Engineering of highly active gene targeting RNase P ribozyme against human cytomegalovirus infection

hLife Pub Date : 2025-05-01 DOI:10.1016/j.hlife.2025.02.006
Yujun Liu , Bin Yan , Isadora Zhang , Fenyong Liu
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Abstract

Sequence-specific ribonuclease (RNase) P ribozymes can be engineered in vitro and are promising gene-targeting agents to knock down gene expression. In this study, we applied an RNase P ribozyme variant to hydrolyze the mRNA of human cytomegalovirus (HCMV) major capsid protein (MCP), which is necessary for viral capsid formation and growth. Functional variant R668-F was about 100 times more efficient in slicing the MCP mRNA in vitro than M1-F, the ribozyme with a natural RNase P ribozyme sequence. In R668-F-expressing cells, a decrease of about 98%–99% in the expression of MCP was detected, and the virus production was reduced by 70,000 folds. However, the expression of inactive control ribozymes in cells resulted in a less than 10% decrease in MCP expression and no apparent decrease in HCMV growth. In cells observed with the ribozyme-mediated reduction of MCP expression, HCMV capsid formation and virus growth were inhibited and the expressions of other viral genes were unaffected. These findings provide the first direct evidence that ribozyme R668-F specifically inhibits MCP expression and blocks HCMV growth. Our results further suggest that the engineered RNase P ribozymes, including R668-F, may act as a novel general gene-targeting strategy to treat infections of viruses, including HCMV.
靶向RNase P核酶的高活性基因工程抗人巨细胞病毒感染
序列特异性核糖核酸酶(RNase) P核酶可以在体外工程化,是很有前途的基因靶向药物,可以降低基因表达。在这项研究中,我们利用RNase P核酶变体水解了人巨细胞病毒(HCMV)主要衣壳蛋白(MCP)的mRNA,这是病毒衣壳形成和生长所必需的。功能变体R668-F在体外切割MCP mRNA的效率是具有天然RNase - P核酶序列的核酶M1-F的100倍左右。在表达r668 - f的细胞中,检测到MCP的表达减少了约98%-99%,病毒产量减少了70,000倍。然而,在细胞中表达无活性的对照核酶导致MCP表达下降不到10%,HCMV生长没有明显下降。在核酶介导的MCP表达降低的细胞中,HCMV衣壳形成和病毒生长受到抑制,其他病毒基因的表达不受影响。这些发现为核酶R668-F特异性抑制MCP表达和阻断HCMV生长提供了第一个直接证据。我们的研究结果进一步表明,工程化的RNase P核酶,包括R668-F,可能作为一种新的通用基因靶向策略来治疗病毒感染,包括HCMV。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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