Human cytomegalovirus clinical strain-specific microRNA miR-UL148D targets the human chemokine RANTES during infection.
Human cytomegalovirus clinical strain-specific microRNA miR-UL148D targets the human chemokine RANTES during infection.
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DOI：
10.1371/journal.ppat.1002577
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Ahn K
中科院分区：
文献类型：
--
作者：
Kim Y;Lee S;Kim S;Kim D;Ahn JH;Ahn K
人巨细胞病毒（HCMV）临床株Toledo和减毒株AD 169在致病力和细胞嗜性方面存在显著差异。毒力托莱多基因组含有一个15 kb的片段，它存在于所有毒力菌株中，但不存在于AD 169基因组中。这两种菌株之间的致病性差异被认为与这一额外的基因组片段有关。在病毒感染期间诱导的细胞因子在涉及免疫和炎症系统的细胞的细胞相互作用的调节中起主要作用，并因此决定感染的致病结果。趋化因子RANTES（调节活化，正常T细胞表达和分泌）在炎症和免疫应答期间吸引免疫细胞，表明RANTES在病毒发病机制中的作用。在这里，我们表明，RANTES下调人包皮成纤维细胞（HFF）细胞在感染后的后期阶段的托莱多株，但不是感染后的AD 169株。miR-UL 148 D是根据Toledo基因组的UL/B'序列预测的唯一一种miRNA，其靶向RANTES的3′-非翻译区，并在感染过程中诱导RANTES mRNA的降解。虽然野生型Toledo抑制HFF细胞中RANTES的表达，但其中miR-UL 148 D被特异性废除的Toledo突变体病毒不抑制RANTES表达。此外，miR-UL 148 D介导的RANTES下调被miR-UL 148 D特异性抑制剂抑制，该抑制剂设计为通过反义机制结合miR-UL 148 D序列，支持反义药物作为针对HCMV的治疗工具的潜在价值。我们的研究结果确定了一种病毒microRNA作为趋化因子RANTES的一种新的负调节因子，并为理解HCMV临床株的发病机制提供了线索。与减毒HCMV毒株AD 169不同，HCMV的临床分离株，包括托莱多毒株，是有毒的，可以在健康成人中引起疾病。Toledo与AD 169的不同之处在于Toledo包含一个15 kb的DNA片段，编码至少19个ORF和一个称为miR-UL 148 D的microRNA。该15 kb片段被认为是托莱多临床菌株毒力和致病性的主要决定因素。CC趋化因子RANTES在病毒感染期间招募免疫细胞，表明它可能在病毒相关疾病中发挥作用。在这里，我们表明，RANTES mRNA降解人包皮成纤维细胞在感染托莱多，但不是在感染AD 169。RANTES mRNA的降解由miR-UL 148 D介导，miR-UL 148 D是从Toledo基因组的15-kb片段预测的唯一病毒microRNA。因此，在用其中miR-UL 148 D缺失的ToledoΔ miR-UL 148 D感染的细胞中分泌的RANTES水平高于用Toledo感染的细胞中分泌的RANTES水平。我们的研究结果表明，病毒microRNA可能是一种新的潜在治疗靶点，并为了解临床和减毒株之间致病潜力的差异提供了重要的见解。
The human cytomegalovirus (HCMV) clinical strain Toledo and the attenuated strain AD169 exhibit a striking difference in pathogenic potential and cell tropism. The virulent Toledo genome contains a 15-kb segment, which is present in all virulent strains but is absent from the AD169 genome. The pathogenic differences between the 2 strains are thought to be associated with this additional genome segment. Cytokines induced during viral infection play major roles in the regulation of the cellular interactions involving cells of the immune and inflammatory systems and consequently determine the pathogenic outcome of infection. The chemokine RANTES (Regulated on activation, normal T-cell expressed and secreted) attracts immune cells during inflammation and the immune response, indicating a role for RANTES in viral pathogenesis. Here, we show that RANTES was downregulated in human foreskin fibroblast (HFF) cells at a later stage after infection with the Toledo strain but not after infection with the AD169 strain. miR-UL148D, the only miRNA predicted from the UL/b' sequences of the Toledo genome, targeted the 3′-untranslated region of RANTES and induced degradation of RANTES mRNA during infection. While wild-type Toledo inhibited expression of RANTES in HFF cells, Toledo mutant virus in which miR-UL148D is specifically abrogated did not repress RANTES expression. Furthermore, miR-UL148D-mediated downregulation of RANTES was inhibited by treatment with a miR-UL148D-specific inhibitor designed to bind to the miR-UL148D sequence via an antisense mechanism, supporting the potential value of antisense agents as therapeutic tools directed against HCMV. Our findings identify a viral microRNA as a novel negative regulator of the chemokine RANTES and provide clues for understanding the pathogenesis of the clinical strains of HCMV. Unlike the attenuated HCMV strain AD169, the clinical isolates of HCMV, including the Toledo strain, are virulent and can cause disease in healthy adults. Toledo differs from AD169 in that Toledo contains a 15-kb DNA segment, encoding at least 19 ORFs and a single microRNA known as miR-UL148D. This 15-kb segment is believed to be a major determinant of the virulence and pathogenicity of the Toledo clinical strain. The CC–chemokine RANTES recruits immune cells during viral infection, suggesting that it may play a role in virus-related diseases. Here, we show that RANTES mRNA was degraded in human foreskin fibroblast cells during infection with Toledo but not during infection with AD169. The degradation of RANTES mRNA was mediated by miR-UL148D, the only viral microRNA predicted from the 15–kb segment of the Toledo genome. Accordingly, the levels of secreted RANTES in infected cells with ToledoΔmiR-UL148D in which miR-UL148D was deleted were higher than those in infected cells with Toledo. Our results reveal that a viral microRNA could be a novel potential therapeutic target and provide important insights into understanding the differences in pathogenic potential between clinical and attenuated strains.
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