Trypsin- and low pH-mediated fusogenicity of avian metapneumovirus fusion proteins is determined by residues at positions 100, 101 and 294.

Bingling Yun,Xiaolu Guan,Honglei Gao,Yongqiang Wang,Yanni Gao,Li Gao,Yulong Gao,Kai Li,Changjun Liu,Hongyu Cui,Yongzhen Liu,Yanping Zhang,Xiaomei Wang,Xiaole Qi

doi:10.1038/srep15584

Abstract

Avian metapneumovirus (aMPV) and human metapneumovirus (hMPV) are members of the genus Metapneumovirus in the subfamily Pneumovirinae. Metapneumovirus fusion (F) protein mediates the fusion of host cells with the virus membrane for infection. Trypsin- and/or low pH-induced membrane fusion is a strain-dependent phenomenon for hMPV. Here, we demonstrated that three subtypes of aMPV (aMPV/A, aMPV/B, and aMPV/C) F proteins promoted cell-cell fusion in the absence of trypsin. Indeed, in the presence of trypsin, only aMPV/C F protein fusogenicity was enhanced. Mutagenesis of the amino acids at position 100 and/or 101, located at a putative cleavage region in aMPV F proteins, revealed that the trypsin-mediated fusogenicity of aMPV F proteins is regulated by the residues at positions 100 and 101. Moreover, we demonstrated that aMPV/A and aMPV/B F proteins mediated cell-cell fusion independent of low pH, whereas the aMPV/C F protein did not. Mutagenesis of the residue at position 294 in the aMPV/A, aMPV/B, and aMPV/C F proteins showed that 294G played a critical role in F protein-mediated fusion under low pH conditions. These findings on aMPV F protein-induced cell-cell fusion provide new insights into the molecular mechanisms underlying membrane fusion and pathogenesis of aMPV.

Highlights

Avian metapneumovirus and human metapneumovirus are members of the genus Metapneumovirus in the subfamily Pneumovirinae
The syncytium induced through Avian metapneumovirus (aMPV)/C F protein after trypsin treatment increased in number and size (Fig. 1a), but these differences were not observed in cells expressing aMPV strains LAH A strain (aMPV/A) and aMPV/B F proteins
We further investigated the impact of trypsin on the fusogenic activity of aMPV F proteins using a quantitative assay, and the addition of trypsin did not result in a notable difference in aMPV/A or aMPV/B F protein fusogenicity compared with the corresponding samples that did not receive trypsin treatment (Fig. 1b,c)

Summary

Introduction

Avian metapneumovirus (aMPV) and human metapneumovirus (hMPV) are members of the genus Metapneumovirus in the subfamily Pneumovirinae. Mutagenesis of the residue at position 294 in the aMPV/A, aMPV/B, and aMPV/C F proteins showed that 294G played a critical role in F protein-mediated fusion under low pH conditions. These findings on aMPV F proteininduced cell-cell fusion provide new insights into the molecular mechanisms underlying membrane fusion and pathogenesis of aMPV. Schickli et al demonstrated that the amino acid substitution S101P in the putative cleavage motif of hMPV F protein is a major determinant for trypsin-independent growth in Vero cells, and the S101P mutant can be cleaved in the absence of trypsin[19]. Wei Y et al reported that trypsin is required for the induction of aMPV/B F protein-mediated cell-cell fusion, but this enzyme is not crucial for the fusion mediated through aMPV/A and aMPV/C F proteins[18]

Methods

Results

Conclusion