Mutagenic Analysis of the 5′ Arm of the Influenza A Virus Virion RNA Promoter Defines the Sequence Requirements for Endonuclease Activity

ABSTRACT Short synthetic influenza virus-like RNAs derived from influenza virus promoter sequences were examined for their ability to stimulate the endonuclease activity of recombinant influenza virus polymerase complexes in vitro, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. An extensive set of point mutants of the 5′ arm of the influenza A virus viral RNA (vRNA) was constructed to determine the cis-acting elements which influenced endonuclease activity. Activity was found to be dependent on three features of the conserved vRNA termini: (i) the presence of the 5′ hairpin loop structure, (ii) the identity of residues at positions 5 and 10 bases from the 5′ terminus, and (iii) the presence of base pair interactions between the 5′ and 3′ segment ends. Further experiments discounted a role for the vRNA U track in endonuclease activation. This study represents the first mutagenic analysis of the influenza virus promoter with regard to endonuclease activity.

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Hairpin Loop Structure in the 3′ Arm of the Influenza A Virus Virion RNA Promoter Is Required for Endonuclease Activity

Journal of Virology ◽

10.1128/jvi.75.15.7042-7049.2001 ◽

2001 ◽

Vol 75 (15) ◽

pp. 7042-7049 ◽

Cited By ~ 32

Author(s):

Michael B. Leahy ◽

Helen C. Dobbyn ◽

George G. Brownlee

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Point Mutations ◽

Nucleotide Position ◽

Loop Structure ◽

Endonuclease Activity ◽

Hairpin Loop ◽

Virus Rna ◽

Rna Promoter

ABSTRACT Previous studies have shown that the 5′ arm of the influenza A virus virion RNA promoter requires a hairpin loop structure for efficient endonuclease activity of influenza virus RNA polymerase, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. Here we examine whether a hairpin loop is also required in the 3′ arm of the viral RNA promoter. We study point mutations at each nucleotide position (1 to 12) within the 3′ arm of the promoter as well as complementary “rescue” mutations which restored base pairing in the stem of a potential hairpin loop. Our results suggest that endonuclease activity is absolutely dependent on the presence of a 3′ hairpin loop structure. This is the first direct evidence for RNA secondary structure within the 3′ arm being required for a specific stage, i.e., endonuclease cleavage, in the influenza virus replicative cycle.

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Differential Activation of Influenza A Virus Endonuclease Activity Is Dependent on Multiple Sequence Differences between the Virion RNA and cRNA Promoters

Journal of Virology ◽

10.1128/jvi.76.4.2019-2023.2002 ◽

2002 ◽

Vol 76 (4) ◽

pp. 2019-2023 ◽

Cited By ~ 15

Author(s):

Michael B. Leahy ◽

Giuseppe Zecchin ◽

George G. Brownlee

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Endonuclease Activity ◽

Multiple Sequence ◽

Differential Activation ◽

Distinctive Features

ABSTRACT Influenza virus endonuclease activity was studied in vitro with model virion RNA (vRNA) and cRNA molecules. We show that endonuclease activity can be partially rescued by transplanting vRNA-like promoter features into the model cRNA promoter. This study defines three distinctive features within the vRNA promoter—absent in the cRNA promoter—that are required for endonuclease cleavage.

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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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Cross-Protection of Influenza A Virus Infection by a DNA Aptamer Targeting the PA Endonuclease Domain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00306-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 4082-4093 ◽

Cited By ~ 23

Author(s):

Shuofeng Yuan ◽

Naru Zhang ◽

Kailash Singh ◽

Huiping Shuai ◽

Hin Chu ◽

...

Keyword(s):

Virus Infection ◽

Influenza A Virus ◽

Influenza A ◽

H5n1 Virus ◽

Influenza Viruses ◽

Cross Protection ◽

Dna Aptamer ◽

Endonuclease Activity ◽

Endonuclease Domain

ABSTRACTAmino acid residues in the N-terminal of the PA subunit (PAN) of the influenza A virus polymerase play critical roles in endonuclease activity, protein stability, and viral RNA (vRNA) promoter binding. In addition, PANis highly conserved among different subtypes of influenza virus, which suggests PANto be a desired target in the development of anti-influenza agents. We selected DNA aptamers targeting the intact PA protein or the PANdomain of an H5N1 virus strain using systematic evolution of ligands by exponential enrichment (SELEX). The binding affinities of selected aptamers were measured, followed by an evaluation ofin vitroendonuclease inhibitory activity. Next, the antiviral effects of enriched aptamers against influenza A virus infections were examined. A total of three aptamers targeting PA and six aptamers targeting PANwere selected. Our data demonstrated that all three PA-selected aptamers neither inhibited endonuclease activity nor exhibited antiviral efficacy, whereas four of the six PAN-selected aptamers inhibited both endonuclease activity and H5N1 virus infection. Among the four effective aptamers, one exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC50) of around 10 nM. Notably, this aptamer was identified at the 5th round but disappeared after the 10th round of selection, suggesting that the identification and evaluation of aptamers at early rounds of selection may be highly helpful for screening effective aptamers. Overall, our study provides novel insights for screening and developing effective aptamers for use as anti-influenza drugs.

