquitination and thereby the efficiency of RNA replication. This study thus identifies a potential antiviral target site and reveals a novel posttranslational mechanism for regulating viral replication. This represents a novel finding in the literature of influenza virus research.

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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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Nonconserved Nucleotides at the 3′ and 5′ Ends of an Influenza A Virus RNA Play an Important Role in Viral RNA Replication

Virology ◽

10.1006/viro.1996.0111 ◽

1996 ◽

Vol 217 (1) ◽

pp. 242-251 ◽

Cited By ~ 70

Author(s):

HONGYONG ZHENG ◽

PETER PALESE ◽

ADOLFO GARCÍA-SASTRE

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Rna Replication ◽

Viral Rna ◽

Virus Rna

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Interaction of the Influenza A Virus Nucleocapsid Protein with the Viral RNA Polymerase Potentiates Unprimed Viral RNA Replication

Journal of Virology ◽

10.1128/jvi.02293-07 ◽

2008 ◽

Vol 83 (1) ◽

pp. 29-36 ◽

Cited By ~ 61

Author(s):

Laura L. Newcomb ◽

Rei-Lin Kuo ◽

Qiaozhen Ye ◽

Yunyun Jiang ◽

Yizhi Jane Tao ◽

...

Keyword(s):

Influenza A Virus ◽

Nucleocapsid Protein ◽

Influenza A ◽

Rna Binding ◽

Direct Interaction ◽

Rna Replication ◽

Binding Activity ◽

Viral Rna ◽

Viral Polymerase

ABSTRACT The influenza A virus polymerase transcribes and replicates the eight virion RNA (vRNA) segments. Transcription is initiated with capped RNA primers excised from cellular pre-mRNAs by the intrinsic endonuclease of the viral polymerase. Viral RNA replication occurs in two steps: first a full-length copy of vRNA is made, termed cRNA, and then this cRNA is copied to produce vRNA. The synthesis of cRNAs and vRNAs is initiated without a primer, in contrast to the initiation of viral mRNA synthesis, and requires the viral nucleocapsid protein (NP). The mechanism of unprimed viral RNA replication is poorly understood. To elucidate this mechanism, we used purified recombinant influenza virus polymerase complexes and NP to establish an in vitro system that catalyzes the unprimed synthesis of cRNA and vRNA using 50-nucleotide-long RNA templates. The purified viral polymerase and NP are sufficient for catalyzing this RNA synthesis without a primer, suggesting that host cell factors are not required. We used this purified in vitro replication system to demonstrate that the RNA-binding activity of NP is not required for the unprimed synthesis of cRNA and vRNA. This result rules out two models that postulate that the RNA-binding activity of NP mediates the switch from capped RNA-primed transcription to unprimed viral RNA replication. Because we showed that NP lacking RNA-binding activity binds directly to the viral polymerase, it is likely that a direct interaction between NP and the viral polymerase results in a modification of the polymerase in favor of unprimed initiation.

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