Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
J Virol. 2012 May;86(10):5660-73. doi: 10.1128/JVI.06338-11. Epub 2012 Mar 21.
Viral drug toxicity, resistance, and an increasing immunosuppressed population warrant continued research into new avenues for limiting diseases associated with human cytomegalovirus (HCMV). In this study, a small interfering RNA (siRNA), siX3, was designed to target coding sequences within shared exon 3 of UL123 and UL122 transcripts encoding IE1 and IE2 immediate-early proteins of HCMV. Pretreatment of cells with siX3 reduced the levels of viral protein expression, DNA replication, and progeny virus production compared to control siRNA. Two siRNAs against UL54 and overlapping transcripts (UL55-57) were compared to siX3 in HCMV infection and were also found to be effective at inhibiting HCMV replication. Further investigation into the effects of the siRNAs on viral replication showed that pretreatment with each of the siRNAs resulted in an inhibition in the formation of mature replication compartments. The ability of these siRNAs to prevent or reduce certain cytopathic effects associated with HCMV infection was also examined. Infected cells pretreated with siX3, but not siUL54, retained promyelocytic leukemia (PML) protein in cellular PML bodies, an essential component of this host intrinsic antiviral defense. DNA damage response proteins, which are localized in nuclear viral replication compartments, were reduced in the siX3- and siUL54-treated cells. siX3, but not siUL54, prevented DNA damage response signaling early after infection. Therapeutic efficacy was demonstrated by treating cells with siRNAs after HCMV replication had commenced. Together, these findings suggest that siRNAs targeting exon 3 of the major IE genes or the UL54-57 transcripts be further studied for their potential development into anti-HCMV therapeutics.
病毒药物毒性、耐药性以及不断增加的免疫抑制人群，都要求我们继续研究新的方法，以限制与人类巨细胞病毒（HCMV）相关的疾病。在这项研究中，设计了一种小干扰 RNA（siRNA），即 siX3，针对 UL123 和 UL122 转录物中共同外显子 3 的编码序列，这些转录物编码 HCMV 的 IE1 和 IE2 即刻早期蛋白。与对照 siRNA 相比，细胞预处理 siX3 可降低病毒蛋白表达、DNA 复制和后代病毒产生的水平。在 HCMV 感染中，比较了针对 UL54 和重叠转录物（UL55-57）的两种 siRNA 与 siX3 的作用，发现它们也能有效地抑制 HCMV 复制。进一步研究这些 siRNA 对病毒复制的影响表明，每种 siRNA 的预处理都会导致成熟复制隔间的形成受到抑制。还检查了这些 siRNA 预防或减少与 HCMV 感染相关的某些细胞病变效应的能力。用 siX3 预处理而不是 siUL54 预处理的感染细胞保留了细胞 PML 体中的早幼粒细胞白血病（PML）蛋白，这是这种宿主固有抗病毒防御的重要组成部分。定位在核病毒复制隔室中的 DNA 损伤反应蛋白在 siX3 和 siUL54 处理的细胞中减少。只有 siX3，而不是 siUL54，可在感染后早期阻止 DNA 损伤反应信号的传递。在 HCMV 复制开始后用 siRNA 处理细胞，证明了治疗功效。综上所述，针对主要 IE 基因的外显子 3 或 UL54-57 转录物的 siRNA 可能会进一步研究，以开发其作为抗 HCMV 治疗药物的潜力。
