Smith Samantha, Reuven Nina, Mohni Kareem N, Schumacher April J, Weller Sandra K
Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut, USA.
Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut, USA
J Virol. 2014 Sep 1;88(17):10146-56. doi: 10.1128/JVI.01723-14. Epub 2014 Jun 25.
The herpes simplex virus 1 (HSV-1) virion DNA contains nicks and gaps, and in this study a novel assay for estimating the size and number of gaps in virion DNA was developed. Consistent with previous reports, we estimate that there are approximately 15 gaps per genome, and we calculate the average gap length to be approximately 30 bases. Virion DNA was isolated and treated with DNA-modifying enzymes in order to fill in the gaps and modify the ends. Interestingly, filling in gaps, blunting the ends, or adding random sequences to the 3' ends of DNA, producing 3' flaps, did not impair the infectivity of treated DNA following transfection of Vero cells. On the other hand, the formation of 5' flaps in the DNA following treatment resulted in a dramatic reduction (95 to 100%) in infectivity. Virion DNA stimulated DNA-PKcs activity in transfected cells, and DNA with 5' flaps stimulated a higher level of DNA-PKcs activity than that observed in cells transfected with untreated virion DNA. The infectivity of 5'-flapped DNA was restored in cells that do not express DNA-PKcs and in cells cotransfected with the immediate early protein ICP0, which degrades DNA-PKcs. These results are consistent with previous reports that DNA-dependent protein kinase (DNA-PK) and the nonhomologous end joining (NHEJ) repair pathway are intrinsically antiviral and that ICP0 can counteract this effect. We suggest that HSV-1 DNA with 5' flaps may induce an antiviral state due to the induction of a DNA damage response, primarily mediated by NHEJ, that renders the HSV-1 genome less efficient for lytic infection.
For productive lytic infection to occur, HSV-1 must counteract a variety of cellular intrinsic antiviral mechanisms, including the DNA damage response (DDR). DDR pathways have been associated with silencing of gene expression, cell cycle arrest, and induction of apoptosis. In addition, the fate of viral genomes is likely to play a role in whether viral genomes adopt a configuration suitable for lytic DNA replication. This study demonstrates that virion DNA activates the cellular DDR kinase, DNA-PK, and that this response is inhibitory to viral infection. Furthermore, we show that HSV-1 ubiquitin ligase, ICP0, plays an important role in counteracting the negative effects of DNA-PK activation. These findings support the notion that DNA-PK is antiviral and suggest that the fate of incoming viral DNA has important consequences for the progression of lytic infection. This study underscores the complex evolutionary relationships between HSV and its host.
单纯疱疹病毒1型（HSV-1）病毒粒子DNA含有切口和缺口，在本研究中，开发了一种用于估计病毒粒子DNA中缺口大小和数量的新方法。与之前的报道一致，我们估计每个基因组大约有15个缺口，并计算出平均缺口长度约为30个碱基。分离病毒粒子DNA并用DNA修饰酶处理，以填补缺口并修饰末端。有趣的是，填补缺口、使末端变平或在DNA的3'末端添加随机序列以产生3'瓣，在转染Vero细胞后，并未损害处理后DNA的感染性。另一方面，处理后DNA中5'瓣的形成导致感染性急剧降低（95%至100%）。病毒粒子DNA刺激转染细胞中的DNA-PKcs活性，带有5'瓣的DNA刺激的DNA-PKcs活性水平高于未处理病毒粒子DNA转染的细胞。在不表达DNA-PKcs的细胞以及与立即早期蛋白ICP0共转染的细胞中，5'瓣DNA的感染性得以恢复，ICP0可降解DNA-PKcs。这些结果与之前的报道一致，即DNA依赖性蛋白激酶（DNA-PK）和非同源末端连接（NHEJ）修复途径本质上具有抗病毒作用，且ICP0可抵消这种作用。我们认为，带有5'瓣的HSV-1 DNA可能由于诱导DNA损伤反应（主要由NHEJ介导）而诱导抗病毒状态，这使得HSV-1基因组在裂解感染中效率降低。
为了发生有效的裂解感染，HSV-1必须抵消多种细胞内在抗病毒机制，包括DNA损伤反应（DDR）。DDR途径与基因表达沉默、细胞周期停滞和凋亡诱导有关。此外，病毒基因组的命运可能在病毒基因组是否采用适合裂解DNA复制的构型中发挥作用。本研究表明，病毒粒子DNA激活细胞DDR激酶DNA-PK，且这种反应对病毒感染具有抑制作用。此外，我们表明HSV-1泛素连接酶ICP0在抵消DNA-PK激活的负面影响中起重要作用。这些发现支持DNA-PK具有抗病毒作用的观点，并表明进入的病毒DNA的命运对裂解感染的进展具有重要影响。本研究强调了HSV与其宿主之间复杂的进化关系。
