马立克氏病病毒立即早期蛋白ICP0抑制宿主PML核小体抗病毒活性的机制研究

The "latency-reactivation" stage is characteristic of Marek's Disease Virus (MDV) infection in chickens. Promyelocytic leukemia protein nuclear bodies (PML NBs) of host cell could exert inhibition to the genome replication of herpesvirus through interaction with immediate early proteins when viral infection is in latent stage. As ICP0 protein is an immediate early transcription factor of MDV, which regulate the viral infectivity, our previous study showed that the deletion of ICP0 could decrease significantly the replication of MDV in CEF, we presume that ICP0 of MDV could also affect the antiviral activity of host PML NBs post infection. However, this mechanism is not clear so far. In this study, the dynamic replication of MDV in T lymphocytes of chicken will be monitored post infection with the MDV deleted ICP0. The role of ICP0 for transcription of MDV particular genes will be investigated in the stage of "latency-reactivation" infection. Then we will analyze the effect of ICP0 on the assembly of host PML NBs by gene over-expression and infection with gene deletion virus, and identify the target proteins and key sites of ICP0 interacting with PML NBs. Next, we will explore the forms and regulatory consequences between ICP0 and its target protein of PML NBs. Finally, we will evaluate whether inhibition of MDV ICP0 to antiviral activity of PML NBs affects MDV proliferation and pathogenicity. The aim of our study is to reveal the somewhat mechanisms of formation of "latency-reactivation" infection. The results will help to develop new strategies against MDV infection.

“潜伏-再活化”期是马立克氏病病毒（MDV）在鸡体内感染的重要特征。疱疹病毒立即早期转录因子通过抑制宿主PML NBs的装配在病毒潜伏再活化过程中发挥重要作用。本团队发现缺失立即早期蛋白ICP0的MDV复制能力显著减弱，表明ICP0是影响MDV感染的重要因素。推测ICP0在MDV感染过程中可能通过抑制PML NBs组装以维持病毒复制，但其机制尚不明确。本课题拟检测ICP0基因缺失MDV感染鸡后在T细胞中的复制动态，分析ICP0在潜伏期和再活化期对病毒关键基因转录的调控作用；研究MDV ICP0对宿主PML NBs组装的影响，筛选并鉴定PML NBs中受ICP0调控的靶蛋白和关键位点，分析ICP0对靶蛋白的调控方式及其对MDV病毒增殖和致病性的影响，阐明MDV抑制PML NBs抗病毒活性的作用机制，为揭示MDV特殊的“潜伏-再活化”感染机制进而研发抗MDV感染的新策略提供依据。

“潜伏-再活化”期是马立克氏病病毒（MDV）在鸡体内感染的重要阶段。α疱疹病毒立即早期转录因子（IE）可以通过抑制宿主细胞PML NBs的装配在病毒感染的潜伏期发挥重要作用。本团队发现缺失ICP0或US3的MDV复制能力显著减弱，表明ICP0、US3均是影响MDV感染的重要因素。推测ICP0、US3在MDV感染的潜伏期也可能通过抑制PML以维持病毒复制。在本研究中我们首次克隆了鸡PML并制备其抗体后验证鸡PML具备抑制MDV病毒复制的能力，而且MDV感染过程中可以在蛋白水平有效抑制宿主PML表达，动物实验结果表明在MDV超强毒株RB1B株感染早期和潜伏感染形成初期宿主PML发挥了重要的防御作用。进一步通过共聚焦和免疫共沉淀实验研究发现，MDV的立即早期蛋白ICP0和US3可以与鸡PML直接互作并通过蛋白酶体途径在蛋白水平降解PML进而有利于MDV的复制。由此我们推断鸡PML在体内形成PML-NBs可以有效抑制MDV病毒复制，但MDV感染后可以通过其立即早期蛋白ICP0以及US3转录蛋白有效降解PML进而下调鸡PML-NBs对MDV复制的抑制作用，从而保证自身复制以及潜伏感染的再活化。本研究中我们首次克隆到了鸡PML分子并验证了其抗病毒功能，为研究鸡的免疫防御系统作用机制提供了新的研究方向，同时筛选到MDV抑制宿主PML防御反应的两个编码蛋白ICP0和US3，并初步阐明了其降解PML的作用方式，这为揭示MDV感染宿主后的免疫逃逸和致病机制提供了新的思路。