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Δ12-Prostaglandin J2 Is a Potent Inhibitor of Influenza A Virus Replication

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.1.200-204.2000 ◽

2000 ◽

Vol 44 (1) ◽

pp. 200-204 ◽

Cited By ~ 21

Author(s):

Francesca Pica ◽

Anna Teresa Palamara ◽

Antonio Rossi ◽

Alessandra De Marco ◽

Carla Amici ◽

...

Keyword(s):

Influenza Virus ◽

Heat Shock ◽

Influenza A Virus ◽

Influenza A ◽

Potent Inhibitor ◽

Influenza Virus Infection ◽

H1n1 Infection ◽

Shock Proteins

ABSTRACT 9-Deoxy-Δ9,Δ12-13,14-dihydro-prostaglandin D2 (Δ12-PGJ2), a natural cyclopentenone metabolite of prostaglandin D2, is shown to possess therapeutic efficacy against influenza A virus A/PR8/34 (H1N1) infection in vitro and in vivo. The results indicate that the antiviral activity is associated with induction of cytoprotective heat shock proteins and suggest novel strategies for treatment of influenza virus infection.

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Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses

Journal of Virology ◽

10.1128/jvi.03101-15 ◽

2016 ◽

Vol 90 (7) ◽

pp. 3684-3693 ◽

Cited By ~ 4

Author(s):

Léa Meyer ◽

Alix Sausset ◽

Laura Sedano ◽

Bruno Da Costa ◽

Ronan Le Goffic ◽

...

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Influenza A Virus ◽

Influenza A ◽

Wild Type Virus ◽

Deletion Mutants ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Wild Type

ABSTRACTThe influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA–PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for furtherin vitroandin vivocharacterization. Four viable mutants with single-codon deletions were generated; all of them exhibited atsphenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serialin vitropassages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stabletsmutants that could be used to design novel attenuated vaccines.IMPORTANCEIn order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA–PB1 dimer to the nucleus, where viral replication occurs. Thesetsdeletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.

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Dinucleotide evolutionary dynamics in influenza A virus

Virus Evolution ◽

10.1093/ve/vez038 ◽

2019 ◽

Vol 5 (2) ◽

Cited By ~ 4

Author(s):

Haogao Gu ◽

Rebecca L Y Fan ◽

Di Wang ◽

Leo L M Poon

Keyword(s):

Influenza Virus ◽

Codon Usage ◽

Influenza A Virus ◽

Codon Usage Bias ◽

Influenza A ◽

Evolutionary Dynamics ◽

Synonymous Codon ◽

Amino Acid Sequences ◽

In Vivo Models

Abstract Significant biases of dinucleotide composition in many RNA viruses including influenza A virus have been reported in recent years. Previous studies have showed that a codon-usage-altered influenza mutant with elevated CpG usage is attenuated in mammalian in vitro and in vivo models. However, the relationship between dinucleotide preference and codon usage bias is not entirely clear and changes in dinucleotide usage of influenza virus during evolution at segment level are yet to be investigated. In this study, a Monte Carlo type method was applied to identify under-represented or over-represented dinucleotide motifs, among different segments and different groups, in influenza viral sequences. After excluding the potential biases caused by codon usage and amino acid sequences, CpG and UpA were found under-represented in all viral segments from all groups, whereas UpG and CpA were found over-represented. We further explored the temporal changes of usage of these dinucleotides. Our analyses revealed significant decrease of CpG frequency in Segments 1, 3, 4, and 5 in seasonal H1 virus after its re-emergence in humans in 1977. Such temporal variations were mainly contributed by the dinucleotide changes at the codon positions 3-1 and 2-3 where silent mutations played a major role. The depletions of CpG and UpA through silent mutations consequently led to over-representations of UpG and CpA. We also found that dinucleotide preference directly results in significant synonymous codon usage bias. Our study helps to provide details on understanding the evolutionary history of influenza virus and selection pressures that shape the virus genome.

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In Vitro System for Modeling Influenza A Virus Resistance under Drug Pressure

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01385-09 ◽

2010 ◽

Vol 54 (8) ◽

pp. 3442-3450 ◽

Cited By ~ 19

Author(s):

Ashley N. Brown ◽

James J. McSharry ◽

Qingmei Weng ◽

Elizabeth M. Driebe ◽

David M. Engelthaler ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Mdck Cells ◽

Influenza Virus Infection ◽

Infection Model ◽

Virus Infections ◽

Drug Pressure

ABSTRACT One of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. It is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the gene coding for the M2 protein. To address this problem, it is critical to develop experimental systems that can accurately model the selection of resistance under drug pressure as seen in humans. We used the hollow-fiber infection model (HFIM) system to examine the effect of amantadine on the replication of influenza virus, A/Albany/1/98 (H3N2), grown in MDCK cells. At 24 and 48 h postinfection, virus replication was inhibited in a dose-dependent fashion. At 72 and 96 h postinfection, virus replication was no longer inhibited, suggesting the emergence of amantadine-resistant virus. Sequencing of the M2 gene revealed that mutations appeared at between 48 and 72 h of drug treatment and that the mutations were identical to those identified in the clinic for amantadine-resistant viruses (e.g., V27A, A30T, and S31N). Interestingly, we found that the type of mutation was strongly affected by the dose of the drug. The data suggest that the HFIM is a good model for influenza virus infection and resistance generation in humans. The HFIM has the advantage of being a highly controlled system where multiplicity parameters can be directly and accurately controlled and measured.

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