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

mBio ◽  
2012 ◽  
Vol 4 (1) ◽  
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.

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

2001 ◽  
Vol 1 ◽  
pp. 812-814 ◽  
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].

scholarly journals Regulation of Influenza A Virus Nucleoprotein Oligomerization by Phosphorylation

2014 ◽  
Vol 89 (2) ◽  
pp. 1452-1455 ◽  
Author(s):  
Lauren Turrell ◽  
Edward C. Hutchinson ◽  
Frank T. Vreede ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Serine Residue ◽  
Reversible Phosphorylation ◽  
Viral Growth ◽  
Ribonucleoprotein Complex ◽  
Infected Cells ◽  
Neighboring Molecule

In the influenza virus ribonucleoprotein complex, the oligomerization of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighboring molecule. In this study, we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerization and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerization in infected cells is regulated by reversible phosphorylation.

scholarly journals Host Shutoff in Influenza A Virus: Many Means to an End

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 475 ◽  
Author(s):  
Rachel Levene ◽  
Marta Gaglia
Keyword(s):  
Immune Responses ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Synthesis ◽  
Viral Proteins ◽  
Host Gene Expression ◽  
Structural Protein ◽  
Infected Cells ◽  
Host Shutoff ◽  
Virus Replication Cycle

Influenza A virus carries few of its own proteins, but uses them effectively to take control of the infected cells and avoid immune responses. Over the years, host shutoff, the widespread down-regulation of host gene expression, has emerged as a key process that contributes to cellular takeover in infected cells. Interestingly, multiple mechanisms of host shutoff have been described in influenza A virus, involving changes in translation, RNA synthesis and stability. Several viral proteins, notably the non-structural protein NS1, the RNA-dependent RNA polymerase and the endoribonuclease PA-X have been implicated in host shutoff. This multitude of host shutoff mechanisms indicates that host shutoff is an important component of the influenza A virus replication cycle. Here we review the various mechanisms of host shutoff in influenza A virus and the evidence that they contribute to immune evasion and/or viral replication. We also discuss what the purpose of having multiple mechanisms may be.

scholarly journals A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription

2015 ◽  
Vol 89 (22) ◽  
pp. 11245-11255 ◽  
Author(s):  
Wen-Chi Su ◽  
Shih-Feng Hsu ◽  
Yi-Yuan Lee ◽  
King-Song Jeng ◽  
Michael M. C. Lai
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Ribosomal Rna ◽  
Rna Processing ◽  
Influenza A ◽  
Host Factors ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Virus Transcription ◽  
Ribosomal Rna Processing

ABSTRACTInfluenza A virus (IAV) undergoes RNA transcription by a unique capped-mRNA-dependent transcription, which is carried out by the viral RNA-dependent RNA polymerase (RdRp), consisting of the viral PA, PB1, and PB2 proteins. However, how the viral RdRp utilizes cellular factors for virus transcription is not clear. Previously, we conducted a genome-wide pooled short hairpin RNA (shRNA) screen to identify host factors important for influenza A virus replication. Ribosomal RNA processing 1 homolog B (RRP1B) was identified as one of the candidates. RRP1B is a nucleolar protein involved in ribosomal biogenesis. Upon IAV infection, part of RRP1B was translocated from the nucleolus to the nucleoplasm, where viral RNA synthesis likely takes place. The depletion of RRP1B significantly reduced IAV mRNA transcription in a minireplicon assay and in virus-infected cells. Furthermore, we showed that RRP1B interacted with PB1 and PB2 of the RdRp and formed a coimmunoprecipitable complex with RdRp. The depletion of RRP1B reduced the amount of capped mRNA in the RdRp complex. Taken together, these findings indicate that RRP1B is a host factor essential for IAV transcription and provide a target for new antivirals.IMPORTANCEInfluenza virus is an important human pathogen that causes significant morbidity and mortality and threatens the human population with epidemics and pandemics every year. Due to the high mutation rate of the virus, antiviral drugs targeting viral proteins might ultimately lose their effectiveness. An alternative strategy that explores the genetic stability of host factors indispensable for influenza virus replication would thus be desirable. Here, we characterized the rRNA processing 1 homolog B (RRP1B) protein as an important cellular factor for influenza A virus transcription. We showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for virus transcription and replication. Furthermore, we reported that RRP1B is crucial for RdRp binding to cellular capped mRNA, which is a critical step of virus transcription. Our study not only provides a deeper understanding of influenza virus-host interplay, but also suggests a potential target for antiviral drug development.

scholarly journals Highly Pathogenic H5N1 Avian Influenza Virus Induces Extracellular Ca2+ Influx, Leading to Apoptosis in Avian Cells

2010 ◽  
Vol 84 (6) ◽  
pp. 3068-3078 ◽  
Author(s):  
Mayo Ueda ◽  
Tomo Daidoji ◽  
Anariwa Du ◽  
Cheng-Song Yang ◽  
Madiha S. Ibrahim ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Highly Pathogenic ◽  
H5n1 Avian Influenza Virus ◽  
H5n1 Avian Influenza ◽  
Infected Cells ◽  
Pathogenic H5n1

