Journal ArticleDOI
The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit
Alexandre Dias,Denis Bouvier,Thibaut Crépin,Andrew A. McCarthy,Darren J. Hart,Florence Baudin,Stephen Cusack,Rob W.H. Ruigrok +7 more
TLDR
In this paper, the amino-terminal 209 residues of the PA subunit contain the active site of the endonuclease active site, which is shown to be strongly activated by manganese ions, matching observations reported for the intact trimeric polymerase.Abstract:
The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism. The PB2 subunit binds the 5' cap of host pre-mRNAs, which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated. These results will facilitate the optimisation of endonuclease inhibitors as potential new anti-influenza drugs.read more
Citations
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Journal ArticleDOI
An Overlapping Protein-Coding Region in Influenza A Virus Segment 3 Modulates the Host Response
Brett W. Jagger,Helen M. Wise,John C. Kash,Kathie-Anne Walters,Norma M. Wills,Yongli Xiao,Rebecca L. Dunfee,Louis M. Schwartzman,Adrian Ozinsky,G. L. Bell,Rosa M. Dalton,Amy C. Y. Lo,Stacey Efstathiou,John F. Atkins,John F. Atkins,Andrew E. Firth,Jeffery K. Taubenberger,Paul Digard +17 more
TL;DR: This work has identified a previously unknown IAV protein that modulates the host response to infection, a finding with important implications for understanding IAV pathogenesis.
Journal ArticleDOI
mRNA capping: biological functions and applications
TL;DR: This review will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells, different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA.
Journal ArticleDOI
Viral subversion of the host protein synthesis machinery.
Derek Walsh,Ian Mohr +1 more
TL;DR: The diverse strategies that viruses use to subvert host protein synthesis functions and regulate mRNA translation in infected cells are discussed.
Journal ArticleDOI
Structure of influenza A polymerase bound to the viral RNA promoter
TL;DR: The crystal structure of the heterotrimeric bat influenza A polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter is presented, laying the basis for an atomic-level mechanistic understanding of the many functions of influenza polymerase and opens new opportunities for anti-influenza drug design.
Journal ArticleDOI
A Complicated Message: Identification of a Novel PB1-Related Protein Translated from Influenza A Virus Segment 2 mRNA
Helen M. Wise,Ágnes Foeglein,Jiechao Sun,Rosa M. Dalton,Sheetal Patel,Wendy A. Howard,Emma C. Anderson,Wendy S. Barclay,Paul Digard +8 more
TL;DR: The influenza A virus proteome includes a 12th primary translation product that (similarly to PB1-F2) is nonessential for virus viability but whose loss, in particular genetic backgrounds, is detrimental to virus replication.
References
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Journal ArticleDOI
Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism.
Lorena S. Beese,Thomas A. Steitz +1 more
TL;DR: The refined crystal structures of the large proteolytic fragment (Klenow fragment) of Escherichia coli DNA polymerase I and its complexes with a deoxynucleoside monophosphate product and a single‐stranded DNA substrate offer a detailed picture of an editing 3′‐5′ exonuclease active site.
Journal ArticleDOI
Thermofluor-based high-throughput stability optimization of proteins for structural studies.
Ulrika B. Ericsson,B. Martin Hallberg,B. Martin Hallberg,George T. DeTitta,Niek Dekker,Pär Nordlund,Pär Nordlund +6 more
TL;DR: A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives, suggesting that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.
Journal ArticleDOI
Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA
TL;DR: This work identifies the polyuridine motif of the HCV genome 3′ non-translated region and its replication intermediate as the PAMP substrate of RIG-I, and shows that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of Rig-I recognition and immune triggering in human and murine cells.
Journal ArticleDOI
A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription
TL;DR: It is shown that virions and purified viral cores contain a unique endonuclease that cleaves RNAs containing a 5' methylated cap structure preferentially at purine residues 10 to 14 nucleotides from the cap, generating fragments with 3'-terminal hydroxyl groups.
Journal ArticleDOI
The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host
TL;DR: Specific mutations in SC35M polymerase considerably increase its activity in mammalian cells, correlating with high virulence in mice, demonstrating convergent evolution in nature and may be a prerequisite for adaptation to a new host paving the way for new pandemic viruses.