A small molecule multi-kinase inhibitor reduces influenza A virus replication by restricting viral RNA synthesis

ABSTRACTInfluenza A viral ribonucleoprotein (vRNP) is responsible for transcription and replication of the viral genome in infected cells and depends on host factors for its functions. Identification of the host factors interacting with vRNP not only improves understanding of virus-host interactions but also provides insights into novel mechanisms of viral pathogenicity and the development of new antiviral strategies. Here, we have identified 80 host factors that copurified with vRNP using affinity purification followed by mass spectrometry. LYAR, a cell growth-regulating nucleolar protein, has been shown to be important for influenza A virus replication. During influenza A virus infection, LYAR expression is increased and partly translocates from the nucleolus to the nucleoplasm and cytoplasm. Furthermore, LYAR interacts with RNP subunits, resulting in enhancing viral RNP assembly, thereby facilitating viral RNA synthesis. Taken together, our studies identify a novel vRNP binding host partner important for influenza A virus replication and further reveal the mechanism of LYAR regulating influenza A viral RNA synthesis by facilitating viral RNP assembly.IMPORTANCEInfluenza A virus (IAV) must utilize the host cell machinery to replicate, but many of the mechanisms of IAV-host interaction remain poorly understood. Improved understanding of interactions between host factors and vRNP not only increases our basic knowledge of the molecular mechanisms of virus replication and pathogenicity but also provides insights into possible novel antiviral targets that are necessary due to the widespread emergence of drug-resistant IAV strains. Here, we have identified LYAR, a cell growth-regulating nucleolar protein, which interacts with viral RNP components and is important for efficient replication of IAVs and whose role in the IAV life cycle has never been reported. In addition, we further reveal the role of LYAR in viral RNA synthesis. Our results extend and improve current knowledge on the mechanisms of IAV transcription and replication.

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A single-nucleotide natural variation (U4 to C4) in an influenza A virus promoter exhibits a large structural change: implications for differential viral RNA synthesis by RNA-dependent RNA polymerase

Nucleic Acids Research ◽

10.1093/nar/gkg214 ◽

2003 ◽

Vol 31 (4) ◽

pp. 1216-1223 ◽

Cited By ~ 23

Author(s):

M.-K. Lee

Keyword(s):

Structural Change ◽

Rna Polymerase ◽

Influenza A Virus ◽

Influenza A ◽

Natural Variation ◽

Rna Synthesis ◽

Viral Rna ◽

Rna Dependent Rna Polymerase ◽

Single Nucleotide ◽

Virus Promoter

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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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Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction

Journal of Virology ◽

10.1128/jvi.00232-15 ◽

2015 ◽

Vol 89 (11) ◽

pp. 6067-6079 ◽

Cited By ~ 39

Author(s):

GuanQun Liu ◽

Hong-Su Park ◽

Hyun-Mi Pyo ◽

Qiang Liu ◽

Yan Zhou

Keyword(s):

