Identification of two mutation sites in spike and envelope proteins mediating optimal cellular infection of porcine epidemic diarrhea virus from different pathways
2017
Sun, Min | Ma, Jiale | Yu, Zeyanqiu | Pan, Zihao | Lu, Chengping | Yao, Huochun
Entry of the α-coronavirus porcine epidemic diarrhea virus (PEDV) requires specific proteases to activate spike (S) protein for the membrane fusion of the virion to the host cell following receptor binding. Herein, PEDV isolate 85-7 could proliferate and induce cell–cell fusion in a trypsin independent manner on Vero cells, and eight homologous mutation strains were screened by continuous proliferation in the absence of trypsin on Vero cells. According to the whole genome sequence comparative analysis, we identified four major variations located in nonstructural protein 2, S, open reading frame 3, and envelope (E) genes, respectively. Comparative analyses of their genomic variations and proliferation characteristics identified a single mutation within the S2′ cleavage site between C30 and C40 mutants: the substitution of conserved arginine (R) by a glycine (G) (R895G). This change resulted in weaker cell–cell fusion, smaller plaque morphology, higher virus titer and serious microfilament condensation. Further analysis confirmed that this mutation was responsible for optimal cell-adaptation, but not the determinant for trypsin-dependent entry of PEDV. Otherwise, a novel variation (16–20 aa deletion and an L25P mutation) in the transmembrane domain of the E protein affected multiple infection processes, including up-regulation of the production of the ER stress indicator GRP78, improving the expression of pro-inflammatory cytokines IL-6 and IL-8, and promoting apoptosis. The results of this study provide a better understanding of the potential mechanisms of viral functional proteins in PEDV replication, infection, and fitness.
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