INTRODUCTION

Avian species are the natural hosts of influenza A viruses, which continuously challenge the poultry industry and human health. Avian influenza viruses (AIVs) must obtain a series of mutations in their genomes to become adapted to mammalian hosts before they can efficiently replicate in and transmit among humans (1). Mutations in the hemagglutinin (HA) and basic polymerase 2 (PB2) proteins are particularly important. Amino acid mutations in HA are known to play important roles in promoting efficient binding to human-type receptors, thereby facilitating the replication and transmission of influenza virus in humans (2–5). Using a ferret model, Lakdawala et al. showed that transmissible influenza viruses with human-type receptor preference are rapidly selected in the soft palate, where long-chain α2,6-sialic acids predominate on the nasopharyngeal surface (6). Different mutations in PB2 have been identified to contribute to the adaptation of influenza viruses to mammalian hosts; these mutations include E627K (7, 8), D701N (9, 10), T271A (11), Q591R/K (12, 13), and E158G (14).

The key role of the PB2 E627K substitution in the mammalian adaptation of AIVs has been well documented, having been shown to enhance polymerase activity, virus replication, pathogenicity, and transmission of AIVs in humans and other mammals (2, 7, 8, 15–19). However, not all AIVs acquire the PB2 E627K mutation upon infection of humans, such as many H5N1 viruses (20, 21). Therefore, understanding the biological mechanism involved in the occurrence of this substitution is important to gain insights into the adaptation and pathogenesis of AIVs in mammals.

In early 2013 in China, a novel H7N9 AIV crossed the species barrier and caused the first human infection (22). To date, five epidemic waves of human infection with H7N9 viruses have caused 1,567 cases, with a fatality rate of approximately 39% (23). Moreover, highly pathogenic H7N9 viruses possessing a multibasic cleavage site motif in HA were detected in 2017 and caused severe threats to the poultry industry and human health (18, 24–26). Remarkably, the PB2 of H7N9 viruses easily acquires the PB2 E627K or D701N adaptive mutation during replication in mammalian hosts (18, 22, 27). In particular, most H7N9 human isolates acquire the PB2 E627K substitution (18). The prominent ability of H7N9 AIVs to acquire the PB2 E627K substitution upon infection of humans provides us with an excellent opportunity to decipher the underlying mechanism by which AIVs rapidly acquire the PB2 E627K substitution during their adaptation to mammalian hosts, including humans.

H7N9 AIV is a triple reassortant virus, with all six internal genes being derived from H9N2 AIVs (19, 28). In this study, we generated a series of reassortant or mutant viruses in the background of an avian 2013 H7N9 virus, harboring genes or mutations from an avian H9N2 virus, and monitored the residue phenotype of PB2 627 during viral passages in mice. We found that the low polymerase activity attributed to the H7N9 viral PA protein is the intrinsic force driving the emergence of the PB2 E627K mutation during the replication of H7N9 AIVs in mammals, a process that also involves interplay with host factors, such as ANP32A.