ALG5对猪流感病毒复制的影响

doi:10.11843/j.issn.0366-6964.2020.07.024

Abstract

Abstract: Earlier in the laboratory, we screened the swine influenza virus (SIV) replication-related host factor ALG5(dolichyl-phosphate beta-glucosyltransferase) through CRISPR/Cas9 genome-wide knockout library in pig kidney cells (PK-15-GeCKO). Under this premise, the relationship between ALG5 and SIV replication was studied in depth. In this study, a plasmid LentiGuide-puro-ALG5 containing ALG5 gene guide RNA (gRNA) was constructed by CRISPR/Cas9, and the newborn pig tracheal epithelial (NPTr) cell line stably expressing Cas9 protein was infected by lentiviral packaging. ALG5 knockout monoclonal cell line was screened by limiting dilution method. The knockout level of ALG5 gene on NPTr cells was confirmed by target genome sequencing and Western blot. The cell viability of knockout cells and wild-type cells was verified by CCK-8 experiment. The effects of ALG5 knockout and overexpression on SIV replication were examined in combination with TCID₅₀ and Western blot experiments. At the same time, the expression of endogenous ALG5 protein was detected after SIV infected NPTr cells. Finally, the effect of ALG5 knockout on replication of swine flu strain F26 was tested. NPTr monoclonal cell line that knocked out ALG5 (ΔALG5) was obtained, and there was no significant difference in cell activity between ALG5 knockout cells and wild-type cells. ALG5 gene knockout significantly inhibited SIV proliferation, and overexpression promoted SIV proliferation. Under viral infection conditions, the protein expression of ALG5 was up-regulated as the virus proliferated. ALG5 gene knockout can also inhibit the replication of swine flu strain F26, without strain specificity. This study indicates that ALG5 is an important host factor affecting the replication process of swine influenza virus. This study lays the foundation for studying the replication and pathogenic mechanism of swine influenza virus.

Key words: ALG5, CRISPR/Cas9, gene knockout, swine influenza virus

CLC Number:

S852.659.5

YANG Shuaike, ZOU Jiahui, JIANG Meijun, ZHAO Yaxin, CAO Jiyue, ZHOU Hongbo. Effect of ALG5 on Swine Influenza Virus Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1719-1727.

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Effect of ALG5 on Swine Influenza Virus Replication

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