Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA.
Department of Dermatology, School of Medicine, University of California, Davis, Sacramento, California, USA.
J Virol. 2020 Aug 17;94(17). doi: 10.1128/JVI.00892-20.
Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, U3. The functions of U3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV U3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV U3 and identified chicken CREB (chCREB) as a substrate of MDV U3. We show that wild-type MDV U3, but not kinase-dead U3 (U3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using U3 deletion and U3 kinase-dead recombinant MDV, we identified U3-responsive MDV genes during infection and found that the majority of U3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some U3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the region, which is also regulated by U3. Furthermore, biochemical studies demonstrated that MDV U3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, kinase studies revealed that Meq is a U3 substrate. MDV U3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression. MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. U3 encoded by alphaherpesviruses is a multifunctional kinase involved in the regulation of various cellular pathways. Using an MDV genome quantitative reverse transcriptase PCR (qRT-PCR) array and chromatin immunoprecipitation, we elucidated the role of MDV U3 in viral and cellular gene regulation. Our results provide insights into how viral kinase regulates host cell signaling pathways to activate both viral and host gene expression. This is an important step toward understanding host-pathogen interaction through activation of signaling cascades.
马立克氏病（MD）是一种由马立克氏病病毒（MDV）引起的鸡肿瘤性疾病，属于亚科。与其他α疱疹病毒一样，MDV 编码一种丝氨酸/苏氨酸蛋白激酶，即 U3。在其他 α疱疹病毒中，U3 的功能已得到广泛研究；然而，MDV U3 及其底物的生物学功能尚未得到详细研究。在这项研究中，我们研究了可能被 MDV U3 调控的细胞途径，并鉴定出鸡 CREB（chCREB）是 MDV U3 的底物。我们发现野生型 MDV U3 而非激酶失活的 U3（U3-K220A）会增加 CREB 的磷酸化，导致磷酸化 CREB（pCREB）募集到 CREB 反应基因的启动子并激活 CREB 靶基因的表达。使用 U3 缺失和 U3 激酶失活的重组 MDV，我们在感染过程中鉴定出 U3 响应的 MDV 基因，并发现大多数 U3 响应的基因位于 MDV 重复区。染色质免疫沉淀测序（ChIP-seq）研究确定了一些 U3 调控的基因与 Meq（一种 MDV 编码的致癌蛋白）募集位点共定位。染色质免疫沉淀-PCR（ChIP-PCR）进一步证实了 Meq 结合到 区域，该区域也受 U3 调控。此外，生化研究表明 MDV U3 在转染细胞和感染 MDV 的鸡胚成纤维细胞中以依赖磷酸化的方式与 Meq 相互作用。最后，激酶研究揭示 Meq 是 U3 的底物。因此，MDV U3 作为 CREB 信号通路的上游激酶，调节 CREB/Meq 异二聚体的转录功能，靶向细胞和病毒基因表达。MDV 是一种具有强大致癌能力的疱疹病毒，可在感染的鸡中诱导 T 细胞淋巴瘤。马立克氏病继续对全球家禽业造成重大经济影响。α疱疹病毒编码的 U3 是一种多功能激酶，参与多种细胞途径的调控。我们使用 MDV 基因组定量逆转录酶 PCR（qRT-PCR）阵列和染色质免疫沉淀，阐明了 MDV U3 在病毒和细胞基因调控中的作用。我们的研究结果提供了关于病毒激酶如何调节宿主细胞信号通路以激活病毒和宿主基因表达的见解。这是了解宿主-病原体相互作用通过激活信号级联的重要一步。
