Rewiring the RNAs of influenza virus to prevent reassortment

Influenza viruses contain segmented, negative-strand RNA genomes. Genome segmentation facilitates reassortment between different influenza virus strains infecting the same cell. This phenomenon results in the rapid exchange of RNA segments. In this study, we have developed a method to prevent the free reassortment of influenza A virus RNAs by rewiring their packaging signals. Specific packaging signals for individual influenza virus RNA segments are located in the 5′ and 3′ noncoding regions as well as in the terminal regions of the ORF of an RNA segment. By putting the nonstructural protein (NS)-specific packaging sequences onto the ORF of the hemagglutinin (HA) gene and mutating the packaging regions in the ORF of the HA, we created a chimeric HA segment with the packaging identity of an NS gene. By the same strategy, we made an NS gene with the packaging identity of an HA segment. This rewired virus had the packaging signals for all eight influenza virus RNAs, but it lost the ability to independently reassort its HA or NS gene. A similar approach can be applied to the other influenza A virus segments to diminish their ability to form reassortant viruses.

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Phylogenetic Evidence against Evolutionary Stasis and Natural Abiotic Reservoirs of Influenza A Virus

Journal of Virology ◽

10.1128/jvi.02207-07 ◽

2008 ◽

Vol 82 (7) ◽

pp. 3769-3774 ◽

Cited By ~ 25

Author(s):

Michael Worobey

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Influenza Viruses ◽

Human Influenza ◽

Human Influenza Virus ◽

Virus Rna ◽

Evolutionary Stasis

ABSTRACT Zhang et al. (G. Zhang, D. Shoham, D. Gilichinsky, S. Davydov, J. D. Castello, and S. O. Rogers, J. Virol. 80:12229-12235, 2006) have claimed to have recovered influenza A virus RNA from Siberian lake ice, postulating that ice might represent an important abiotic reservoir for the persistence and reemergence of this medically important pathogen. A rigorous phylogenetic analysis of these influenza A virus hemagglutinin gene sequences, however, indicates that they originated from a laboratory reference strain derived from the earliest human influenza A virus isolate, WS/33. Contrary to Zhang et al.'s assertions that the Siberian “ice viruses” are most closely related either to avian influenza virus or to human influenza virus strains from Asia from the 1960s (Zhang et al., J. Virol. 81:2538 [erratum], 2007), they are clearly contaminants from the WS/33 positive control used in their laboratory. There is thus no credible evidence that environmental ice acts as a biologically relevant reservoir for influenza viruses. Several additional cases with findings that seem at odds with the biology of influenza virus, including modern-looking avian influenza virus RNA sequences from an archival goose specimen collected in 1917 (T. G. Fanning, R. D. Slemons, A. H. Reid, T. A. Janczewski, J. Dean, and J. K. Taubenberger, J. Virol. 76:7860-7862, 2002), can also be explained by laboratory contamination or other experimental errors. Many putative examples of evolutionary stasis in influenza A virus appear to be due to laboratory artifacts.

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Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus, Respiratory Syncytial Virus and Influenza A Virus

Viruses ◽

10.3390/v13020234 ◽

2021 ◽

Vol 13 (2) ◽

pp. 234

Author(s):

Sarah Al-Beltagi ◽

Cristian Alexandru Preda ◽

Leah V. Goulding ◽

Joe James ◽

Juan Pu ◽

...

Keyword(s):

Influenza Virus ◽

Respiratory Syncytial Virus ◽

Influenza A Virus ◽

Broad Spectrum ◽

Influenza A ◽

Respiratory Viruses ◽

Influenza Viruses ◽

Virus Challenge ◽

2009 H1n1 ◽

Syncytial Virus

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG’s antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.

