蛋白酶体抑制剂PS-341抑制人肠道病毒D68复制研究

Proteasome inhibitor bortezomib inhibits replication of Enterovirus D68

目的探究蛋白酶体抑制剂PS-341对肠道病毒属病毒复制的抑制作用。方法利用MTT实验分析药物毒性。利用实时荧光定量PCR技术,从核酸水平检测PS-341处理对宿主细胞内肠道病毒 D68 型(enterovirus D68 ,EV-D68)、柯萨奇 B3 型(coxsackieviros B3 , CV-B3 )复制的影响。通过Western blot检测细胞内病毒蛋白表达水平。结果 PS-341处理EV-D68或CV-B3感染后的细胞,细胞内病毒RNA含量分别被抑制50%?70%和60%~90%。EV-D68感染细胞后,加入PS-341, RD细胞内病毒滴度下调90.23%,上清中病毒滴度下调83.4%,HeLa细胞内病毒滴度下调93.08%,上清中病毒滴度下调90%。但PS-341对病毒吸附、进入及释放无影响。当PS-341与凋亡抑制剂Ac-YVADCHO共处理EV-D68感染的细胞,与PS-341处理组相比,PS-341对病毒RNA复制抑制率为10%~30%,同时病毒蛋白表达水平提高,PS-341对病毒复制抑制作用减弱。结论 PS-341能够抑制肠道病毒属病毒在细胞内复制及组装,但对病毒吸附、进入及释放无影响。PS-341可能通过调节蛋白酶体通路,诱导细胞凋亡,抑制EV-D68在宿主体内的基因复制抑制病毒增殖。此外,PS-341对小RNA病毒科肠道病毒属病毒可能具有广谱抗病毒作用。

Objective To investigate the inhibitory effect of bortezomib ( PS-341 ) on enterovirus replication. Methods The methyl thiazolyl tetrazolium ( MTT) assay was used to value cell viability in response to PS-341 treatment. The protein and viral gene mRNAs were measured by real-time quantitative PCR ( qRT-PCR). Results Our result show that after enterovirus ( EV)-D68 or coxsackievirus B3 ( CVB3) infected cells were treated with PS-341 , compared with the control group, the inhibition rate of the intracellular viral RNA reached 50%~ 70% or 60%~ 90%. PS-341 was added after RD cells were infectd with EV-D68, the intracellular virus titer was down-regulated by 90. 23% and 83. 40% in the supernatant, the intracellular virus titer was down-regulated by 93% and 90% in the supernatant and in RD cells. PS-341 had no effect on virus adsorption and importing. The cells were treated with PS-341 and apoptosis-inhibiting agent Ac-YVAD-CHO, the viral RNA replication inhibition rate reached 10%-30%, and the expression of viral protein was increased, which indicated that the inhibitory effect of PS-341 on viral replication wasattenuated. Conclusions According to the result of the study, PS-341 could reduce apoptosis by regulating the proteasome pathway, inhibiting the gene replication and assemble, without effect on virus adsorption, entry and release. In addition, PS-341 also inhibited the replication of CV-B3 in cells, which suggest that PS?341 has a broad spectrum anti-EVs effects.