Regulation of the influenza A virus polymerase activity by the viral nuclear export protein (NEP)
Final Report Abstract
Human influenza A viruses (IAV) infections cause respiratory febrile illnesses, resulting in significant morbidity and mortality in the population. However, to cross the human species barrier, both avian and swine IAV have to adapt to the new host environment by acquiring so called adaptive mutations in all gene products, including the viral polymerase subunits. Although the polymerase complex of these viruses generates all viral RNA species, we could provide evidence that the viral nuclear export protein (NEP) is a polymerase cofactor required for efficient viral polymerase activity by stimulating vRNA and cRNA synthesis. We could further show that a single adaptive mutation in the NEP of a human H5N1 isolate that is absent in avian H5N1 isolates increases the replication efficiency of the viral polymerase and the pathogenicity of this isolate in mice. Furthermore, we obtained evidence that the C-terminal half of NEP is sufficient to stimulate the polymerase activity, whereas the N-terminal part is required for regulating NEP activity by back folding to the C-terminus. Although it was believed that NEP phosphorylation at the N-terminus might regulate its polymerase cofactor activity, cell culture studies and in vivo experiments using suitable NEP mutant viruses revealed only a minor role of this posttranslational modification on NEP activity. Based on these NEP mutant viruses we developed stable reporterexpressing viruses that harbored the gene of interest in the NS segment. We succeeded to generate a Cre-expressing virus that allowed the visualization of IAV infected cells or cells that survived acute infection.
Publications
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Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells. Nat Commun. 3, 802 (2012)
Manz B, Brunotte L, Reuther P, Schwemmle M
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Adaptation of avian influenza A virus polymerase in mammals to overcome the host species barrier. J Virol. 87, 7200-7209 (2013)
Manz B, Schwemmle M, Brunotte L
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Adaptive mutations in the nuclear export protein of human-derived H5N1 strains facilitate a polymerase activity-enhancing conformation. J Virol. 88, 263-271 (2014)
Reuther P, et al.
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Phosphorylation of highly conserved serine residues in the influenza A virus nuclear export protein NEP plays a minor role in viral growth in human cells and mice. J Virol. 88, 7668-7673 (2014)
Reuther P, Giese S, Gotz V, Riegger D, Schwemmle M
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Generation of a variety of stable Influenza A reporter viruses by genetic engineering of the NS gene segment. Sci Rep. 5, 11346 (2015)
Reuther P, Gopfert K, Dudek AH, Heiner M, Herold S, Schwemmle M