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Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

Journal of Virology ◽

10.1128/jvi.75.17.8127-8136.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 8127-8136 ◽

Cited By ~ 78

Author(s):

Daniel R. Perez ◽

Ruben O. Donis

Keyword(s):

Amino Acids ◽

Influenza Virus ◽

Amino Acid ◽

Viral Replication ◽

Influenza A Virus ◽

Influenza A ◽

Amino Acid Sequences ◽

Influenza Viruses ◽

Virus Growth ◽

Transcription And Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

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Cathepsin W Is Required for Escape of Influenza A Virus from Late Endosomes

mBio ◽

10.1128/mbio.00297-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 13

Author(s):

Thomas O. Edinger ◽

Marie O. Pohl ◽

Emilio Yángüez ◽

Silke Stertz

Keyword(s):

Influenza Virus ◽

Rna Interference ◽

Influenza A Virus ◽

Proteolytic Activity ◽

Influenza A ◽

Antiviral Drugs ◽

Influenza Viruses ◽

Late Endosomes ◽

Genome Wide ◽

Cathepsin W

ABSTRACT Human cathepsin W (CtsW) is a cysteine protease, which was identified in a genome-wide RNA interference (RNAi) screen to be required for influenza A virus (IAV) replication. In this study, we show that reducing the levels of expression of CtsW reduces viral titers for different subtypes of IAV, and we map the target step of CtsW requirement to viral entry. Using a set of small interfering RNAs (siRNAs) targeting CtsW, we demonstrate that knockdown of CtsW results in a decrease of IAV nucleoprotein accumulation in the nuclei of infected cells at 3 h postinfection. Assays specific for the individual stages of IAV entry further show that attachment, internalization, and early endosomal trafficking are not affected by CtsW knockdown. However, we detected impaired escape of viral particles from late endosomes in CtsW knockdown cells. Moreover, fusion analysis with a dual-labeled influenza virus revealed a significant reduction in fusion events, with no detectable impact on endosomal pH, suggesting that CtsW is required at the stage of viral fusion. The defect in IAV entry upon CtsW knockdown could be rescued by ectopic expression of wild-type CtsW but not by the expression of a catalytically inactive mutant of CtsW, suggesting that the proteolytic activity of CtsW is required for successful entry of IAV. Our results establish CtsW as an important host factor for entry of IAV into target cells and suggest that CtsW could be a promising target for the development of future antiviral drugs. IMPORTANCE Increasing levels of resistance of influenza viruses to available antiviral drugs have been observed. Development of novel treatment options is therefore of high priority. In parallel to the classical approach of targeting viral enzymes, a novel strategy is pursued: cell-dependent factors of the virus are identified with the aim of developing small-molecule inhibitors against a cellular target that the virus relies on. For influenza A virus, several genome-wide RNA interference (RNAi) screens revealed hundreds of potential cellular targets. However, we have only limited knowledge on how these factors support virus replication, which would be required for drug development. We have characterized cathepsin W, one of the candidate factors, and found that cathepsin W is required for escape of influenza virus from the late endosome. Importantly, this required the proteolytic activity of cathepsin W. We therefore suggest that cathepsin W could be a target for future host cell-directed antiviral therapies.

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Mismatches in the Influenza A Virus RNA Panhandle Prevent Retinoic Acid-Inducible Gene I (RIG-I) Sensing by Impairing RNA/RIG-I Complex Formation

Journal of Virology ◽

10.1128/jvi.01671-15 ◽

2015 ◽

Vol 90 (1) ◽

pp. 586-590 ◽

Cited By ~ 10

Author(s):

Stéphanie Anchisi ◽

Jessica Guerra ◽

Geneviève Mottet-Osman ◽

Dominique Garcin

Keyword(s):

Influenza Virus ◽

Retinoic Acid ◽

Complex Formation ◽

Influenza A Virus ◽

Rna Structure ◽

Influenza A ◽

Double Stranded Rna ◽

Virus Rna ◽

Inducible Gene

Influenza virus RNA (vRNA) promoter panhandle structures are believed to be sensed by retinoic acid-inducible gene I (RIG-I). The occurrence of mismatches in this double-stranded RNA structure raises questions about their effect on innate sensing. Our results suggest that mismatches in vRNA promoters decrease binding to RIG-Iin vivo, affecting RNA/RIG-I complex formation and preventing RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral strategy to escape RIG-I sensing.

