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Screening and characterization of inhibitory vNAR targeting nanodisc-assembled influenza M2 proteins.

ABSTRACT: Influenza A virus poses a constant challenge to human health. The highly conserved influenza matrix-2 (M2) protein is an attractive target for the development of a universal antibody-based drug. However, screening using antigens with subphysiological conformation in a nonmembrane environment significantly reduces the generation of efficient antibodies. Here, M2(1-46) was incorporated into nanodiscs (M2-nanodiscs) with M2 in a membrane-embedded tetrameric conformation, closely resembling its natural physiological state in the influenza viral envelope. M2-nanodisc generation, an antigen, was followed by Chiloscyllium plagiosum immunization. The functional vNARs were selected by phage display panning strategy from the shark immune library. One of the isolated vNARs, AM2H10, could specifically bind to tetrameric M2 instead of monomeric M2e (the ectodomain of M2 protein). Furthermore, AM2H10 blocked ion influx through amantadine-sensitive and resistant M2 channels. Our findings indicated the possibility of developing functional shark nanobodies against various influenza viruses by targeting the M2 protein.

SUBMITTER: Yu C

PROVIDER: S-EPMC9771723 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Screening and characterization of inhibitory vNAR targeting nanodisc-assembled influenza M2 proteins.

Yu Chuandi C Ding Wen W Zhu Lei L Zhou Yuhang Y Dong Yingkui Y Li Ling L Liu Juanjuan J Wang Yizhuo Y Li Zehua Z Zhu Lina L Chen FaJun F Ruan Maosen M Qian Dongming D Wang Yujuan Y Wu Bo B Xu Huangtao H Li Ming M Bi Yunchen Y Wang Hao H Wang Weiqian W Chao Peng P Xing Lei L Shen Bing B Dai Han H Zha Lisha L Zhao Hongxin H Wang Junfeng J

iScience 20221205 1

Influenza A virus poses a constant challenge to human health. The highly conserved influenza matrix-2 (M2) protein is an attractive target for the development of a universal antibody-based drug. However, screening using antigens with subphysiological conformation in a nonmembrane environment significantly reduces the generation of efficient antibodies. Here, M2(1-46) was incorporated into nanodiscs (M2-nanodiscs) with M2 in a membrane-embedded tetrameric conformation, closely resembling its natu ...[more]

PMID: 36570769

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Project description:The influenza A virus (IAV) M2 protein has proton channel activity, which plays a role in virus uncoating and may help to preserve the metastable conformation of the IAV hemagglutinin (HA). In contrast to the highly conserved M2 proteins of conventional IAV, the primary sequences of bat IAV H17N10 and H18N11 M2 proteins show remarkable divergence, suggesting that these proteins may differ in their biological function. We, therefore, assessed the proton channel activity of bat IAV M2 proteins and investigated its role in virus replication. Here, we show that the M2 proteins of bat IAV did not fully protect acid-sensitive HA of classical IAV from low pH-induced conformational change, indicating low proton channel activity. Interestingly, the N31S substitution not only rendered bat IAV M2 proteins sensitive to inhibition by amantadine but also preserved the metastable conformation of acid-sensitive HA to a greater extent. In contrast, the acid-stable HA of H18N11 did not rely on such support by M2 protein. When mutant M2(N31S) protein was expressed in the context of chimeric H18N11/H5N1(6:2) encoding HA and NA of avian IAV H5N1, amantadine significantly inhibited virus entry, suggesting that ion channel activity supported virus uncoating. Finally, the cytoplasmic domain of the H18N11 M2 protein mediated rapid internalization of the protein from the plasma membrane leading to low-level expression at the cell surface. However, cell surface levels of H18N11 M2 protein were significantly enhanced in cells infected with the chimeric H18N11/H5N1(6:2) virus. The potential role of the N1 sialidase in arresting M2 internalization is discussed. IMPORTANCE Bat IAV M2 proteins not only differ from the homologous proteins of classical IAV by their divergent primary sequence but are also unable to preserve the metastable conformation of acid-sensitive HA, indicating low proton channel activity. This unusual feature may help to avoid M2-mediated cytotoxic effects and inflammation in bats infected with H17N10 or H18N11. Unlike classical M2 proteins, bat IAV M2 proteins with the N31S substitution mediated increased protection of HA from acid-induced conformational change. This remarkable gain of function may help to understand how single point mutations can modulate proton channel activity. In addition, the cytoplasmic domain was found to be responsible for the low cell surface expression level of bat IAV M2 proteins. Given that the M2 cytoplasmic domain of conventional IAV is well known to participate in virus assembly at the plasma membrane, this atypical feature might have consequences for bat IAV budding and egress.

Dataset Information

Screening and characterization of inhibitory vNAR targeting nanodisc-assembled influenza M2 proteins.

Publications

Screening and characterization of inhibitory vNAR targeting nanodisc-assembled influenza M2 proteins.

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