Proteomics Analysis of Herpes Simplex Virus Type 1‑Infected Cells Reveals Dynamic Changes of Viral Protein Expression, Ubiquitylation, and Phosphorylation

Herpesviruses are among the most complex and widespread human viruses and cause a number of diseases ranging from cold sores to genital infections and encephalitis. While the composition of viral particles has been studied, less is known about the expression of the whole viral proteome in infected cells. Here, we analyzed the proteome of the prototypical Herpes Simplex Virus type 1 (HSV1) in infected cells by mass spectrometry. Using a high sensitivity LTQ-Orbitrap, we achieved a very high level of protein coverage and identified a total of 67 structural and nonstructural viral proteins. We also identified 90 novel phosphorylation sites and 10 novel ubiquitylation sites on different viral proteins. Ubiquitylation was observed on nine HSV1 proteins. We identified phosphorylation sites on about half of the detected viral proteins; many of the highly phosphorylated ones are known to regulate gene expression. Treatment with inhibitors of DNA replication induced changes of both viral protein abundance and modifications, highlighting the interdependence of viral proteins during the life cycle. Given the importance of expression dynamics, ubiquitylation, and phosphorylation for protein function, these findings will serve as important tools for future studies on herpesvirus biology.

疱疹病毒（Herpesviruses）是人类病毒中结构最复杂、分布最广泛的类群之一，可引发多种疾病，涵盖唇疱疹、生殖器感染乃至脑炎。尽管学界已对病毒颗粒的组成展开研究，但对于感染细胞内完整病毒蛋白质组（proteome）的表达情况，目前所知仍较为有限。本研究通过质谱（mass spectrometry）技术，分析了经典的1型单纯疱疹病毒（Herpes Simplex Virus type 1, HSV1）在感染细胞内的蛋白质组。采用高灵敏度LTQ-Orbitrap质谱仪，本研究实现了极高的蛋白质覆盖度，共鉴定出67种病毒结构蛋白与非结构蛋白。此外，我们还在不同病毒蛋白上鉴定出90个全新的磷酸化位点（phosphorylation sites）与10个全新的泛素化位点（ubiquitylation sites），另有9种1型单纯疱疹病毒蛋白被检测到存在泛素化修饰。我们在约半数已鉴定的病毒蛋白上发现了磷酸化位点，其中多数高磷酸化蛋白已知可调控基因表达。使用DNA复制抑制剂处理细胞后，病毒蛋白的丰度与修饰模式均发生改变，这凸显了病毒蛋白在生命周期中的相互依存关系。鉴于蛋白质表达动态、泛素化与磷酸化修饰对蛋白功能的重要性，本研究成果将为未来疱疹病毒生物学相关研究提供重要的参考工具。