Asp149 and Asp152 in chicken and human ANP32A play an essential role in the interaction with influenza viral polymerase

Abstract

The acidic nuclear phosphoprotein 32 family member A (ANP32A) is a cellular host factor that determines the host tropism of the viral polymerase (vPol) of avian influenza viruses (AIVs). Compared with human ANP32A (hANP32A), chicken ANP32A contains an additional 33 amino acid residues (176-208) duplicated from amino acid residues 149-175 (27 residues), suggesting that these residues could be involved in increasing vPol activity by strengthening interactions between ANP32A and vPol. However, the molecular interactions and functional roles of the 27 residues within hANP32A during AIV vPol activity remain unclear. Here, we examined the functional role of 27 residues of hANP32A based on comparisons with other human (h) ANP32 family members. It was notable that unlike hANP32A and hANP32B, hANP32C could not support vPol activity or replication of AIVs, despite the fact that hANP32C shares a higher sequence identity with hANP32A than hANP32B.Pairwise comparison between hANP32A and hANP32C revealed that Asp149 (D149) and Asp152 (D152) are involved in hydrogen bonding and electrostatic interactions, respectively, which support vPol activity. Mutation of these residues reduced the interaction between hANP32A and vPol. Finally, we demonstrated that precise substitution of the identified residues within chicken ANP32A via homology-directed repair using the CRISPR/Cas9 system resulted in a marked reduction of viral replication in chicken cells. These results increase our understanding of ANP32A function 2 of 11 | PARK et Al.

Structures of influenza A and B replication complexes explain avian to human host adaption and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase

“…Consistently, hANP32A/K153E and PA/E493K individually decreased FluPol-binding to hANP32A, but showed increased binding-levels when tested in combination (Figure S4E). This is in line with the interactions shown in Figure 2C,D and with previous results showing that this region of hANP32A is critical for its interactions with the polymerase (Park et al, 2021).…”

“…showing that this region of hANP32A is critical for its interactions with the polymerase (Park et al, 2021).…”

Creating resistance to avian influenza infection through genome editing of the ANP32 gene family

“…One of the polymerase substitutions, PA-T639I, is located in a region of PA in the encapsidating polymerase opposite LRR5 but is not a contact residue with amino acids 129 or 130 in the ICV complex. Another of the PA substitutions, at amino acid 556 is also situated in the encapsidating polymerase in the vicinity of the central region of ANP32A essential for supporting polymerase activity 10 , 26 . The PB2 residue 631 is located close to the prototypic host-range determining residue 627 and within the PB2-627 domain which is thought to interact with the unstructured LCAR of ANP32A 27 .…”

Strategies for the Generation of Gene Modified Avian Models: Advancement in Avian Germline Transmission, Genome Editing, and Applications

“…The 99 nucleotides of chicken ANP32A encoding the additional 33 amino acids in birds have been deleted in chicken DF1 cells and viral polymerase (vPol) activity was significantly reduced [ 162 , 170 , 171 ]. Recently, the critical residues (aspartate 149 and 152) for interactions with viral protein in the additional 33 amino acids of chicken ANP32A were revealed [ 172 ]. In addition, chickens lost Retinoic inducible gene 1 ( RIG-I ), a major IAV RNA sensor in mammals; they only harbor Melanoma differentiation associated protein 5 ( MDA5 ), a member of RIG-I like receptor (RLR) family [ 173 ].…”