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

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.

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

Journal of Virology ◽

10.1128/jvi.02293-07 ◽

2008 ◽

Vol 83 (1) ◽

pp. 29-36 ◽

Cited By ~ 61

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.

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Proteomics Analysis of the Tombusvirus Replicase: Hsp70 Molecular Chaperone Is Associated with the Replicase and Enhances Viral RNA Replication

Journal of Virology ◽

10.1128/jvi.80.5.2162-2169.2006 ◽

2006 ◽

Vol 80 (5) ◽

pp. 2162-2169 ◽

Cited By ~ 149

Author(s):

Saulius Serva ◽

Peter D. Nagy

Keyword(s):

Rna Binding ◽

Rna Virus ◽

Rna Replication ◽

Viral Rna ◽

Mass Spectrometry Analysis ◽

Host Proteins ◽

Two Dimensional Gel Electrophoresis ◽

Viral Replicase

ABSTRACT Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown ∼35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (ssa1 ssa2) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned ssa1 ssa2 mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1009336 ◽

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.

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Influenza A Virus Inhibits RSV Infection via a Two-Wave Expression of IFIT Proteins

Viruses ◽

10.3390/v12101171 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1171

Author(s):

Yaron Drori ◽

Jasmine Jacob-Hirsch ◽

Rakefet Pando ◽

Aharona Glatman-Freedman ◽

Nehemya Friedman ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Influenza Viruses ◽

Mass Spectrometry Analysis ◽

Influenza A Viruses ◽

Rsv Infection ◽

Syncytial Virus ◽

Mouse Lungs

Influenza viruses and respiratory syncytial virus (RSV) are respiratory viruses that primarily circulate worldwide during the autumn and winter seasons. Seasonal surveillance has shown that RSV infection generally precedes influenza. However, in the last four winter seasons (2016–2020) an overlap of the morbidity peaks of both viruses was observed in Israel, and was paralleled by significantly lower RSV infection rates. To investigate whether the influenza A virus inhibits RSV, human cervical carcinoma (HEp2) cells or mice were co-infected with influenza A and RSV. Influenza A inhibited RSV growth, both in vitro and in vivo. Mass spectrometry analysis of mouse lungs infected with influenza A identified a two-wave pattern of protein expression upregulation, which included members of the interferon-induced protein with the tetratricopeptide (IFITs) family. Interestingly, in the second wave, influenza A viruses were no longer detectable in mouse lungs. In addition, knockdown and overexpression of IFITs in HEp2 cells affected RSV multiplicity. In conclusion, influenza A infection inhibits RSV infectivity via upregulation of IFIT proteins in a two-wave modality. Understanding the immune system involvement in the interaction between influenza A and RSV viruses will contribute to the development of future treatment strategies against these viruses.

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Laninamivir-Interferon Lambda 1 Combination Treatment Promotes Resistance by Influenza A Virus More Rapidly than Laninamivir Alone

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00301-20 ◽

2020 ◽

Vol 64 (7) ◽

Author(s):

Simone E. Adams ◽

Vladimir Y. Lugovtsev ◽

Anastasia Kan ◽

Nicolai V. Bovin ◽

Raymond P. Donnelly ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Combination Treatment ◽

Influenza A ◽

Influenza Virus Infection ◽

The United States ◽

Epithelial Cell Line ◽

Drug Resistant ◽

Interferon Lambda

ABSTRACT Each year, 5% to 20% of the population of the United States becomes infected with influenza A virus. Combination therapy with two or more antiviral agents has been considered a potential treatment option for influenza virus infection. However, the clinical results derived from combination treatment with two or more antiviral drugs have been variable. We examined the effectiveness of cotreatment with two distinct classes of anti-influenza drugs, i.e., neuraminidase (NA) inhibitor, laninamivir, and interferon lambda 1 (IFN-λ1), against the emergence of drug-resistant virus variants in vitro. We serially passaged pandemic A/California/04/09 [A(H1N1)pdm09] influenza virus in a human lung epithelial cell line (Calu-3) in the presence or absence of increasing concentrations of laninamivir or laninamivir plus IFN-λ1. Surprisingly, laninamivir used in combination with IFN-λ1 promoted the emergence of the E119G NA mutation five passages earlier than laninamivir alone (passage 2 versus passage 7, respectively). Acquisition of this mutation resulted in significantly reduced sensitivity to the NA inhibitors laninamivir (∼284-fold) and zanamivir (∼1,024-fold) and decreased NA enzyme catalytic activity (∼5-fold) compared to the parental virus. Moreover, the E119G NA mutation emerged together with concomitant hemagglutinin (HA) mutations (T197A and D222G), which were selected more rapidly by combination treatment with laninamivir plus IFN-λ1 (passages 2 and 3, respectively) than by laninamivir alone (passage 10). Our results show that treatment with laninamivir alone or in combination with IFN-λ1 can lead to the emergence of drug-resistant influenza virus variants. The addition of IFN-λ1 in combination with laninamivir may promote acquisition of drug resistance more rapidly than treatment with laninamivir alone.

