Danqi Bao,a Chenyang Lu,a,b Tianxin Ma,a Guanlong Xu,c Yaqing Mao,c Lingxiang Xin,c Shiqi Niu,a Zihua Wu,a Xuesong Li,aQiaoyang Teng,a Zejun Li,a Qinfang Liuaa
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, ChinabGuangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Guangxi Province, People’s Republic of ChinacChina Institute of Veterinary Drug Control, Beijing, People’s Republic of China
Journal of Virology，May 2022 Volume 96 Issue 9
ABSTRACT M2 protein of influenza virus plays an important role in
virus budding,including membrane scission and vRNP packaging. Three hydrophobic
amino acids (91F,92V, and 94I) at the intracellular domain of the M2 protein
constitute a hydrophobicmotif, also known as the LC3-interacting region (LIR),
whereas the role of this motif remains largely unclear. To explore the role of
the 91–94 hydrophobic motif for influenza virus, all three
hydrophobic amino acids were mutated to either hydrophilic S or hydrophobic A,
resulting in two mutant viruses (WSN-M2/SSS and WSN-M2/AAA) in the background
of WSN/H1N1. The results showed that the budding ability of the M2/SSS protein
was inhibited and the bilayer membrane integrity of the WSN-M2/SSS virion was impaired
based on transmission electron microscopy (TEM), which in turn abolished the resistance
to trypsin treatment. Moreover, the mutant WSN-M2/SSS was dramatically attenuated
in mice. In contrast, the AAA mutations did not have a significant effect on the
budding of the M2 proteins or the bilayer membrane integrity of the viruses,
and the mutant WSN-M2/AAA was still lethal to mice. In addition, although the
91–94 motif is an LIR, knocking out of the LC3 protein of
A549 cells did not significantly affect the membrane integrity of the influenza
viruses propagated on the LC3KO cells, which suggested that the 91–94 hydrophobic motif affected
the viral membrane integrity and budding is independent of the LC3 protein.
Overall, the hydrophobicity of the 91–94 motif is crucial for the
budding of M2, bilayer membrane integrity, and pathogenicity of the influenza
viruses.
KEYWORDS matrix protein 2, budding, hydrophobicity, membrane
integrity,LC3-interacting region