A mechanism for prime-realignment during influenza A virus replication

ABSTRACTThe influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2, and PA. To copy the viral RNA (vRNA) genome segments and the cRNA segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two differentde novoinitiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3′ terminus, while on the cRNA promoter, the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular, the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into priming and realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly.IMPORTANCEInfluenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome by using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and that abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.

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The host factor ANP32A is required for influenza A virus vRNA and cRNA synthesis

Journal of Virology ◽

10.1128/jvi.02092-21 ◽

2021 ◽

Author(s):

Benjamin E. Nilsson-Payant ◽

Benjamin R. tenOever ◽

Aartjan J.W. te Velthuis

Keyword(s):

Avian Influenza ◽

Rna Polymerase ◽

Viral Replication ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Host Factor ◽

Rna Polymerases ◽

Viral Rna ◽

Influenza A Viruses

Influenza A viruses are negative-sense RNA viruses that rely on their own viral replication machinery to replicate and transcribe their segmented single-stranded RNA genome. The viral ribonucleoprotein complexes in which viral RNA is replicated consist of a nucleoprotein scaffold around which the RNA genome is bound, and a heterotrimeric RNA-dependent RNA polymerase that catalyzes viral replication. The RNA polymerase copies the viral RNA (vRNA) via a replicative intermediate, called the complementary RNA (cRNA), and subsequently uses this cRNA to make more vRNA copies. To ensure that new cRNA and vRNA molecules are associated with ribonucleoproteins in which they can be amplified, the active RNA polymerase recruits a second polymerase to encapsidate the cRNA or vRNA. Host factor ANP32A has been shown to be essential for viral replication and to facilitate the formation of a dimer between viral RNA polymerases. Differences between mammalian and avian ANP32A proteins are sufficient to restrict viral replication. It has been proposed that ANP32A is only required for the synthesis of vRNA molecules from a cRNA, but not vice versa. However, this view does not match recent molecular evidence. Here we use minigenome assays, virus infections, and viral promoter mutations to demonstrate that ANP32A is essential for both vRNA and cRNA synthesis. Moreover, we show that ANP32 is not only needed for the actively replicating polymerase, but also for the polymerase that is encapsidating nascent viral RNA products. Overall, these results provide new insights into influenza A virus replication and host adaptation. IMPORTANCE Zoonotic avian influenza A viruses pose a constant threat to global health, and they have the potential to cause pandemics. Species variations in host factor ANP32A play a key role in supporting the activity of avian influenza A virus RNA polymerases in mammalian hosts. Here we show that ANP32A acts at two stages in the influenza A virus replication cycle, supporting recent structural experiments, in line with its essential role. Understanding how ANP32A supports viral RNA polymerase activity and how it supports avian polymerase function in mammalian hosts is important for understanding influenza A virus replication and the development of antiviral strategies against influenza A viruses.

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The host factor ANP32A is required for influenza A virus vRNA and cRNA synthesis

10.1101/2021.04.30.442228 ◽

2021 ◽

Author(s):

Benjamin E Nilsson-Payant ◽

Benjamin R. tenOever ◽

Aartjan J.W. te Velthuis

Keyword(s):

Rna Polymerase ◽

Viral Replication ◽

Influenza A Virus ◽

Influenza A ◽

Host Factor ◽

Viral Rna ◽

Molecular Evidence ◽

Influenza A Viruses ◽

Ribonucleoprotein Complexes ◽

Rna Genome

Influenza A viruses are negative-sense RNA viruses that rely on their own viral replication machinery to replicate and transcribe their segmented single-stranded RNA genome. The viral ribonucleoprotein complexes in which viral RNA is replicated consist of a nucleoprotein scaffold around which the RNA genome is bound, and a heterotrimeric RNA-dependent RNA polymerase that catalyzes viral replication. The RNA polymerase copies the viral RNA (vRNA) via a replicative intermediate, called the complementary RNA (cRNA), and subsequently uses this cRNA to make more vRNA copies. To ensure that new cRNA and vRNA molecules are associated with ribonucleoproteins in which they can be amplified, the active RNA polymerase recruits a second polymerase to encapsidate the cRNA or vRNA. Host factor ANP32A has been shown to be essential for viral replication and to facilitate the formation of a dimer between viral RNA polymerases and differences between mammalian and avian ANP32A proteins are sufficient to restrict viral replication. It has been proposed that ANP32A is only required for the synthesis of vRNA molecules from a cRNA, but not vice versa. However, this view does not match recent molecular evidence. Here we use minigenome assays, virus infections, and viral promoter mutations to demonstrate that ANP32A is essential for both vRNA and cRNA synthesis. Moreover, we show that ANP32 is not only needed for the actively replicating polymerase, but also for the polymerase that is encapsidating nascent viral RNA products. Overall, these results provide new insights into influenza A virus replication and host adaptation.

