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）是一类结构最为复杂、传播最为广泛的人类病毒，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开展了深入研究，但对于感染宿主细胞内完整病毒蛋白质组的表达特征，当前认知仍相对匮乏。本研究采用质谱技术，对原型病毒1型单纯疱疹病毒（Herpes Simplex Virus type 1, HSV1）感染的细胞蛋白质组进行了系统分析。借助高灵敏度LTQ-Orbitrap质谱仪，我们实现了极高的蛋白覆盖度，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发现了90个全新磷酸化位点与10个全新泛素化位点，其中9种HSV1蛋白存在泛素化修饰。我们在约半数被检测到的病毒蛋白上鉴定到了磷酸化位点；其中多数高磷酸化蛋白已知可调控基因表达。经DNA复制抑制剂处理后，病毒蛋白的丰度与修饰模式均发生显著改变，这凸显了病毒蛋白在生命周期中的相互依存关系。鉴于表达动态、泛素化与磷酸化修饰对蛋白质功能的关键作用，本研究所得结果将为未来疱疹病毒生物学的相关研究提供重要的支撑工具。