Sec62/Sec63复合物对乙型脑炎病毒在HEK-293细胞中复制影响的研究

Effection of knocking out SEC62,SEC63 gene of HEK-293 cells on replication of Japanese encephalitis virus

Sec62/Sec63是位于内质网膜上的跨膜蛋白复合物,辅助新合成的前体蛋白或新生多肽链转运至内质网,进行后期的折叠加工。为研究Sec62/Sec63复合物对乙型脑炎病毒(JEV)复制的影响,本研究利用CRISPR/Cas9基因编辑技术分别构建SEC62、SEC63基因敲除的HEK-293细胞系,经测序鉴定以及western blot检测,获得两个基因敲除细胞系,分别命名为SEC62 KO HEK-293细胞和SEC63 KO HEK-293细胞。分别以MOI为1、0.05的JEV感染HEK-293、SEC62 KO HEK-293、SEC63 KO HEK-293细胞后,利用间接免疫荧光试验检测Sec62/Sec63复合物对JEV复制的影响。结果显示,不同剂量JEV感染SEC62 KO HEK-293细胞与SEC63 KO HEK-293细胞中荧光信号阳性细胞均少于HEK-293细胞对照组;以MOI为1的JEV感染HEK-293、SEC62 KO HEK-293、SEC63 KO HEK-293细胞后,经western blot检测,结果显示于感染后12 h、24 h和48 h,SEC62 KO HEK-293、SEC63 KO HEK-293细胞中均未检测出JEV E蛋白条带,而对照HEK-293细胞中JEV E蛋白的表达量逐渐增加。以MOI为1的JEV感染上述3种细胞后检测细胞培养液中病毒滴度,结果显示JEV感染12 h后SEC62 KO HEK-293、SEC63 KO HEK-293细胞与HEK-293细胞病毒滴度无差异(p>0.05),而感染后24 h与48 h时,SEC62 KO HEK-293、SEC63 KO HEK-293细胞培养液中病毒滴度均显著低于HEK-293细胞对照组(p<0.01)。以MOI为1、0.5、0.05的JEV感染上述3种细胞后检测48 h后细胞培养液中病毒滴度,结果显示SEC62 KO HEK-293、SEC63 KO HEK-293细胞培养液中病毒滴度均显著低于HEK-293细胞对照组(p<0.001)。以上结果表明,SEC62、SEC63基因敲除后均抑制了JEV的复制,也表明Sec62、Sec63是影响JEV在细胞内复制的重要宿主因子。本研究为进一步探究Sec62、Sec63等宿主因子对JEV复制的影响机制提供了参考依据。

The Sec62/Sec63 complex is a transmembrane protein complex locates at the endoplasmic reticulum.The complex mediates the transport of newly synthesized polypeptides for later folding process.In order to study the effect of Sec62/Sec63 complex on the replication of JEV(Japanese encephalitis virus,JEV),SEC62 or SEC63 gene knock-out HEK-293 cells were prepared by CRISPR/Cas9 genome editing technology.Knock-out was verified by genomic sequencing and western blot and the resultant cells were named as SEC62 KO HEK-293 and SEC63 KO HEK-293 cells respectively.The efficiency of JEV replication was determined by indirect immunofluorescence test in JEV infected HEK-293,SEC62 KO HEK-293 and SEC63 KO HEK-293 cells at a MOI of 1 or 0.05.Results showed that fluorescence positive cells in JEV infected SEC62 KO HEK-293 cells and SEC63 KO HEK-293 cells were lower than that of HEK-293 cells.Western blot results showed that the expression of E protein in JEV infected SEC62 KO HEK-293 and SEC63 KO HEK-293 cells(MOI=1)could not be detected at 12h,24h or 48h,while the expression of JEV E protein in HEK-293 increased.Virus titers were determined after JEV infection of the above three cell lines by JEV at MOI=1.Results showed that there were no significant differences in virus titers between SEC62 KO HEK-293,SEC63 KO HEK-293 cells and HEK-293 cells at 12h after infection(p>0.05),while the differences were significant at 24h and 48h after infection.Virus titers in supernatant of JEV infected SEC62 KO HEK-293 and SEC63 KO HEK-293 cells were significantly lower than that of HEK-293 cells(p<0.01).Virus titers at 48h were determined after infecting the above three cell lines by JEV on MOI=1,0.5 and 0.05.The results showed that virus titers in supernatant of SEC62 KO HEK-293 and SEC63 KO HEK-293 cells were significantly lower than those in HEK-293 cells(p<0.001).These results showed that knock-out of SEC62 or SEC63 gene decreased the replication of JEV in HEK-293 cells.Our results demonstrate Sec62/Sec63 complex is a host factor required for JEV infection in vitro.