Functional characterization of BHV-1 tegument proteins

疱疹病毒由四个同心的区室组成：围绕DNA的核蛋白核心、衣壳、被膜和包膜。被皮包含至少15种蛋白质，这些蛋白质在病毒感染中起着重要作用，因为它们首先与细胞内环境相互作用，并在后期结合产生新的病毒粒子。该项目的长期目标是对牛疱疹病毒-1 （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。本研究的具体目的是进一步表征BHV-1的主要被膜蛋白VP8的功能。其他被膜蛋白，特别是那些与VP8相互作用的蛋白将采用类似的方法进行研究。缺乏VP8的突变病毒在体外受损，而在体内无毒，这表明VP8除了具有结构作用外，还具有重要的早期功能，可能调节先天免疫反应。我们的假设是VP8在建立病毒有效复制的条件和促进病毒组装方面发挥作用。我们将进行如下测试：i)由于VP8具有RNA结合活性，我们将研究VP8对病毒mRNA转运的早期影响；ii)有趣的是，VP8与DNA损伤结合蛋白-1和culllin4A相互作用，因此我们将确定VP8是否劫持cullin-4A-DDB1 E3泛素连接酶来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)在没有VP8的情况下，我们观察到成熟病毒粒子的数量减少，因此我们将评估VP8在病毒组装中的作用；iv)由于VP8被磷酸化，我们将定义VP8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，这将对预防和控制疱疹病毒感染具有重要意义。牛作为体内试验的天然宿主，不仅为BHV-1，而且为相关疱疹病毒的疫苗和疗法的开发提供了优势，这将有利于人类健康以及加拿大的畜牧业。

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.