Viral Replication ◽

Influenza A Virus ◽

Promoter Region ◽

Influenza A ◽

Rna Synthesis ◽

Nucleotide Composition ◽

Viral Rna ◽

Viral Transcription ◽

Wild Type ◽

Viral Promoter

ABSTRACTRetinoic acid-inducible gene I (RIG-I) is an important innate immune sensor that recognizes viral RNA in the cytoplasm. Its nonself recognition largely depends on the unique RNA structures imposed by viral RNA. The panhandle structure residing in the influenza A virus (IAV) genome, whose primary function is to serve as the viral promoter for transcription and replication, has been proposed to be a RIG-I agonist. However, this has never been proved experimentally. Here, we employed multiple approaches to determine if the IAV panhandle structure is directly involved in RIG-I activation and type I interferon (IFN) induction. First, in porcine alveolar macrophages, we demonstrated that the viral genomic coding region is dispensable for RIG-I-dependent IFN induction. Second, usingin vitro-synthesized hairpin RNA, we showed that the IAV panhandle structure could directly bind to RIG-I and stimulate IFN production. Furthermore, we investigated the contributions of the wobble base pairs, mismatch, and unpaired nucleotides within the wild-type panhandle structure to RIG-I activation. Elimination of these destabilizing elements within the panhandle structure promoted RIG-I activation and IFN induction. Given the function of the panhandle structure as the viral promoter, we further monitored the promoter activity of these panhandle variants and found that viral replication was moderately affected, whereas viral transcription was impaired dramatically. In all, our results indicate that the IAV panhandle promoter region adopts a nucleotide composition that is optimal for balanced viral RNA synthesis and suboptimal for RIG-I activation.IMPORTANCEThe IAV genomic panhandle structure has been proposed to be an RIG-I agonist due to its partial complementarity; however, this has not been experimentally confirmed. Here, we provide direct evidence that the IAV panhandle structure is competent in, and sufficient for, RIG-I activation and IFN induction. By constructing panhandle variants with increased complementarity, we demonstrated that the wild-type panhandle structure could be modified to enhance RIG-I activation and IFN induction. These panhandle variants posed moderate influence on viral replication but dramatic impairment of viral transcription. These results indicate that the IAV panhandle promoter region adopts a nucleotide composition to achieve optimal balance of viral RNA synthesis and suboptimal RIG-I activation. Our results highlight the multifunctional role of the IAV panhandle promoter region in the virus life cycle and offer novel insights into the development of antiviral agents aiming to boost RIG-I signaling or virus attenuation by manipulating this conserved region.

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A mechanism for prime-realignment during influenza A virus replication

10.1101/138487 ◽

2017 ◽

Cited By ~ 2

Author(s):

Judith Oymans ◽

Aartjan J.W. te Velthuis

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Viral Replication ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

De Novo ◽

Severe Disease ◽

Viral Rna ◽

Insight Into

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 complementary RNA (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 prime-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 using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and 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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A Mechanism for Priming and Realignment during Influenza A Virus Replication

Journal of Virology ◽

10.1128/jvi.01773-17 ◽

2017 ◽

Vol 92 (3) ◽

Cited By ~ 8

Author(s):

Judith Oymans ◽

Aartjan J. W. te Velthuis

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Viral Replication ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

De Novo ◽

Severe Disease ◽

Viral Rna ◽

Insight Into

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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Model Suggesting that Replication of Influenza Virus Is Regulated by Stabilization of Replicative Intermediates

Journal of Virology ◽

10.1128/jvi.78.17.9568-9572.2004 ◽

2004 ◽

Vol 78 (17) ◽

pp. 9568-9572 ◽

Cited By ~ 142

Author(s):

Frank T. Vreede ◽

Tanis E. Jung ◽

George G. Brownlee

Keyword(s):

Rna Polymerase ◽

Influenza A Virus ◽

Influenza A ◽

Rna Synthesis ◽

Viral Rna ◽

Mrna Transcription ◽

Rna Dependent Rna Polymerase ◽

Replicative Intermediates ◽

Negative Sense ◽

Transcription And Replication

ABSTRACT The RNA-dependent RNA polymerase of influenza A virus is responsible for both transcription and replication of negative-sense viral RNA. It is thought that a “switching” mechanism regulates the transition between these activities. We demonstrate that, in the presence of preexisting viral RNA polymerase and nucleoprotein (NP), influenza A virus synthesizes both mRNA (transcription) and cRNA (replication) early in infection. We suggest that there may be no switch regulating the initiation of RNA synthesis and present a model suggesting that nascent cRNA is degraded by host cell nucleases unless it is stabilized by newly synthesized viral RNA polymerase and NP.

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Engineered Small-Molecule Control of Influenza A Virus Replication

Journal of Virology ◽

10.1128/jvi.01677-18 ◽

2018 ◽

Vol 93 (1) ◽

Cited By ~ 1

Author(s):

Elizabeth J. Fay ◽

Stephanie L. Aron ◽

Ian A. Stone ◽

Barbara M. Waring ◽

Richard K. Plemper ◽

...

