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      Synergistic effect of PB2 283M and 526R contributes to enhanced virulence of H5N8 influenza viruses in mice

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          Abstract

          Highly pathogenic avian influenza (HPAI) H5N8 virus has caused considerable economic losses to poultry industry and poses a great threat to public health. Our previous study revealed two genetically similar HPAI H5N8 viruses displaying completely different virulence in mice. However, the molecular basis for viral pathogenicity to mammals remains unknown. Herein, we generated a series of reassortants between the two viruses and evaluated their virulence in mice. We demonstrated that 283M in PB2 is a new mammalian virulence marker for H5 viruses and that synergistic effect of amino acid residues 283M and 526R in PB2 is responsible for high virulence of the HPAI H5N8 virus. Analysis of available PB2 sequences showed that PB2 283M is highly conserved among influenza A viruses, while PB2 526R presents in most of human H3N2 and H5N1 isolates. Further study confirmed that the residues 283M and 526R had similar impacts on an HPAI H5N1 virus, suggesting that influenza viruses with both residues may replicate well in mammalian hosts. Together, these results present new insights for synergistic effect of 283M and 526R in PB2 of H5 HPAI virus on virulence to mammalian host, furthering our understanding of the pathogenesis of influenza A virus.

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          Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses.

          M Hatta (2001)
          In 1997, an H5N1 influenza A virus was transmitted from birds to humans in Hong Kong, killing 6 of the 18 people infected. When mice were infected with the human isolates, two virulence groups became apparent. Using reverse genetics, we showed that a mutation at position 627 in the PB2 protein influenced the outcome of infection in mice. Moreover, high cleavability of the hemagglutinin glycoprotein was an essential requirement for lethal infection.
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            A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.

            The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.
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              The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit.

              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.
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                Author and article information

                Contributors
                1007094902@qq.com
                chensj@yzu.edu.cn
                1067721448@qq.com
                871965306@qq.com
                1805404649@qq.com
                267206059@qq.com
                wma@vet.k-state.edu
                pengdx@yzu.edu.cn
                xfliu@yzu.edu.cn
                Journal
                Vet Res
                Vet. Res
                Veterinary Research
                BioMed Central (London )
                0928-4249
                1297-9716
                25 October 2017
                25 October 2017
                2017
                : 48
                : 67
                Affiliations
                [1 ]GRID grid.268415.c, College of Veterinary Medicine, , Yangzhou University, ; Yangzhou, Jiangsu 225009 PR China
                [2 ]Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou, Jiangsu China
                [3 ]Jiangsu Research Centre of Engineering and Technology for Prevention and Control of Poultry Disease, Yangzhou, Jiangsu China
                [4 ]ISNI 0000 0001 0737 1259, GRID grid.36567.31, Department of Diagnostic Medicine/Pathobiology, , Kansas State University, ; Manhattan, KS USA
                Author information
                http://orcid.org/0000-0001-6773-5149
                Article
                471
                10.1186/s13567-017-0471-0
                5657129
                29070059
                06990377-a30d-421a-8a66-d10a897445b3
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 30 July 2017
                : 9 October 2017
                Funding
                Funded by: Important National Science & Technology Specific Projects
                Award ID: 2016YFD0500202
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31372450
                Award ID: 31402229
                Award Recipient :
                Funded by: Agricultural Science & Technology Independent Innovation Fund of Jiangsu Province
                Award ID: CX(15)1065
                Award Recipient :
                Funded by: Yangzhou University Funding for Scientific Research
                Award ID: KYLX15_1380
                Award Recipient :
                Funded by: Priority Academic Program Development of Jiangsu Higher Education Institutions
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2017

                Veterinary medicine
                Veterinary medicine

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