Infection of mammals by avian influenza viruses requires adaptive mutations to achieve high-level replication in the new host. However, the basic mechanism underlying this adaptation process is still unknown. Here we show that avian polymerases, lacking the human signature PB2-E627K, are incapable of generating usable complementary RnA templates in cultured human cells and therefore require adaptation. Characterization of the highly pathogenic human H5n1 isolate A/Thailand/1(KAn-1)/2004 that retained the avian PB2-E627 reveals that the defect in RnA replication is only partially compensated by mutations in the polymerase. Instead, mutations in the nuclear export protein are required for efficient polymerase activity. We demonstrate that adaptive mutations in nuclear export proteins of several human isolates enhance the polymerase activity of avian polymerases in human cultured cells. In conclusion, when crossing the species barrier, avian influenza viruses acquire adaptive mutations in nuclear export protein to escape restricted viral genome replication in mammalian cells.

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Influenza A viruses (IAV) pose a constant threat to the human population and therefore a better understanding of their fundamental biology and identification of novel therapeutics is of upmost importance. Various reporter-encoding IAV were generated to achieve these goals, however, one recurring difficulty was the genetic instability especially of larger reporter genes. We employed the viral NS segment coding for the non-structural protein 1 (NS1) and nuclear export protein (NEP) for stable expression of diverse reporter proteins. This was achieved by converting the NS segment into a single open reading frame (ORF) coding for NS1, the respective reporter and NEP. To allow expression of individual proteins, the reporter genes were flanked by two porcine Teschovirus-1 2A peptide (PTV-1 2A)-coding sequences. The resulting viruses encoding luciferases, fluorescent proteins or a Cre recombinase are characterized by a high genetic stability in vitro and in mice and can be readily employed for antiviral compound screenings, visualization of infected cells or cells that survived acute infection.

Background: NEP stimulates viral RNA synthesis and nuclear vRNP export. Results: NEP is required to stabilize M1-vRNP binding for nuclear export. Deletion of the last three amino acids of NEP abrogates nuclear export and the polymerase-enhancing function of NEP. Conclusion:The polymerase-enhancing function and the nuclear export function of NEP are linked functionally. Significance: This study provides new insights into the assembly of the nuclear export complex.