Keyword(s):

Influenza A Virus ◽

Virus Replication ◽

Small Molecule ◽

Human Population ◽

Influenza A ◽

Health Concern ◽

Highly Pathogenic ◽

Significant Safety

ABSTRACT Influenza A virus (IAV) remains a global health concern despite the availability of a seasonal vaccine. It is difficult to predict which strains will circulate during influenza season, and therefore, it is extremely challenging to test novel vaccines in the human population. To overcome this obstacle, new vaccines must be tested in challenge studies. This approach poses significant safety problems, since current pharmacological interventions for IAV are poorly efficacious. New methods are needed to enhance the safety of these challenge studies. In this study, we have generated a virus expressing a small-molecule-assisted shutoff (SMASh) tag as a safety switch for IAV replication. The addition of the SMASh tag to an essential IAV protein allows for small-molecule-mediated inhibition of replication. Treatment with this drug controls the replication of a SMASh-tagged virus in vitro and in vivo. This model for restriction of viral replication has potential for broad applications in vaccine studies, virotherapy, and basic virus research. IMPORTANCE Influenza A virus (IAV) causes significant morbidity and mortality annually worldwide, despite the availability of new formulations of the vaccine each season. There is a critical need to develop more-efficacious vaccines. However, testing novel vaccines in the human population in controlled studies is difficult due to the limited availability and efficacy of intervention strategies should the vaccine fail. There are also significant safety concerns for work with highly pathogenic IAV strains in the laboratory. Therefore, novel strategies are needed to improve the safety of vaccine studies and of research on highly pathogenic IAV. In this study, we developed an IAV strain engineered to contain a small-molecule-mediated safety switch. This tag, when attached to an essential viral protein, allows for the regulation of IAV replication in vitro and in vivo. This strategy provides a platform for the regulation of virus replication without targeting viral proteins directly.

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Biochemical and Structural Evidence in Support of a Coherent Model for the Formation of the Double-Helical Influenza A Virus Ribonucleoprotein

mBio ◽

10.1128/mbio.00467-12 ◽

2012 ◽

Vol 4 (1) ◽

Cited By ~ 24

Author(s):

Qiaozhen Ye ◽

Tom S. Y. Guu ◽

Douglas A. Mata ◽

Rei-Lin Kuo ◽

Bartram Smith ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Rna Synthesis ◽

Viral Rna ◽

Helical Structures ◽

Structural Element ◽

X Ray Crystallography ◽

Multiple Copies ◽

Infected Cells

ABSTRACTInfluenza A virions contain eight ribonucleoproteins (RNPs), each comprised of a negative-strand viral RNA, the viral polymerase, and multiple nucleoproteins (NPs) that coat the viral RNA. NP oligomerization along the viral RNA is mediated largely by a 28-amino-acid tail loop. Influenza viral RNPs, which serve as the templates for viral RNA synthesis in the nuclei of infected cells, are not linear but rather are organized in hairpin-like double-helical structures. Here we present results that strongly support a coherent model for the assembly of the double-helical influenza virus RNP structure. First, we show that NP self-associates much more weakly in the absence of RNA than in its presence, indicating that oligomerization is very limited in the cytoplasm. We also show that once NP has oligomerized, it can dissociate in the absence of bound RNA, but only at a very slow rate, indicating that the NP scaffold remains intact when viral RNA dissociates from NPs to interact with the polymerase during viral RNA synthesis. In addition, we identify a previously unknown NP-NP interface that is likely responsible for organizing the double-helical viral RNP structure. This identification stemmed from our observation that NP lacking the oligomerization tail loop forms monomers and dimers. We determined the crystal structure of this NP dimer, which reveals this new NP-NP interface. Mutation of residues that disrupt this dimer interface does not affect oligomerization of NPs containing the tail loop but does inactivate the ability of NPs containing the tail loop to support viral RNA synthesis in minigenome assays.IMPORTANCEInfluenza A virus, the causative agent of human pandemics and annual epidemics, contains eight RNA gene segments. Each RNA segment assumes the form of a rod-shaped, double-helical ribonucleoprotein (RNP) that contains multiple copies of a viral protein, the nucleoprotein (NP), which coats the RNA segment along its entire length. Previous studies showed that NP molecules can polymerize via a structural element called the tail loop, but the RNP assembly process is poorly understood. Here we show that influenza virus RNPs are likely assembled from NP monomers, which polymerize through the tail loop only in the presence of viral RNA. Using X-ray crystallography, we identified an additional way that NP molecules interact with each other. We hypothesize that this new interaction is responsible for organizing linear, single-stranded influenza virus RNPs into double-helical structures. Our results thus provide a coherent model for the assembly of the double-helical influenza virus RNP structure.

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