卡波氏肉瘤相关疱疹病毒ORF45介导ERK-RSK持续活化的新功能研究

Infection of Kaposi's sarcoma associated herpesvirus (KSHV) causes severe malignancy in AIDS patients and immunosuppressive populations, and its persistent infection and pathogenesis require a continuous low level of lytic replication. Studies have revealed that KSHV immediate early protein ORF45 induces sustained activation of ERK-RSK pathway, promotes lytic gene expressions during late stage and viral lytic replication, however, the effects on cellular gene expression and activity of host cells remain unknown. Recently we found that ORF45-RSK pathway activates cellular transcription factor c-Fos to accelerate viral transcription during late lytic replication, and induces the phosphorylation of Filamin A. Based on our preliminary results, we hypothesized that the sustained ERK-RSK activation by ORF45 control the gene expression, cellular adhesion and survival of host cells through targeting specific substrates. This project is planning to study the regulation and important function of ORF45-RSK in cellular gene transcription through c-Fos (Aim 1), regulation in cell morphology and cell adhesion through Filamin A (Aim2), the protective effects of ORF45-RSK in cell survival (Aim3) during KSHV lytic replication. This project will expect to elucidate how KSHV lytic lifecycle utilize ORF45-mediated ERK-RSK activation to modulate cellular gene expression and cellular physiological processes to protect cell survival and facilitate viral replication, and to further reveal the novel function and significance of RSK in KSHV lytic infection and KSHV-related diseases.

卡波氏肉瘤相关疱疹病毒KSHV在HIV患者和免疫功能低下的人群可显著诱发恶性肿瘤。KSHV持续感染和致病需要裂解复制，即早期蛋白ORF45诱导ERK-RSK持续活化，促进病毒的基因表达和复制，但该途径对宿主细胞的影响和机制尚不清楚。最近我们发现ORF45-RSK途径激活转录因子c-Fos和诱导Filamin A磷酸化，我们推测ORF45-RSK途径通过这些底物调控细胞基因表达和细胞活动。本项目将深入探讨ORF45-RSK途径通过c-Fos对宿主基因转录的调控作用和功能，以及通过Filamin A改变Integrin功能、降低细胞粘附和相关信号，调控KSHV裂解复制细胞的存活和锚定粘附性；阐明KSHV裂解复制如何利用ORF45介导的ERK-RSK持续活化改变宿主细胞形态和保护细胞存活以利于复制和子代病毒释放，进一步揭示RSK靶点对KSHV感染细胞的重要功能和在KSHV相关疾病治疗中的重要意义

KSHV是与三种已知人类肿瘤（MCD、PEL、KS）相关的淋巴嗜性疱疹病毒。在体外KSHV能感染大多数人体细胞但效率不高.感染以细胞结合病毒（cell associated virus）的形式发生比以游离病毒的形式具有显著更高的效率，以此跨越胞膜屏障、逃避免疫实现终生携带。这种病毒依赖细胞-细胞接触的传播使感染细胞的脱落、迁移、凋亡抑制、黏附的调控对病毒生存和肿瘤发生显得尤为重要。本项研究主要集中于了解KSHV的即早期调控蛋白ORF45诱导 ERK-MAPK通路持续活化与细胞粘附和趋化运动调节之间的关系及其中的分子机制。研究结果揭示了细胞结合病毒具有和游离病毒不同的感染效率、细胞嗜性和复制特性，ORF45在过表达和重组病毒中的确诱导感染细胞的黏附下降和促进迁移。高通量蛋白测序筛选发现至少ERK通路底物细胞微丝结合蛋白Filamin A在其中起到关键作用。研究其中的机制发现ORF45通过RSK在病毒复制过程中诱导Filamin A的持续磷酸化，细胞Filamin A及其磷酸化为细胞的粘附与生长所必需但不是病毒复制所必需。通过一种不影响产毒复制的ORF45核定位重组病毒，我们发现这种病毒不激活胞质骨架蛋白Filamin A的磷酸化，和ORF45缺失突变病毒一样不能促进裂解复制细胞的迁移，为ORF45促进细胞迁移提供了进一步的实验证据。我们的发现阐明ORF45通过ERK/RSK诱导Filamin A磷酸化促进细胞的趋化迁移，促进病毒复制和体内传播，逃避免疫识别,揭示了KSHV相关肿瘤体内转移的新机制。