ABSTRACT In this study, we show that the highly pathogenic H5N1 avian influenza virus (AIV) (A/crow/Kyoto/53/04 and A/chicken/Egypt/CL6/07) induced apoptosis in duck embryonic fibroblasts (DEF). In contrast, apoptosis was reduced among cells infected with low-pathogenic AIVs (A/duck/HK/342/78 [H5N2], A/duck/HK/820/80 [H5N3], A/wigeon/Osaka/1/01 [H7N7], and A/turkey/Wisconsin/1/66 [H9N2]). Thus, we investigated the molecular mechanisms of apoptosis induced by H5N1-AIV infection. Caspase-dependent and -independent pathways contributed to the cytopathic effects. We further showed that, in the induction of apoptosis, the hemagglutinin of H5N1-AIV played a major role and its cleavage sequence was not critical. We also observed outer membrane permeabilization and loss of the transmembrane potential of the mitochondria of infected DEF, indicating that mitochondrial dysfunction was caused by the H5N1-AIV infection. We then analyzed Ca2+ dynamics in the infected cells and demonstrated an increase in the concentration of Ca2+ in the cytosol ([Ca2+]i) and mitochondria ([Ca2+]m) after H5N1-AIV infection. Regardless, gene expression important for regulating Ca2+ efflux from the endoplasmic reticulum did not significantly change after H5N1-AIV infection. These results suggest that extracellular Ca2+ may enter H5N1-AIV-infected cells. Indeed, EGTA, which chelates extracellular free Ca2+, significantly reduced the [Ca2+]i, [Ca2+]m, and apoptosis induced by H5N1-AIV infection. In conclusion, we identified a novel mechanism for influenza A virus-mediated cell death, which involved the acceleration of extracellular Ca2+ influx, leading to mitochondrial dysfunction and apoptosis. These findings may be useful for understanding the pathogenesis of H5N1-AIV in avian species as well as the impact of Ca2+ homeostasis on influenza A virus infection.

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

2015 ◽  
Vol 89 (11) ◽  
pp. 6067-6079 ◽  
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.

scholarly journals NF-κB Signaling Differentially Regulates Influenza Virus RNA Synthesis

2008 ◽  
Vol 82 (20) ◽  
pp. 9880-9889 ◽  
Author(s):  
Naveen Kumar ◽  
Zhong-tao Xin ◽  
Yuhong Liang ◽  
Hinh Ly ◽  
Yuying Liang
Keyword(s):  
Influenza Virus ◽  
Signaling Pathway ◽  
Virus Replication ◽  
Influenza A ◽  
Time Course ◽  
Rna Synthesis ◽  
Nucleocytoplasmic Trafficking ◽  
Rna Transcription ◽  
Virus Rna ◽  
Infected Cells

ABSTRACT The NF-κB signaling pathway has previously been shown to be required for efficient influenza A virus replication, although the molecular mechanism is not well understood. In this study, we identified a specific step of the influenza virus life cycle that is influenced by NF-κB signaling by using two known NF-κB inhibitors and a variety of influenza virus-specific assays. The results of time course experiments suggest that the NF-κB inhibitors Bay11-7082 and ammonium pyrrolidinedithiocarbamate inhibited an early postentry step of viral infection, but they did not appear to affect the nucleocytoplasmic trafficking of the viral ribonucleoprotein complex. Instead, we found that the levels of influenza virus genomic RNA (vRNA), but not the corresponding cRNA or mRNA, were specifically reduced by the inhibitors in virus-infected cells, indicating that NF-κB signaling is intimately involved in the vRNA synthesis. Furthermore, we showed that the NF-κB inhibitors specifically diminished influenza virus RNA transcription from the cRNA promoter but not from the vRNA promoter in a reporter assay, a result which is consistent with data obtained from virus-infected cells. The overexpression of the p65 NF-κB molecule could not only eliminate the inhibition but also activate influenza virus RNA transcription from the cRNA promoter. Finally, using p65-specific small interfering RNA, we have shown that p65 knockdown reduced the levels of influenza virus replication and vRNA synthesis. In summary, we have provided evidence showing, for the first time, that the NF-κB host signaling pathway can differentially regulate influenza virus RNA synthesis, which may also offer some new perspectives into understanding the host regulation of RNA synthesis by other RNA viruses.

scholarly journals Phosphatidylserine-Mediated Phagocytosis of Influenza A Virus-Infected Cells by Mouse Peritoneal Macrophages

2000 ◽  
Vol 74 (19) ◽  
pp. 9240-9244 ◽  
Author(s):  
Akiko Shiratsuchi ◽  
Masako Kaido ◽  
Takenori Takizawa ◽  
Yoshinobu Nakanishi
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Cell Lines ◽  
Hela Cells ◽  
Influenza A ◽  
Peritoneal Macrophages ◽  
Animal Tissues ◽  
Cultured Cell ◽  
Infected Cells ◽  
Mouse Peritoneal Macrophages

ABSTRACT Influenza virus induces apoptosis in cultured cell lines as well as in animal tissues. HeLa cells were infected with influenza virus A/Udon/72 (H3N2) under conditions resulting in almost 100% infection. Such cells underwent typical caspase-dependent apoptosis and were efficiently phagocytosed by macrophages prepared from peritoneal fluids of thioglycolate-treated mice. The membrane phospholipid phosphatidylserine appeared on the surfaces of virus-infected cells at around the time efficient phagocytosis became detectable. In fact, the phagocytosis was almost completely inhibited in the presence of liposomes containing phosphatidylserine, which did not influence the antibody-dependent uptake of zymosan particles by the same macrophages. These results indicate that macrophages phagocytose influenza virus-infected HeLa cells in a manner mediated by phosphatidylserine that appears on the surfaces of infected cells during the process of apoptosis.

scholarly journals A mechanism for prime-realignment during influenza A virus replication

2017 ◽  
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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