Effective inhibition of HCMV UL49 gene expression and viral replication by oligonucleotide external guide sequences and RNase P.
Effective inhibition of HCMV UL49 gene expression and viral replication by oligonucleotide external guide sequences and RNase P.
复制标题
寡核苷酸外部引导序列和RNase P有效抑制HCMV UL49基因表达和病毒复制
DOI：
10.1186/1743-422x-7-100
复制
发表时间：
2010-05-18
期刊：
影响因子：
4.8
通讯作者：
Zhou T
中科院分区：
文献类型：
--
作者：
Zhang W;Li H;Li Y;Zeng Z;Li S;Zhang X;Zou Y;Zhou T
背景人巨细胞病毒（human cytomegalovirus，HCMV）是一种普遍存在的疱疹病毒，在健康个体中通常引起无症状感染，但在新生儿和免疫缺陷个体中可能导致严重并发症。HCMV耐药株的出现提出了开发新药物和治疗策略的需求。反义分子是一种很有前途的基因靶向药物，可用于基因表达的特异性调控。外部引导序列（EGS）是由与靶mRNA互补的序列组成的寡核苷酸，并且募集细胞内RNA酶P用于靶RNA的特异性降解。HCMV的UL 49缺失BAC在人包皮成纤维细胞中的生长明显缺陷。因此，UL 49基因可能作为一个潜在的目标，为新的药物开发，以打击HCMV感染。本研究以人巨细胞病毒（human cytomegalovirus，HCMV）的UL 49 mRNA为靶点，合成了一系列以DNA为基础的EGS分子。结果体外切割活性测定表明，以UL 49 mRNA翻译起始密码子下游324 nt为靶点的EGS（EGS 324）能有效地引导人RNase P切割靶mRNA序列。当EGS 324被外源性地施用到HCMV感染的人包皮成纤维细胞（HFFs）中时，与用对照EGSs转染的细胞相比，UL 49基因的mRNA和蛋白质表达显著降低~76%和~80%。此外，约330倍的HCMV感染的HFFs中观察到的EGS.ConclusionsThese结果表明，UL 49基因是必不可少的HCMV复制的减少。此外，我们的研究提供的证据表明，外源性管理的DNA为基础的EGS可以用作一个潜在的治疗方法，抑制基因表达和复制的人病毒。
BackgroundHuman cytomegalovirus (HCMV) is a ubiquitous herpesvirus that typically causes asymptomatic infections in healthy individuals but may lead to serious complications in newborns and immunodeficient individuals. The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and treatment strategies. Antisense molecules are promising gene-targeting agents for specific regulation of gene expression. External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. The UL49-deletion BAC of HCMV was significantly defective in growth in human foreskin fibroblasts. Therefore, UL49 gene may serve as a potential target for novel drug development to combat HCMV infection. In this study, DNA-based EGS molecules were synthesized to target the UL49 mRNA of human cytomegalovirus (HCMV).ResultsBy cleavage activity assessingin vitro, the EGS aimed to the cleavage site 324 nt downstream from the translational initiation codon of UL49 mRNA (i.e. EGS324) was confirmed be efficient to direct human RNase P to cleave the target mRNA sequence. When EGS324 was exogenously administered into HCMV-infected human foreskin fibroblasts (HFFs), a significant reduction of ~76% in the mRNA and ~80% in the protein expression of UL49 gene, comparing with the cells transfected with control EGSs. Furthermore, a reduction of about 330-fold in HCMV growth were observed in HCMV-infected HFFs treated with the EGS.ConclusionsThese results indicated that UL49 gene was essential for replication of HCMV. Moreover, our study provides evidence that exogenous administration of a DNA-based EGS can be used as a potential therapeutic approach for inhibiting gene expression and replication of a human virus.