| Summary: | An avian virus can become adapted to humans by mutating or recombining with currently circulating human viruses. These viruses have the potential to cause pandemics in an immunologically naïve human population. Host restriction involves multiple determinants, however, influenza polymerase is considered to play an important role. The heterotrimeric polymerase complex (PA, PB1 and PB2) associates with viral RNA and nucleoprotein (NP) to form a ribonucleoprotein (RNP) complex responsible for viral replication and transcription. Host specific genetic signatures have been identified on all of the polymerase subunits and on NP, but the PB2 protein arguably carries the dominant determinants of host range. Avian-origin influenza polymerase activity can be dramatically increased in human cells with the PB2 E627K substitution. This has been suggested to stabilise the interaction between the NP and PB2 components of the vRNP complex in the nuclei of infected cells. However, we demonstrate that a variety of adaptive PB2 substitutions including E627K did not enhance the stability of the vRNP in human cells, but rather increased the amount of replicated RNA, and that resulted in more PB2-NP co-precipitation. The introduction of many adaptive PB2 mutations enhances avian influenza polymerase activity in an in vitro reconstituted polymerase assay. However, only some of these mutations have been detected in viruses that are found circulating in nature. We explored whether the polymerase assay truly predicts viral growth and investigated viral selection pressures that might favour some adaptive mutations over others. We used reverse genetics to create a series of viral variants carrying mutations in the PB2 gene and carried out virological assays and also analysed the effects of the mutations in vivo. Some mutations that increased in vitro polymerase activity led to attenuated virus replication and resulted in an increase in interferon activation. These data increase our understanding of the host range barrier and why certain adaptive mutations may or may not have emerged.
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