Molecular Mechanisms For The Emergency Of The Mammalian-adaptive PB2 E627K Substitution Of H7N9 Avian Influenza Virus

In early 2013 in China,a novel H7N9 avian influenza virus crossed the species barrier and caused human infections.The results of genetic evolution analysis indicated that the H7N9 AIV is a triple reassortant virus,with all six internal genes being derived from H9N2 AIVs.To date,five epidemic waves of human infection with H7N9 viruses have caused 1568 cases,with a fatality rate of approximately 39%.Moreover,highly pathogenic H7N9 viruses possessing a multi-basic cleavage site motif in the viral hemagglutinin(HA)emerged in 2017,posing a major threat to both human public health and animal industry.Avian influenza viruses(AIVs)must acquire mammalian-adaptive mutations before they can efficiently replicate in and transmit among humans.The PB2 E627K mutation is known to play a prominent role in the mammalian adaptation of AIVs.The H7N9 AIVs emerged in 2013 in China easily acquired the PB2 E627K mutation upon replication in humans.Here,we generate a series of reassortant or mutant H7N9 AIVs and test them in mice.We show that the PA gene carried by H7N9 virus is responsible for driving the emergence of the PB2 E627K mutation in mice,and the low polymerase activity attributed to the viral PA protein is the intrinsic driving force behind the emergence of PB2 E627K during H7N9 AIV replication in mice.Four residues in the N-terminal of PA are critical in mediating the PB2 E627K acquisition.Notably,due to the identity of viral PA protein,the polymerase activity and growth of H7N9 AIV are highly sensitive to changes in expression levels of human ANP32A protein.Furthermore,the impaired viral polymerase activity of H7N9 AIV caused by the depletion of ANP32A led to reduced virus replication in Anp32c-/-.mice,abolishing the acquisition of PB2 E627K mutation and instead driving the virus to acquire the alternative PB2 D70IN mutation.Taken together,our findings show that the emergence of the PB2 E627K mutation of H7N9 AIV is driven by the intrinsic low polymerase activity conferred by the viral PA protein,which also involves the engagement of mammalian ANP32A.The study explained the molecular mechanism of the rapid acquisition of the viral PB2 E627K mutation in humans infected with H7N9 avian influenza virus,from both the "internal cause" of the virus itself and the "external cause" of the host.Our results deepened the understanding of the mechanism of cross-species transmission of avian influenza virus.