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

2008 ◽  
Vol 83 (1) ◽  
pp. 29-36 ◽  
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.

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

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
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.

scholarly journals Viral RNA-binding ability conferred by SUMOylation at PB1 K612 of influenza A virus is essential for viral pathogenesis and transmission

2021 ◽  
Vol 17 (2) ◽  
pp. e1009336
Author(s):  
Junping Li ◽  
Libin Liang ◽  
Li Jiang ◽  
Qian Wang ◽  
Xia Wen ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Posttranslational Modifications ◽  
Influenza A ◽  
Cellular Localization ◽  
Rna Binding ◽  
Viral Rna ◽  
Complex Activity ◽  
Sumoylation Site

Posttranslational modifications, such as SUMOylation, play specific roles in the life cycle of invading pathogens. However, the effect of SUMOylation on the adaptation, pathogenesis, and transmission of influenza A virus (IAV) remains largely unknown. Here, we found that a conserved lysine residue at position 612 (K612) of the polymerase basic protein 1 (PB1) of IAV is a bona fide SUMOylation site. SUMOylation of PB1 at K612 had no effect on the stability or cellular localization of PB1, but was critical for viral ribonucleoprotein (vRNP) complex activity and virus replication in vitro. When tested in vivo, we found that the virulence of SUMOylation-defective PB1/K612R mutant IAVs was highly attenuated in mice. Moreover, the airborne transmission of a 2009 pandemic H1N1 PB1/K612R mutant virus was impaired in ferrets, resulting in reversion to wild-type PB1 K612. Mechanistically, SUMOylation at K612 was essential for PB1 to act as the enzymatic core of the viral polymerase by preserving its ability to bind viral RNA. Our study reveals an essential role for PB1 K612 SUMOylation in the pathogenesis and transmission of IAVs, which can be targeted for the design of anti-influenza therapies.

scholarly journals Long-Range RNA-RNA Interactions Circularize the Dengue Virus Genome

2005 ◽  
Vol 79 (11) ◽  
pp. 6631-6643 ◽  
Author(s):  
Diego E. Alvarez ◽  
María F. Lodeiro ◽  
Silvio J. Ludueña ◽  
Lía I. Pietrasanta ◽  
Andrea V. Gamarnik
Keyword(s):  
Dengue Virus ◽  
Rna Binding ◽  
Rna Replication ◽  
Virus Genome ◽  
Viral Rna ◽  
Physical Interaction ◽  
Virus Rna ◽  
Rna Elements ◽  
Rna Interaction

ABSTRACT Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5′ and 3′ ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5′ and 3′ CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5′ and 3′ untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5′ and 3′ UAR (upstream AUG region). In order to investigate the functional role of 5′-3′ UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5′ or 3′ UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

scholarly journals An in vitro network of intermolecular interactions between viral RNA segments of an avian H5N2 influenza A virus: comparison with a human H3N2 virus

2012 ◽  
Vol 41 (2) ◽  
pp. 1241-1254 ◽  
Author(s):  
Cyrille Gavazzi ◽  
Catherine Isel ◽  
Emilie Fournier ◽  
Vincent Moules ◽  
Annie Cavalier ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Intermolecular Interactions ◽  
Influenza A ◽  
Viral Rna ◽  
H3n2 Virus

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

2019 ◽  
Vol 93 (13) ◽  
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.

scholarly journals Regulation of Poliovirus 3C Protease by the 2C Polypeptide

2004 ◽  
Vol 78 (17) ◽  
pp. 9243-9256 ◽  
Author(s):  
Rajeev Banerjee ◽  
Mary K. Weidman ◽  
Angela Echeverri ◽  
Pallob Kundu ◽  
Asim Dasgupta
Keyword(s):  
Cell Lines ◽  
Inhibitory Activity ◽  
Rna Binding ◽  
Rna Replication ◽  
Viral Rna ◽  
Physical Interaction ◽  
Viral Protease ◽  
Hela Cell Lines ◽  
Protease Inhibitory Activity

ABSTRACT Poliovirus-encoded nonstructural polypeptide 2C is a multifunctional protein that plays an important role in viral RNA replication. 2C interacts with both intracellular membranes and virus-specific RNAs and has ATPase and GTPase activities. Extensive computer analysis of the 2C sequence revealed that in addition to the known ATPase-, GTPase-, membrane-, and RNA-binding domains it also contains several “serpin” (serine protease inhibitor) motifs. We provide experimental evidence suggesting that 2C is indeed capable of regulating virus-encoded proteases. The purified 2C protein inhibits 3Cpro-catalyzed cleavage of cellular transcription factors at Q-G sites in vitro. It also inhibits cleavage of a viral precursor by the other viral protease, 2Apro. However, at least three cellular proteases appear not to be inhibited by 2C in vitro. The 2C-associated protease inhibitory activity can be depleted by anti-2C antibody. A physical interaction between 2C and His-tagged 3Cpro can be demonstrated in vitro by coimmunoprecipitation of 2C with anti-His antibody. Deletion analysis suggests that the 2C central and C-terminal domains that include several serpin motifs are important for 3Cpro-inhibitory activity. To examine the 2C protease inhibitory activity in vivo, stable HeLa cell lines were made that express 2C in an inducible fashion. Infection of 2C-expressing cells with poliovirus led to incomplete (or inefficient) processing of viral precursor polypeptides compared to control cell lines containing the vector alone. These results suggest that 2C can negatively regulate the viral protease 3Cpro. The possible role of the 2C protease inhibitory activity in viral RNA replication is discussed.

Antiviral activity of double‐stranded RNA‐binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase

2018 ◽  
Vol 32 (8) ◽  
pp. 4380-4393 ◽  
Author(s):  
Chi‐Ping Chan ◽  
Chun‐Kit Yuen ◽  
Pak‐Hin Hinson Cheung ◽  
Sin‐Yee Fung ◽  
Pak‐Yin Lui ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Viral Rna ◽  
Double Stranded Rna

scholarly journals Mutational Analysis of Influenza A Virus Nucleoprotein: Identification of Mutations That Affect RNA Replication

1999 ◽  
Vol 73 (2) ◽  
pp. 1186-1194 ◽  
Author(s):  
Ignacio Mena ◽  
Enrique Jambrina ◽  
Carmen Albo ◽  
Beatriz Perales ◽  
Juan Ortín ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Intracellular Localization ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Virus Mutant

ABSTRACT The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.

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