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Type-IInterferon-Inducible SERTAD3 Inhibits Influenza A Virus Replication by Blocking the Assembly of Viral RNA Polymerase Complex

Cell Reports ◽

10.1016/j.celrep.2020.108342 ◽

2020 ◽

Vol 33 (5) ◽

pp. 108342

Author(s):

Nina Sun ◽

Chunfeng Li ◽

Xiao-Feng Li ◽

Yong-Qiang Deng ◽

Tao Jiang ◽

...

Keyword(s):

Rna Polymerase ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Viral Rna ◽

Polymerase Complex

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1SA3-01 Structural insight into essential subunit contacts of the influenza virus RNA polymerase(1SA3 Novel findings of influenza A virus RNA dependent RNA polymerase,The 47th Annual Meeting of the Biophysical Society of Japan)

Seibutsu Butsuri ◽

10.2142/biophys.49.s5_2 ◽

2009 ◽

Vol 49 (supplement) ◽

pp. S5

Author(s):

Eiji Obayashi ◽

Kanako Sugiyama ◽

Hisashi Yoshida ◽

Fumihiro Kawai ◽

Atsushi Kawaguchi ◽

...

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Influenza A Virus ◽

Influenza A ◽

Rna Dependent Rna Polymerase ◽

Virus Rna ◽

Biophysical Society ◽

Structural Insight ◽

Essential Subunit ◽

Insight Into

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Viral RNA Polymerase: A Promising Antiviral Target for Influenza A Virus

Current Medicinal Chemistry ◽

10.2174/09298673113209990208 ◽

2013 ◽

Vol 20 (31) ◽

pp. 3923-3934 ◽

Cited By ~ 14

Author(s):

Fangyuan Shi ◽

Yuanchao Xie ◽

Lifang Shi ◽

Wenfang Xu

Keyword(s):

Rna Polymerase ◽

Influenza A Virus ◽

Influenza A ◽

Viral Rna

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Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

Journal of Virology ◽

10.1128/jvi.75.17.8127-8136.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 8127-8136 ◽

Cited By ~ 78

Author(s):

Daniel R. Perez ◽

Ruben O. Donis

Keyword(s):

Amino Acids ◽

Influenza Virus ◽

Amino Acid ◽

Viral Replication ◽

Influenza A Virus ◽

Influenza A ◽

Amino Acid Sequences ◽

Influenza Viruses ◽

Virus Growth ◽

Transcription And Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

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The Fight against the Influenza A Virus H1N1: Synthesis, Molecular Modeling, and Biological Evaluation of Benzofurazan Derivatives as Viral RNA Polymerase Inhibitors

ChemMedChem ◽

10.1002/cmdc.201300378 ◽

2013 ◽

Vol 9 (1) ◽

pp. 129-150 ◽

Cited By ~ 16

Author(s):

Mafalda Pagano ◽

Daniele Castagnolo ◽

Martina Bernardini ◽

Anna Lucia Fallacara ◽

Ilaria Laurenzana ◽

...

Keyword(s):

Molecular Modeling ◽

Rna Polymerase ◽

Influenza A Virus ◽

Influenza A ◽

Biological Evaluation ◽

Viral Rna ◽

Virus H1n1 ◽

Polymerase Inhibitors

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Role of Initiating Nucleoside Triphosphate Concentrations in the Regulation of Influenza Virus Replication and Transcription

Journal of Virology ◽

10.1128/jvi.00627-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 6902-6910 ◽

Cited By ~ 27

Author(s):

Frank T. Vreede ◽

Hugh Gifford ◽

George G. Brownlee

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Influenza A ◽

De Novo ◽

Nucleoside Triphosphate ◽

Ribonucleoprotein Complexes ◽

Influenza Virus Replication ◽

High Concentrations

ABSTRACT The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3′-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.

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