Sun Xu, Chen Weijie, He Lingling, Sheng Jingxue, Liu Yujun, Vu Gia-Phong, Yang Zhu, Li Wei, Trang Phong, Wang Yu, Hai Rong, Zhu Hua, Lu Sangwei, Liu Fenyong
College of Life Science and Technology, Jinan University, Guangzhou, Guangdong, China.
School of Public Health, University of California, Berkeley, CA, United States of America.
PLoS One. 2017 Oct 23;12(10):e0186791. doi: 10.1371/journal.pone.0186791. eCollection 2017.
We have previously engineered new RNase P-based ribozyme variants with improved in vitro catalytic activity. In this study, we employed a novel engineered variant to target a shared mRNA region of human cytomegalovirus (HCMV) immediate early proteins 1 (IE1) and 2 (IE2), which are essential for the expression of viral early and late genes as well as viral growth. Ribozyme F-R228-IE represents a novel variant that possesses three unique base substitution point mutations at the catalytic domain of RNase P catalytic RNA. Compared to F-M1-IE that is the ribozyme derived from the wild type RNase P catalytic RNA sequence, the functional variant F-R228-IE cleaved the target mRNA sequence in vitro at least 100 times more efficiently. In cultured cells, expression of F-R228-IE resulted in IE1/IE2 expression reduction by 98-99% and in HCMV production reduction by 50,000 folds. In contrast, expression of F-M1-IE resulted in IE1/IE2 expression reduction by less than 80% and in viral production reduction by 200 folds. Studies of the ribozyme-mediated antiviral effects in cultured cells suggest that overall viral early and late gene expression and viral growth were inhibited due to the ribozyme-mediated reduction of HCMV IE1 and IE2 expression. Our results provide direct evidence that engineered RNase P ribozymes, such as F-R228-IE, can serve as a novel class of inhibitors for the treatment and prevention of HCMV infection. Moreover, these results suggest that F-R228-IE, with novel and unique mutations at the catalytic domain to enhance ribozyme activity, can be a candidate for the construction of effective agents for anti-HCMV therapy.
我们之前构建了基于核糖核酸酶P(RNase P)的新型核酶变体,其体外催化活性有所提高。在本研究中,我们采用了一种新型工程变体来靶向人巨细胞病毒(HCMV)即刻早期蛋白1(IE1)和2(IE2)的共享mRNA区域,这两种蛋白对于病毒早期和晚期基因的表达以及病毒生长至关重要。核酶F-R228-IE是一种新型变体,在RNase P催化RNA的催化结构域具有三个独特的碱基替代点突变。与源自野生型RNase P催化RNA序列的核酶F-M1-IE相比,功能性变体F-R228-IE在体外切割靶mRNA序列的效率至少高100倍。在培养细胞中,F-R228-IE的表达导致IE1/IE2表达降低98 - 99%,HCMV产生减少50000倍。相比之下,F-M1-IE的表达导致IE1/IE2表达降低不到80%,病毒产生减少200倍。对培养细胞中核酶介导的抗病毒作用的研究表明,由于核酶介导的HCMV IE1和IE2表达降低,总体病毒早期和晚期基因表达以及病毒生长受到抑制。我们的结果提供了直接证据,即工程化的RNase P核酶,如F-R228-IE,可作为治疗和预防HCMV感染的新型抑制剂。此外,这些结果表明,在催化结构域具有新颖独特突变以增强核酶活性的F-R228-IE,可作为构建抗HCMV治疗有效药物的候选物。