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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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RNA-Sequencing-Based Transcriptomic Analysis Reveals a Role for Annexin-A1 in Classical and Influenza A Virus-Induced Autophagy

Cells ◽

10.3390/cells9061399 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1399 ◽

Cited By ~ 1

Author(s):

Jianzhou Cui ◽

Dhakshayini Morgan ◽

Dao Han Cheng ◽

Sok Lin Foo ◽

Gracemary L. R. Yap ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Influenza Infection ◽

Critical Role ◽

Mtor Inhibitors ◽

Influenza Viruses ◽

Annexin A1 ◽

Underlying Mechanisms

Influenza viruses have been shown to use autophagy for their survival. However, the proteins and mechanisms involved in the autophagic process triggered by the influenza virus are unclear. Annexin-A1 (ANXA1) is an immunomodulatory protein involved in the regulation of the immune response and Influenza A virus (IAV) replication. In this study, using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated protein 9) deletion of ANXA1, combined with the next-generation sequencing, we systematically analyzed the critical role of ANXA1 in IAV infection as well as the detailed processes governing IAV infection, such as macroautophagy. A number of differentially expressed genes were uniquely expressed in influenza A virus-infected A549 parental cells and A549 ∆ANXA1 cells, which were enriched in the immune system and infection-related pathways. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed the role of ANXA1 in autophagy. To validate this, the effect of mechanistic target of rapamycin (mTOR) inhibitors, starvation and influenza infection on autophagy was determined, and our results demonstrate that ANXA1 enhances autophagy induced by conventional autophagy inducers and influenza virus. These results will help us to understand the underlying mechanisms of IAV infection and provide a potential therapeutic target for restricting influenza viral replication and infection.

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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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Immunomic Analysis of the Repertoire of T-Cell Specificities for Influenza A Virus in Humans

Journal of Virology ◽

10.1128/jvi.01563-08 ◽

2008 ◽

Vol 82 (24) ◽

pp. 12241-12251 ◽

Cited By ~ 135

Author(s):

Erika Assarsson ◽

Huynh-Hoa Bui ◽

John Sidney ◽

Qing Zhang ◽

Jean Glenn ◽

...

Keyword(s):

Influenza Virus ◽

T Cell ◽

Influenza A Virus ◽

Influenza A ◽

T Cell Responses ◽

Class Ii ◽

Influenza Viruses ◽

Class I ◽

Cell Responses ◽

Virus Strains

ABSTRACT Continuing antigenic drift allows influenza viruses to escape antibody-mediated recognition, and as a consequence, the vaccine currently in use needs to be altered annually. Highly conserved epitopes recognized by effector T cells may represent an alternative approach for the generation of a more universal influenza virus vaccine. Relatively few highly conserved epitopes are currently known in humans, and relatively few epitopes have been identified from proteins other than hemagglutinin and nucleoprotein. This prompted us to perform a study aimed at identifying a set of human T-cell epitopes that would provide broad coverage against different virus strains and subtypes. To provide coverage across different ethnicities, seven different HLA supertypes were considered. More than 4,000 peptides were selected from a panel of 23 influenza A virus strains based on predicted high-affinity binding to HLA class I or class II and high conservancy levels. Peripheral blood mononuclear cells from 44 healthy human blood donors were tested for reactivity against HLA-matched peptides by using gamma interferon enzyme-linked immunospot assays. Interestingly, we found that PB1 was the major target for both CD4+ and CD8+ T-cell responses. The 54 nonredundant epitopes (38 class I and 16 class II) identified herein provided high coverage among different ethnicities, were conserved in the majority of the strains analyzed, and were consistently recognized in multiple individuals. These results enable further functional studies of T-cell responses during influenza virus infection and provide a potential base for the development of a universal influenza vaccine.

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To Conquer the Host, Influenza Virus Is Packing It In: Interferon-Antagonistic Strategies beyond NS1

Journal of Virology ◽

10.1128/jvi.00041-16 ◽

2016 ◽

Vol 90 (19) ◽

pp. 8389-8394 ◽

Cited By ~ 10

Author(s):

Michaela Weber-Gerlach ◽

Friedemann Weber

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Host Defense ◽

Influenza A ◽

Type I Interferon ◽

Current Knowledge ◽

Nonstructural Protein ◽

Structural Proteins ◽

Type I ◽

Interferon System

The nonstructural protein NS1 is well established as a virulence factor of influenza A virus counteracting induction of the antiviral type I interferon system. Recent studies now show that viral structural proteins, their derivatives, and even the genome itself also contribute to keeping the host defense under control. Here, we summarize the current knowledge on these NS1-independent interferon escape strategies.

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