A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

The NS1 protein is a nonstructural protein encoded by the influenza A virus. It is responsible for many alterations produced in the cellular metabolism upon infection by the virus and for modulation of virus virulence. The NS1 protein is able to perform a large variety of functions due to its ability to bind various types of RNA molecules, from both viral and nonviral origin, and to interact with several cell factors. With the aim of exploring whether the binding of NS1 protein to viral RNA (vRNA) could constitute a novel target for the search of anti-influenza drugs, a filter-binding assay measuring the specific interaction between the recombinant His-NS1 protein from influenza A virus and a radiolabeled model vRNA ( 32P-vNSZ) was adapted to a format suitable for screening and easy automation. Flashplate® technology (PerkinElmer, Waltham, MA), either in 96- or 384-well plates, was used. The Flashplate® wells were precoated with the recombinant His-NS1 protein, and the binding of His-NS1 to a 35S-vNSZ probe was measured. A pilot screening of a collection of 27,520 mixtures of synthetic chemical compounds was run for inhibitors of NS1 binding to vRNA. We found 3 compounds in which the inhibition of NS1 binding to vRNA, observed at submicromolar concentrations, was correlated with a reduction of the cytopathic effect during the infection of cell cultures with influenza virus. These results support the hypothesis that the binding of NS1 to vRNA could be a novel target for the development of anti-influenza drugs. ( Journal of Biomolecular Screening 2008:581-590)

Download Full-text

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

Download Full-text

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.

Download Full-text

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.

Download Full-text

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

Antiviral Research ◽

10.1016/j.antiviral.2013.07.010 ◽

2013 ◽

Vol 100 (1) ◽

pp. 29-37 ◽

Cited By ~ 8

Author(s):

Luis Martinez-Gil ◽

Judith G. Alamares-Sapuay ◽

M.V. Ramana Reddy ◽

Peter H. Goff ◽

E. Premkumar Reddy ◽

...

Keyword(s):

Influenza A Virus ◽

Virus Replication ◽

Small Molecule ◽

Influenza A ◽

Rna Synthesis ◽

Kinase Inhibitor ◽

Viral Rna

Download Full-text

The Nucleolar Protein LYAR Facilitates Ribonucleoprotein Assembly of Influenza A Virus

Journal of Virology ◽

10.1128/jvi.01042-18 ◽

2018 ◽

Vol 92 (23) ◽

Cited By ~ 7

Author(s):

Cha Yang ◽

Xiaokun Liu ◽

Qingxia Gao ◽

Tailang Cheng ◽

Rong Xiao ◽

...

Keyword(s):

Cell Growth ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Rna Synthesis ◽

Nucleolar Protein ◽

Host Factors ◽

Viral Rna ◽

A Cell ◽

Transcription And Replication

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.

Download Full-text

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.

Download Full-text

Autophagy Promotes Replication of Influenza A VirusIn Vitro

Journal of Virology ◽

10.1128/jvi.01984-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 15

Author(s):

