A functional sequence-specific interaction between influenza A virus genomic RNA segments

The NS1 protein is a nonstructural protein encoded by the influenza A virus. It is responsible for many alterations produced in the cellular metabolism upon infection by the virus and for modulation of virus virulence. The NS1 protein is able to perform a large variety of functions due to its ability to bind various types of RNA molecules, from both viral and nonviral origin, and to interact with several cell factors. With the aim of exploring whether the binding of NS1 protein to viral RNA (vRNA) could constitute a novel target for the search of anti-influenza drugs, a filter-binding assay measuring the specific interaction between the recombinant His-NS1 protein from influenza A virus and a radiolabeled model vRNA ( 32P-vNSZ) was adapted to a format suitable for screening and easy automation. Flashplate® technology (PerkinElmer, Waltham, MA), either in 96- or 384-well plates, was used. The Flashplate® wells were precoated with the recombinant His-NS1 protein, and the binding of His-NS1 to a 35S-vNSZ probe was measured. A pilot screening of a collection of 27,520 mixtures of synthetic chemical compounds was run for inhibitors of NS1 binding to vRNA. We found 3 compounds in which the inhibition of NS1 binding to vRNA, observed at submicromolar concentrations, was correlated with a reduction of the cytopathic effect during the infection of cell cultures with influenza virus. These results support the hypothesis that the binding of NS1 to vRNA could be a novel target for the development of anti-influenza drugs. ( Journal of Biomolecular Screening 2008:581-590)

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Organization of the Influenza A Virus Genomic RNA in the Viral Replication Cycle—Structure, Interactions, and Implications for the Emergence of New Strains

Pathogens ◽

10.3390/pathogens9110951 ◽

2020 ◽

Vol 9 (11) ◽

pp. 951

Author(s):

Julita Piasecka ◽

Aleksandra Jarmolowicz ◽

Elzbieta Kierzek

Keyword(s):

Viral Replication ◽

Influenza A Virus ◽

Rna Structure ◽

Influenza A ◽

Respiratory Infections ◽

Specific Interaction ◽

Replication Cycle ◽

Genome Packaging ◽

Functional Roles ◽

Interaction Sites

The influenza A virus is a human pathogen causing respiratory infections. The ability of this virus to trigger seasonal epidemics and sporadic pandemics is a result of its high genetic variability, leading to the ineffectiveness of vaccinations and current therapies. The source of this variability is the accumulation of mutations in viral genes and reassortment enabled by its segmented genome. The latter process can induce major changes and the production of new strains with pandemic potential. However, not all genetic combinations are tolerated and lead to the assembly of complete infectious virions. Reports have shown that viral RNA segments co-segregate in particular circumstances. This tendency is a consequence of the complex and selective genome packaging process, which takes place in the final stages of the viral replication cycle. It has been shown that genome packaging is governed by RNA–RNA interactions. Intersegment contacts create a network, characterized by the presence of common and strain-specific interaction sites. Recent studies have revealed certain RNA regions, and conserved secondary structure motifs within them, which may play functional roles in virion assembly. Growing knowledge on RNA structure and interactions facilitates our understanding of the appearance of new genome variants, and may allow for the prediction of potential reassortment outcomes and the emergence of new strains in the future.

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Interaction network linking the human H3N2 influenza A virus genomic RNA segments

Vaccine ◽

10.1016/j.vaccine.2012.09.079 ◽

2012 ◽

Vol 30 (51) ◽

pp. 7359-7367 ◽

Cited By ~ 58

Author(s):

Emilie Fournier ◽

Vincent Moules ◽

Boris Essere ◽

Jean-Christophe Paillart ◽

Jean-Daniel Sirbat ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Interaction Network ◽

Genomic Rna ◽

H3n2 Influenza

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Universal and strain specific structure features of segment 8 genomic RNA of influenza A virus – application of 4-thiouridine photocrosslinking

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.101245 ◽

2021 ◽

pp. 101245

Author(s):

Marta Soszynska-Jozwiak ◽

Maciej Pszczola ◽

Julita Piasecka ◽

Jake M. Peterson ◽

Walter N. Moss ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Specific Structure ◽

Genomic Rna ◽

Segment 8

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Defective Assembly of Influenza A Virus due to a Mutation in the Polymerase Subunit PA

Journal of Virology ◽

10.1128/jvi.80.1.252-261.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 252-261 ◽

Cited By ~ 50

Author(s):

John F. Regan ◽

Yuying Liang ◽

Tristram G. Parslow

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Virus Assembly ◽

Functional Characterization ◽

Genomic Rna ◽

Viral Mrnas ◽

Virus Particles ◽

Essential Components ◽

Viral Genomic Rna

ABSTRACT The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.

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Detection and Characterization of Influenza A Virus PA-PB2 Interaction through a Bimolecular Fluorescence Complementation Assay

Journal of Virology ◽

10.1128/jvi.02300-08 ◽

2009 ◽

Vol 83 (8) ◽

pp. 3944-3955 ◽

Cited By ~ 55

Author(s):

Joseph N. Hemerka ◽

Dan Wang ◽

Yuejin Weng ◽

Wuxun Lu ◽

Radhey S. Kaushik ◽

...

Keyword(s):

Influenza A Virus ◽

Protein Interactions ◽

Influenza A ◽

Specific Interaction ◽

Polymerase Activity ◽

Bimolecular Fluorescence Complementation ◽

Polymerase Complex ◽

Binary Complexes ◽

Fluorescence Complementation ◽

Bimolecular Fluorescence

ABSTRACT The influenza virus polymerase complex, consisting of the PA, PB1, and PB2 subunits, is required for the transcription and replication of the influenza A viral genome. Previous studies have shown that PB1 serves as a core subunit to incorporate PA and PB2 into the polymerase complex by directly interacting with PA and PB2. Despite numerous attempts, largely involving biochemical approaches, a specific interaction between PA and PB2 subunits has yet to be detected. In the current study, we developed and utilized bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in the assembly of the influenza A virus polymerase complex. Proof-of-concept experiments demonstrated that BiFC can specifically detect PA-PB1 interactions in living cells. Strikingly, BiFC demonstrated an interaction between PA and PB2 that has not been reported previously. Deletion-based BiFC experiments indicated that the N-terminal 100 amino acid residues of PA are responsible for the PA-PB2 interaction observed in BiFC. Furthermore, a detailed analysis of subcellular localization patterns and temporal nuclear import of PA-PB2 binary complexes suggested that PA and PB2 subunits interacted in the cytoplasm initially and were subsequently transported as a dimer into the nucleus. Taken together, results of our studies reveal a previously unknown PA-PB2 interaction and provide a framework for further investigation of the biological relevance of the PA-PB2 interaction in the polymerase activity and viral replication of influenza A virus.

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