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The mechanism of resistance to favipiravir in influenza

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1811345115 ◽

2018 ◽

Vol 115 (45) ◽

pp. 11613-11618 ◽

Cited By ~ 87

Author(s):

Daniel H. Goldhill ◽

Aartjan J. W. te Velthuis ◽

Robert A. Fletcher ◽

Pinky Langat ◽

Maria Zambon ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

H1n1 Influenza ◽

Viral Fitness ◽

Virus Infections ◽

Virus Rna ◽

Evolution Of Resistance ◽

Emergence Of Resistance

Favipiravir is a broad-spectrum antiviral that has shown promise in treatment of influenza virus infections. While emergence of resistance has been observed for many antiinfluenza drugs, to date, clinical trials and laboratory studies of favipiravir have not yielded resistant viruses. Here we show evolution of resistance to favipiravir in the pandemic H1N1 influenza A virus in a laboratory setting. We found that two mutations were required for robust resistance to favipiravir. We demonstrate that a K229R mutation in motif F of the PB1 subunit of the influenza virus RNA-dependent RNA polymerase (RdRP) confers resistance to favipiravir in vitro and in cell culture. This mutation has a cost to viral fitness, but fitness can be restored by a P653L mutation in the PA subunit of the polymerase. K229R also conferred favipiravir resistance to RNA polymerases of other influenza A virus strains, and its location within a highly conserved structural feature of the RdRP suggests that other RNA viruses might also acquire resistance through mutations in motif F. The mutations identified here could be used to screen influenza virus-infected patients treated with favipiravir for the emergence of resistance.

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Antiviral Activity of Aspalathus linearis against Human Influenza Virus

Natural Product Communications ◽

10.1177/1934578x1701200432 ◽

2017 ◽

Vol 12 (4) ◽

pp. 1934578X1701200 ◽

Cited By ~ 1

Author(s):

Ratika Rahmasari ◽

Takahiro Haruyama ◽

Siriwan Charyasriwong ◽

Tomoki Nishida ◽

Nobuyuki Kobayashi

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Time Course ◽

Influenza Viruses ◽

Replication Process ◽

Human Influenza Virus ◽

Alkaline Extract ◽

Aspalathus Linearis

Influenza A viruses are responsible for annual epidemics and occasional pandemics, which cause significant morbidity and mortality. The limited protection offered by influenza vaccination, and the emergence of drug-resistant influenza strains, highlight the urgent need for the development of novel anti-influenza drugs. However, the search for antiviral substances from the library of low molecular weight chemical compounds is limited. Thus, because of their natural diversity and accessibility, plants or plant-derived materials are rapidly becoming valuable sources for the discovery and development of new antiviral drugs. In this study, crude extracts of Aspalathus linearis, a plant reported to have anti-HIV activity, were evaluated in vitro for their activity against the influenza A virus. Of the extracts tested, an alkaline extract of Aspalathus linearis demonstrated the strongest inhibition against influenza A virus and could also inhibit different types of influenza viruses, including Oseltamivir-resistant influenza viruses A and B. Our time course of addition studies indicated that the alkaline extract of Aspalathus linearis exerts its antiviral effect predominantly during the late stages of the influenza virus replication process.

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

Journal of Virology ◽

10.1128/jvi.01487-15 ◽

2015 ◽

Vol 89 (22) ◽

pp. 11245-11255 ◽

Cited By ~ 10

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.

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

Journal of Virology ◽

10.1128/jvi.79.11.6631-6643.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 6631-6643 ◽

Cited By ~ 238

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.

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