Ruifang Wang ◽

Yinxing Zhu ◽

Jiachang Zhao ◽

Chenwei Ren ◽

Peng Li ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Rna Synthesis ◽

Virus Production ◽

Viral Rna ◽

Host Cells ◽

Progeny Virus ◽

Mtor Signaling Pathway ◽

Autophagy Pathway ◽

Progeny Virus Production

ABSTRACTInfluenza A virus (IAV) infection could induce autophagosome accumulation. However, the impact of the autophagy machinery on IAV infection remains controversial. Here, we showed that induction of cellular autophagy by starvation or rapamycin treatment increases progeny virus production, while disruption of autophagy using a small interfering RNA (siRNA) and pharmacological inhibitor reduces progeny virus production. Further studies revealed that alteration of autophagy significantly affects the early stages of the virus life cycle or viral RNA synthesis. Importantly, we demonstrated that overexpression of both the IAV M2 and NP proteins alone leads to the lipidation of LC3 to LC3-II and a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Intriguingly, both M2 and NP colocalize and interact with LC3 puncta during M2 or NP transfection alone and IAV infection, leading to an increase in viral ribonucleoprotein (vRNP) export and infectious viral particle formation, which indicates that the IAV-host autophagy interaction plays a critical role in regulating IAV replication. We showed that NP and M2 induce the AKT-mTOR-dependent autophagy pathway and an increase in HSP90AA1 expression. Finally, our studies provided evidence that IAV replication needs an autophagy pathway to enhance viral RNA synthesis via the interaction of PB2 and HSP90AA1 by modulating HSP90AA1 expression and the AKT-mTOR signaling pathway in host cells. Collectively, our studies uncover a new mechanism that NP- and M2-mediated autophagy functions in different stages of virus replication in the pathogenicity of influenza A virus.IMPORTANCEAutophagy impacts the replication cycle of many viruses. However, the role of the autophagy machinery in IAV replication remains unclear. Therefore, we explored the detailed mechanisms utilized by IAV to promote its replication. We demonstrated that IAV NP- and M2-mediated autophagy promotes IAV replication by regulating the AKT-mTOR signaling pathway and HSP90AA1 expression. The interaction of PB2 and HSP90AA1 results in the increase of viral RNA synthesis first; subsequently the binding of NP to LC3 favors vRNP export, and later the interaction of M2 and LC3 leads to an increase in the production of infectious viral particles, thus accelerating viral progeny production. These findings improve our understanding of IAV pathogenicity in host cells.

Download Full-text

Structure of Influenza A Virus Promoter and its Implications for Viral RNA Synthesis

The Scientific World JOURNAL ◽

10.1100/tsw.2001.459 ◽

2001 ◽

Vol 1 ◽

pp. 812-814 ◽

Cited By ~ 2

Author(s):

Sung-Hun Bae ◽

Byong-Seok Choi

Keyword(s):

Influenza Virus ◽

Hong Kong ◽

Influenza A Virus ◽

Influenza A ◽

Rna Synthesis ◽

Viral Rna ◽

Influenza Outbreak ◽

Spanish Influenza ◽

Virus Promoter

Since the worst worldwide pandemic ever recorded — the 1918 Spanish influenza outbreak that killed more than 20 million people — we have achieved significant advances in understanding the influenza virus. However, the fear of such a pandemic remains strong. For example, in 1997, when a lethal influenza variant afflicted eight people in Hong Kong, contributing to the death of six, officials feared the next wave had begun. They managed to solve the problem quickly, however, by destroying all of the poultry in Hong Kong[1].

Download Full-text

Studies of an Influenza A Virus Temperature-Sensitive Mutant Identify a Late Role for NP in the Formation of Infectious Virions

Journal of Virology ◽

10.1128/jvi.01424-08 ◽

2008 ◽

Vol 83 (2) ◽

pp. 562-571 ◽

Cited By ~ 26

Author(s):

Sarah L. Noton ◽

Martha Simpson-Holley ◽

Elizabeth Medcalf ◽

Helen M. Wise ◽

Edward C. Hutchinson ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Rna Synthesis ◽

Reverse Genetics ◽

Rna Binding ◽

Virus Genome ◽

Viral Rna ◽

Temperature Sensitive ◽

Single Mutation ◽

Nucleocytoplasmic Trafficking

ABSTRACT The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperature-sensitive (ts) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited an abnormal morphology. Reverse-genetics studies in which A/PR/8/34 segment 7 was swapped with sequences from other strains of virus revealed a profound incompatibility between the M239L mutation and the A/Udorn/72 M1 gene, suggesting that the ts mutation affects M1-NP interactions. Thus, we have identified a late-acting defect in NP that, separate from its function in RNA synthesis, indicates a role for the polypeptide in virion assembly, most likely involving M1 as a partner.

Download Full-text