Optimized PCR amplification of influenza A virus RNA using Tth DNA polymerase, incorporating uracil N glycosylase (UNG) in a single tube reaction

ABSTRACTAL18, an inhibitor of human cytomegalovirus DNA polymerase, was serendipitously found to also block the interaction between the PB1 and PA polymerase subunits of influenza A virus. Furthermore, AL18 effectively inhibited influenza A virus polymerase activity and the overall replication of influenza A and B viruses. A molecular model to explain the binding of AL18 to both cytomegalovirus and influenza targets is proposed. Thus, AL18 represents an interesting lead for the development of new antivirals.

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Artificial Hybrids of Influenza A Virus RNA Polymerase Reveal PA Subunit Modulates Its Thermal Sensitivity

PLoS ONE ◽

10.1371/journal.pone.0015140 ◽

2010 ◽

Vol 5 (12) ◽

pp. e15140 ◽

Cited By ~ 14

Author(s):

Takahito Kashiwagi ◽

Koyu Hara ◽

Yoko Nakazono ◽

Nobuyuki Hamada ◽

Hiroshi Watanabe

Keyword(s):

Rna Polymerase ◽

Influenza A Virus ◽

Influenza A ◽

Thermal Sensitivity ◽

Virus Rna

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Rewiring the RNAs of influenza virus to prevent reassortment

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0908897106 ◽

2009 ◽

Vol 106 (37) ◽

pp. 15891-15896 ◽

Cited By ~ 52

Author(s):

Qinshan Gao ◽

Peter Palese

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Nonstructural Protein ◽

Influenza Viruses ◽

Ha Gene ◽

Ns Gene ◽

Virus Rna ◽

Reassortant Viruses ◽

Packaging Signals

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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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 role of the priming loop in influenza A virus RNA synthesis

Nature Microbiology ◽

10.1038/nmicrobiol.2016.29 ◽

2016 ◽

Vol 1 (5) ◽

Cited By ~ 42

Author(s):

Aartjan J. W. te Velthuis ◽

Nicole C. Robb ◽

Achillefs N. Kapanidis ◽

Ervin Fodor

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Rna Synthesis ◽

Virus Rna

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Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing

PLoS Pathogens ◽

10.1371/journal.ppat.1003460 ◽

2013 ◽

Vol 9 (6) ◽

pp. e1003460 ◽

Cited By ~ 46

Author(s):

Pei-Ling Tsai ◽

Ni-Ting Chiou ◽

Sharon Kuss ◽

Adolfo García-Sastre ◽

Kristen W. Lynch ◽

...

Keyword(s):

Influenza A Virus ◽

Rna Splicing ◽

Influenza A ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Hnrnp K ◽

Virus Rna

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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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