小干扰 RNA 靶向感染细胞多肽 4 抑制视网膜色素上皮细胞中单纯疱疹病毒 1 复制。
Small interfering RNA targeting for infected-cell polypeptide 4 inhibits herpes simplex virus type 1 replication in retinal pigment epithelial cells.
机构信息
Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, Guangdong, China.
出版信息
Clin Exp Ophthalmol. 2012 Mar;40(2):195-204. doi: 10.1111/j.1442-9071.2011.02668.x. Epub 2011 Oct 20.
BACKGROUND
This study sought to inhibit herpes simplex virus type 1 replication using small interfering RNA which targeting infected-cell polypeptide 4 genes to mediate transcription of early and late viral genes in herpes simplex virus type 1 lytic (productive) infection in retina epithelial cells.
METHODS
After pre- or post-infecting with herpes simplex virus type 1, small interfering RNAs were transfected into retina epithelial cells. The antiviral effects of small interfering RNA were evaluated by Western blot, plaque assays, indirect immunofluorescence and reverse transcription polymerase chain reaction. The viral titre was detected by the 50% tissue culture infective dose method.
RESULTS
Small interfering RNA decreased infected-cell polypeptide 4 expression in retina epithelial cells that were infected with herpes simplex virus type 1 before or after small interfering RNA transfection. Compared with herpes simplex virus type 1 infection alone or transfection with negative control small interfering RNA, the viral titre and the retina epithelial cell cytopathic effect were significantly decreased in retina epithelial cells transfected with infected-cell polypeptide 4-targeting small interfering RNA (50 and 100nM) (P<0.05). The small interfering RNA effectively silenced herpes simplex virus type 1 infected-cell polypeptide 4 expression on both mRNA and the protein levels (P<0.05). The inhibition of infected-cell polypeptide 4-targeting small interfering RNA on infected-cell polypeptide 4 protein expression was also verified by Western blot in herpes simplex virus type 1 infected human cornea epithelial cell, human trabecular meshwork cells and Vero cells.
CONCLUSIONS
Infected-cell polypeptide 4-targeting small interfering RNA can inhibit herpes simplex virus type 1 replication in retina epithelial cells, providing a foundation for development of RNA interference as an antiviral therapy.
背景
本研究旨在通过靶向感染细胞多肽 4 基因的小干扰 RNA 抑制单纯疱疹病毒 1 复制，以介导单纯疱疹病毒 1 裂解（产毒）感染中早期和晚期病毒基因的转录。
方法
在感染单纯疱疹病毒 1 之前或之后，将小干扰 RNA 转染到视网膜上皮细胞中。通过 Western blot、噬斑试验、间接免疫荧光和逆转录聚合酶链反应评估小干扰 RNA 的抗病毒作用。通过 50%组织培养感染剂量法检测病毒滴度。
结果
小干扰 RNA 降低了感染单纯疱疹病毒 1 之前或之后转染的视网膜上皮细胞中感染细胞多肽 4 的表达。与单纯疱疹病毒 1 感染或转染阴性对照小干扰 RNA 相比，转染感染细胞多肽 4 靶向小干扰 RNA（50 和 100nM）的视网膜上皮细胞中病毒滴度和视网膜上皮细胞细胞病变效应明显降低（P<0.05）。小干扰 RNA 有效地沉默了感染细胞多肽 4 靶向小干扰 RNA 在 mRNA 和蛋白水平上对单纯疱疹病毒 1 感染细胞多肽 4 的表达（P<0.05）。在感染单纯疱疹病毒 1 的人角膜上皮细胞、人小梁网细胞和 Vero 细胞中，Western blot 也验证了感染细胞多肽 4 靶向小干扰 RNA 对感染细胞多肽 4 蛋白表达的抑制作用。
结论
感染细胞多肽 4 靶向小干扰 RNA 可抑制视网膜上皮细胞中单纯疱疹病毒 1 的复制，为 RNA 干扰作为抗病毒治疗提供了基础。
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