scholarly journals Differential splicing of ANP32A in birds alters its ability to stimulate RNA synthesis by restricted influenza polymerase

2018 ◽  
Author(s):  
Steven F. Baker ◽  
Mitchell P. Ledwith ◽  
Andrew Mehle
Keyword(s):  
Influenza Virus ◽  
Host Range ◽  
Rna Synthesis ◽  
Splice Variants ◽  
Polymerase Activity ◽  
Genomic Rna ◽  
Differential Splicing ◽  
Exon Duplication ◽  
Species Specific ◽  
Viral Genomic Rna

AbstractAdaptation of viruses to their host can result in specialization and a restricted host range. Species-specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently been identified as a cellular co-factor impacting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation amongst birds differentially impacts viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.

scholarly journals pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Kenji Sugiyama ◽  
Atsushi Kawaguchi ◽  
Mitsuru Okuwaki ◽  
Kyosuke Nagata
Keyword(s):  
Influenza Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Free Form ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
Nuclear Extracts ◽  
Virus Rna ◽  
Viral Genomic Rna

Replication of influenza viral genomic RNA (vRNA) is catalyzed by viral RNA-dependent RNA polymerase (vRdRP). Complementary RNA (cRNA) is first copied from vRNA, and progeny vRNAs are then amplified from the cRNA. Although vRdRP and viral RNA are minimal requirements, efficient cell-free replication could not be reproduced using only these viral factors. Using a biochemical complementation assay system, we found a novel activity in the nuclear extracts of uninfected cells, designated IREF-2, that allows robust unprimed vRNA synthesis from a cRNA template. IREF-2 was shown to consist of host-derived proteins, pp32 and APRIL. IREF-2 interacts with a free form of vRdRP and preferentially upregulates vRNA synthesis rather than cRNA synthesis. Knockdown experiments indicated that IREF-2 is involved in in vivo viral replication. On the basis of these results and those of previous studies, a plausible role(s) for IREF-2 during the initiation processes of vRNA replication is discussed.

scholarly journals Species specific differences in use of ANP32 proteins by influenza A virus

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Jason S Long ◽  
Alewo Idoko-Akoh ◽  
Bhakti Mistry ◽  
Daniel Goldhill ◽  
Ecco Staller ◽  
...  
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Host Range ◽  
Influenza A Virus ◽  
Support Function ◽  
Influenza A ◽  
Polymerase Activity ◽  
Influenza A Viruses ◽  
The Poor ◽  
Species Specific

Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that the gene currently designated as avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV relies solely on chicken ANP32A to support its replication in chicken cells. Amino acids 129I and 130N, accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resilient genome edited chickens.

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

2006 ◽  
Vol 80 (1) ◽  
pp. 252-261 ◽  
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.

scholarly journals Effect of the fourth nucleotide at the 3′ end of neuraminidase and matrix viral genomic RNA on the pathogenicity of influenza virus A/PR/8/34

2017 ◽  
Vol 18 (S1) ◽  
pp. 307 ◽  
Author(s):  
Chung-Young Lee ◽  
Hyuk-Joon Kwon ◽  
Thanh Trung Nguyen ◽  
Ilhwan Kim ◽  
Hyung-Kwan Jang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Genomic Rna ◽  
Influenza Virus A ◽  
Viral Genomic Rna

scholarly journals Phosphorylation controls RNA binding and transcription by the influenza virus polymerase

2020 ◽  
Author(s):  
Anthony R. Dawson ◽  
Gary M. Wilson ◽  
Elyse C. Freiberger ◽  
Arindam Mondal ◽  
Joshua J. Coon ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Genome ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Polymerase Activity ◽  
Genome Replication ◽  
Replication Machinery ◽  
Biochemical Analyses ◽  
Successful Replication ◽  
Infectious Cycle

AbstractThe influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. We previously showed that phosphorylation regulates genome replication by controlling assembly of the replication machinery (Mondal, et al. 2017). However, it remained unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle.

scholarly journals Insights into species-specific regulation of ANP32A on the mammalian-restricted influenza virus polymerase activity

2019 ◽  
Vol 8 (1) ◽  
pp. 1465-1478 ◽  
Author(s):  
Zhenwei Bi ◽  
Hongliu Ye ◽  
Xingbo Wang ◽  
An Fang ◽  
Tianqi Yu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Polymerase Activity ◽  
Specific Regulation ◽  
Species Specific

scholarly journals A species-specific signature residue in the PB2 subunit of the bat influenza virus polymerase restricts viral RNA synthesis.

2021 ◽  
Author(s):  
Saptarshi Banerjee ◽  
Aratrika De ◽  
Nandita Kedia ◽  
Lin-Fa Wang ◽  
Arindam Mondal
Keyword(s):  
Influenza Virus ◽  
Cell Lines ◽  
Influenza A ◽  
Polymerase Activity ◽  
Influenza Viruses ◽  
Influenza A Viruses ◽  
Bat Virus ◽  
Influenza A H1n1 ◽  
Chimeric Rna ◽  
Species Specific

Bat influenza A viruses (H17N10 and H18N11) are genetically distant from conventional influenza A viruses and replicates poorly in non-bat hosts species. However, the reason behind the lower replication fitness of these viruses are yet to be elucidated. In this work, we have identified species-specific signature residues, present in viral PB2 protein, which is a major determinant of polymerase fitness in human, avian and bat cell lines. Through extensive sequence and structural comparison between the bat and non-bat influenza virus RNA polymerases, we have identified a previously uncharacterized PB2-282 residue, which is serine in bat virus PB2 protein but harbours highly conserved glutamic acid in conventional influenza A viruses. Introduction of these bat specific signatures in the polymerase of a human adapted strain of influenza A/H1N1 virus drastically reduces its polymerase activity and replication efficiency in cell lines of human, bat and canine origin. In contrast, introduction of the human-specific signatures in bat virus PB2 (H17N10), significantly enhances its function in the context of a chimeric RNA polymerase. Interestingly, the PB2-282 resides within an evolutionary conserved 'S-E-S' motif present across different genera of influenza viruses but is replaced with a 'S-S-T' motif in bat influenza viruses, indicating that this E to S transition may serve as a species-specific adaptation signature that modulates the activity of bat virus polymerase in other host species.

scholarly journals Avian ANP32B does not support influenza A virus polymerase and influenza A virus relies exclusively on ANP32A in chicken cells

2019 ◽  
Author(s):  
Jason S. Long ◽  
Alewo Idoko-Akoh ◽  
Bhakti Mistry ◽  
Daniel H. Goldhill ◽  
Ecco Staller ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Host Range ◽  
Influenza A Virus ◽  
Support Function ◽  
Influenza A ◽  
Polymerase Activity ◽  
Human Cells ◽  
Influenza A Viruses ◽  
The Poor

SummaryInfluenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV does not replicate in chicken cells that lack ANP32A. Amino acid differences in LRR5 domain accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resistant genome edited chickens.

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