A unique feature of swine ANP32A provides susceptibility to avian influenza virus infection in pigs

Abstract

Both the replication and transcription of the influenza virus are catalyzed by the viral polymerase complex. The polymerases of most avian influenza A viruses have poor performance in mammalian cells, which is considered to be one of the important species barriers. Pigs have been long considered as important intermediate hosts for interspecies transmission of the avian influenza virus, because of their susceptibility to infection with both avian and mammalian influenza viruses. However, the molecular basis of influenza polymerase adaptation in pigs remains largely unknown. ANP32A and ANP32B proteins have been identified as playing fundamental roles in influenza virus replication and host range determination. In this study, we found that swine ANP32A (swANP32A), unlike swine ANP32B or other mammalian ANP32A or B, shows stronger supporting activity to avian viral polymerase. Knockout of ANP32A in pig cells PK15 dramatically reduced avian influenza polymerase activity and viral infectivity, suggesting a unique feature of swANP32A in supporting avian influenza viral polymerase. This species-specific activity is mapped to two key sites, 106V and 156S, in swANP32A. Interestingly, the amino acid 106V is unique to pigs among all the vertebrate species studied, and when combined with 156S, exhibits positive epistasis in pigs. Mutation of 106V and 156S to the signature found in ANP32As from other mammalian species weakened the interaction between swANP32A and chicken viral polymerase, and reduced polymerase activity. Understanding the molecular basis of ANP32 proteins may help to discover new antiviral targets and design avian influenza resistant genome edited pigs.

Swine ANP32A Supports Avian Influenza Virus Polymerase

“…A recent study from Zhang and colleagues independently corroborated the superior ability of swine ANP32A amongst mammalian ANP32 proteins to support avian influenza virus polymerase activity (29). Moreover, they also correlated this phenotype with amino acids at position 106 and 156 that increased the strength of interactions between the host factor and the viral polymerase complex.…”

Mammalian ANP32A and ANP32B proteins drive alternative avian influenza virus polymerase adaptations

“…In this study we showed that several different mutations in PB2 avian-origin influenza polymerases to use the shorter ANP32 proteins found in mammalian cells but that the most well-known of these PB2-E627K, strongly biases polymerases towards reliance on mammalian ANP32B. While ANP32A and ANP32B serve redundant proviral roles in many mammals, ANP32B is the dominant proviral factor in humans and mice whereas in most other relevant mammalian hosts, such as pigs, horses and dogs, ANP32A proteins is the more potent (Peacock et al, 2020, Zhang et al, 2020, Zhang et al, 2019. This pattern shapes the adaptive evolution of avian viruses in these different mammalian hosts.…”

“…These subtle variations are due to polymorphisms between the ANP32 orthologues in different species. For example, the potent pro-viral activity of swine ANP32A is due to polymorphisms at positions 106 and 156 (Peacock et al, 2020, Zhang et al, 2020. Similarly, the weak pro-viral activity of dog (as well as bat and seal) ANP32B is attributed to residue 153 that is glutamine in the human ANP32B, but arginine in the canine orthologue (Supplementary Figure S3A-C)…”

“…Incursions of avian influenza viruses into pigs rarely showed adaptation at any of these three sites ( Figure 4A, right panel). This might be explained by the observation that swine ANP32A is somewhat supportive of non-adapted avian influenza virus polymerases (Peacock et al, 2020, Zhang et al, 2020. It is also noteworthy that the number of viruses with multiple ANP32-specific PB2 adaptations simultaneously is very low, suggesting these mutations are partially redundant (although the more poorly adaptive Q591R/K and D701N mutations were occasionally found together).…”

“…One exception is mice in which only ANP32B can efficiently support influenza polymerase due to a polymorphism in murine ANP32A at position 130 -a residue critical for the interaction between ANP32 proteins and viral polymerase (Beck, Zickler et al, 2020, Zhang et al, 2019. It has recently been shown that, although both swine ANP32A and ANP32B can support mammalian-adapted polymerases, swine ANP32A has the more potent pro-viral function and can even partially support avian polymerases (Peacock et al, 2020, Zhang, Li et al, 2020. Similarly in horses, dogs, seals and bats, ANP32A appears to be a more potent pro-viral factor than ANP32B (Peacock et al, 2020).…”