DDX21 regulates the biogenesis of rRNA and transcription of ribonucleoprotein genes. Recently, it has been reported that DDX21 regulates the growth of some RNA viruses through various mechanisms, such as inhibiting viral genome replication, suppressing virion assembly and release, and modulating antiviral immune responses (Chen et al., Cell Host Microbe 15:484-493, 2014, https://doi.org/10.1016/j.chom.2014.03.002; Dong et al., Biophys Res Commun, 473:648-653, 2016, https://doi.org/10.1016/j.bbrc.2016.03.120; and Watanabe et al., PLoS Pathog 5:e1000654, 2009, https://doi.org/10.1371/journal.ppat.1000654). The relationship between DDX21 and DNA viruses has not yet been explored. In this study, we used human cytomegalovirus (HCMV), a large human DNA virus, to investigate the potential role of DDX21 in DNA virus replication. We found that HCMV infection prevented the repression of DDX21 at protein and mRNA levels. Knockdown of DDX21 inhibited HCMV growth in human fibroblast cells (MRC5). Immunofluorescence and quantitative PCR (qPCR) results showed that knockdown of DDX21 did not affect viral DNA replication or the formation of the viral replication compartment but did significantly inhibit viral late gene transcription. Some studies have reported that DDX21 knockdown promotes the accumulation of R-loops that could restrain RNA polymerase II elongation and inhibit the transcription of certain genes. Thus, we used the DNA-RNA hybrid-specific S9.6 antibody to stain R-loops and observed that more R-loops formed in DDX21-knockdown cells than in control cells. Moreover, an DNA-RNA immunoprecipitation assay showed that more R-loops accumulated on a viral late gene in DDX21-knockdown cells. Altogether, these results suggest that DDX21 knockdown promotes the accumulation of R-loops, which prevents viral late gene transcription and consequently results in the suppression of HCMV growth. This finding provides new insight into the relationship between DDX21 and DNA virus replication.IMPORTANCE Previous studies have confirmed that DDX21 is vital for the regulation of various aspects of RNA virus replication. Our research is the first report on the role of DDX21 in HCMV DNA virus replication. We identified that DDX21 knockdown affected HCMV growth and viral late gene transcription. In order to elucidate how DDX21 regulated this transcription, we applied DNA-RNA immunoprecipitation by using the DNA-RNA hybrid-specific S9.6 antibody to test whether more R-loops accumulated on the viral late gene. Consistent with our expectation, more R-loops were detected on the viral late gene at late HCMV infection time points, which demonstrated that the accumulation of R-loops caused by DDX21 knockdown prevented viral late gene transcription and consequently impaired HCMV replication. These results reveal that DDX21 plays an important role in regulating HCMV replication and also provide a basis for investigating the role of DDX21 in regulating other DNA viruses.

中文翻译：

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剖析 DDX21 在调节人类巨细胞病毒复制中的作用。


DDX21 调节 rRNA 的生物合成和核糖核蛋白基因的转录。最近，有报道称DDX21通过多种机制调节一些RNA病毒的生长，例如抑制病毒基因组复制、抑制病毒粒子组装和释放以及调节抗病毒免疫反应(Chen等，Cell Host Microbe 15:484- 493，2014，https://doi.org/10.1016/j.chom.2014.03.002；Dong 等人，Biophys Res Commun，473:648-653，2016，https://doi.org/10.1016/j .bbrc.2016.03.120；以及 Watanabe 等人，PLoS Pathog 5：e1000654，2009，https://doi.org/10.1371/journal.ppat.1000654）。 DDX21 和 DNA 病毒之间的关系尚未被探索。在本研究中，我们使用人类巨细胞病毒 (HCMV)（一种大型人类 DNA 病毒）来研究 DDX21 在 DNA 病毒复制中的潜在作用。我们发现 HCMV 感染阻止了 DDX21 在蛋白质和 mRNA 水平上的抑制。 DDX21 的敲除可抑制人成纤维细胞 (MRC5) 中的 HCMV 生长。免疫荧光和定量PCR (qPCR)结果表明，DDX21的敲低并不影响病毒DNA复制或病毒复制区室的形成，但确实显着抑制病毒晚期基因转录。一些研究报告称，DDX21敲低会促进R环的积累，从而抑制RNA聚合酶II的延伸并抑制某些基因的转录。因此，我们使用 DNA-RNA 杂交特异性 S9.6 抗体对 R 环进行染色，并观察到 ​​DDX21 敲低细胞中形成的 R 环比对照细胞中更多。此外，DNA-RNA免疫沉淀测定表明，在DDX21敲低细胞中，病毒晚期基因上积累了更多的R环。 总而言之，这些结果表明 DDX21 敲除促进 R 环的积累，从而阻止病毒晚期基因转录，从而抑制 HCMV 生长。这一发现为DDX21与DNA病毒复制之间的关系提供了新的见解。 重要性 先前的研究已证实DDX21对于RNA病毒复制各个方面的调节至关重要。我们的研究是第一份关于 DDX21 在 HCMV DNA 病毒复制中的作用的报告。我们发现 DDX21 敲低影响 HCMV 生长和病毒晚期基因转录。为了阐明 DDX21 如何调节这种转录，我们通过使用 DNA-RNA 杂合特异性 S9.6 抗体应用 DNA-RNA 免疫沉淀来测试病毒晚期基因上是否积累了更多的 R 环。与我们的预期一致，在HCMV感染晚期时间点，在病毒晚期基因上检测到更多的R环，这表明DDX21敲低引起的R环积累阻止了病毒晚期基因转录，从而损害了HCMV复制。这些结果揭示了DDX21在调节HCMV复制中发挥重要作用，也为研究DDX21在调节其他DNA病毒中的作用提供了基础。