Functional characterization of BHV-1 tegument proteins

疱疹病毒由四个同心隔室组成，即围绕DNA的核蛋白核心、衣壳、被膜和包膜。被膜包含至少15种蛋白质，它们在病毒感染中起重要作用，因为它们是第一个与细胞内环境相互作用的蛋白质，并且在稍后阶段，它们结合以产生新的病毒体。本项目的长期目标是对牛疱疹病毒1型（BHV-1）的被膜蛋白进行功能表征，BHV-1是牛呼吸道疾病的主要病原体之一。本提案的具体目标是进一步表征VP 8的功能，VP 8是BHV-1中的主要被膜蛋白。其他的被膜蛋白，特别是那些与VP 8相互作用的将按照类似的方法进行研究。缺乏VP 8的突变体病毒在体外受损，在体内无毒力，这表明，除了结构作用，VP 8具有重要的早期功能，可能调节先天性免疫反应。我们的假设是，VP 8在建立有效的病毒复制条件和促进病毒组装中发挥作用。我们将测试如下：i）由于VP 8具有RNA结合活性，我们将检查VP 8对病毒mRNA转运的早期作用; ii）有趣的是，VP 8与DNA损伤结合蛋白-1和cullin 4A相互作用，因此我们将确定VP 8是否劫持cullin-4A-DDB 1 E3泛素连接酶以控制感染细胞中的蛋白质降解，并可能阻断干扰素信号传导; iii）在不存在VP 8的情况下，我们观察到成熟病毒体的数量减少，因此我们将评估VP 8在病毒组装中的作用; iv）由于VP 8被磷酸化，我们将确定VP 8磷酸化对其功能特性的影响。通过表征BHV-1的被膜蛋白，我们将深入了解复杂病毒的复制和形态发生，这将对疱疹病毒感染的预防和控制产生影响。牛作为体内试验的天然宿主的可用性为疫苗和治疗剂的开发提供了优势，不仅针对BHV-1，而且针对相关疱疹病毒，这将有利于加拿大的人类健康和畜牧业。

Herpesviruses are composed of four concentric compartments, the nucleoprotein core surrounding the DNA, the capsid, the tegument and the envelope. The tegument contains at least 15 proteins, which play an important role in viral infection, because they are the first to interact with the intracellular environment and at a later stage they associate to create new virions. The long-term objective of this program is to perform a functional characterization of the tegument proteins of bovine herpesvirus-1 (BHV-1), one of the major pathogens in bovine respiratory disease. The specific objective of this proposal is to further characterize the functions of VP8, which is the major tegument protein in BHV-1. Additional tegument proteins, in particular those that interact with VP8 will be studied following similar approaches. A mutant virus lacking VP8 was impaired in vitro and non-virulent in vivo, which suggests that in addition to a structural role VP8 has an important early function, possibly modulating innate immune responses. Our hypothesis is that VP8 plays a role both in establishing conditions for efficient viral replication and in promoting virus assembly. We will test this as follows: i) Since VP8 has RNA binding activity, we will examine the early effects of VP8 on viral mRNA transport; ii) Interestingly, VP8 interacts with DNA damage-binding protein-1 and culllin4A, so we will determine whether VP8 hijacks the cullin-4A-DDB1 E3 ubiquitin ligase to control protein degradation in infected cells, and possibly block interferon signaling; iii) In the absence of VP8 we observed reduced numbers of mature virions, so we will assess the role of VP8 in virus assembly; iv) As VP8 is phosphorylated, we will define the impact of VP8 phosphorylation on its functional properties. By characterizing tegument proteins of BHV-1, we will gain insight into the replication and morphogenesis of a complex virus, which will have implications for prevention and control of herpesvirus infections. The availability of cattle as the natural host for in vivo testing provides an advantage for the development of vaccines and therapeutics, not only for BHV-1 but also for related herpesviruses, which will benefit human health as well as the livestock industry in Canada.