Restriction of KSHV by cellular RNA decay pathways

项目摘要 卡波西肉瘤相关疱疹病毒(KSHV)是疱疹病毒亚科的一员，是 与几种恶性肿瘤的发展有因果关系，包括卡波西肉瘤和原发肿瘤 渗出性淋巴瘤(PEL)。病毒建立潜伏期以及重新激活的能力对于 KSHV相关疾病的发展。尽管有这些疾病进展的要求，但仍有 关于限制KSHV生命周期的细胞内在机制的知识存在显著差距。虽然与生俱来 限制通常被认为是在抗病毒免疫反应的背景下，越来越多的证据表明 细胞RNA质量控制途径具有抗病毒作用。无意义介导的RNA衰退(NMD)是一种 进化上保守的RNA衰变途径，促进核糖体所在RNA的降解 被认为异常地终止了翻译。新出现的证据表明，NMD在抗病毒方面发挥了作用 然而，限制具有显著作用的限制正链RNA病毒的复制，NMD在 DNA病毒限制，如KSHV，是未知的。我们最近报道了NMD是一个细胞的发现- DNA病毒的内在限制机制，并证明它对 KSHV在PEL细胞中的生命周期。我们的数据表明，依赖NMD的限制与这两种调控都有关 未折叠蛋白反应(UPR)以及主要KSHV转录因子的靶向降解， RTA。基于这些观察，我们的中心假设是NMD通过以下方式限制KSHV的重新激活 靶向关键的病毒mRNAs以及对病毒基因表达至关重要的细胞转录本 该病毒通过病毒编码的机制对抗NMD。为了检验这一假设，我们提出了一个 旨在确定NMD和KSHV之间相互作用的一系列综合实验。在目标1中，我们 将研究NMD依赖的UPR途径调节如何调节KSHV的生命周期。在目标2中，我们 将研究一类KSHV mRNAs，它们逃脱了NMD，尽管含有应该 使他们对国家导弹防御系统敏感。在目标3中，我们将确定KSHV编码蛋白的机制 抑制NMD。这些研究的完成有望确定NMD如何限制KSHV的生命周期 因为病毒用来对抗它的机制。这些将代表着对如何 DNA病毒感染是由细胞内在机制调节的，可以被利用来开发新的 治疗策略。

Project Summary Kaposi’s sarcoma-associated herpesvirus (KSHV) is a member of the subfamily gammaherpesvirinae and is causally associated with the development of several malignancies including Kaposi’s sarcoma and primary effusion lymphoma (PEL). The virus’ ability to establish latency as well as reactivate are essential for the development of KSHV-associated disease. Despite these requirements for disease progression there are significant gaps in knowledge regarding cell-intrinsic mechanisms that restrict the KSHV lifecycle. While innate restriction is typically thought of in the context of antiviral immune responses, growing evidence suggests that cellular RNA quality control pathways have an antiviral role. Nonsense-mediated RNA decay (NMD) is an evolutionarily conserved RNA decay pathway that facilitates degradation of RNAs on which ribosomes are deemed to terminate translation aberrantly. Emerging evidence has pointed to a role for NMD in antiviral restriction with a prominent role limiting replication of positive-stranded RNA viruses, however, a role for NMD in DNA virus restriction, such as KSHV, was unknown. We recently reported the discovery that NMD is a cell- intrinsic restriction mechanism for DNA viruses and demonstrated that it imposes a significant restriction on the KSHV lifecycle in PEL cells. Our data demonstrate that NMD-dependent restriction is linked both to the regulation of the unfolded protein response (UPR) as well as targeted degradation of the main KSHV transcription factor, RTA. Building upon these observations our central hypothesis is that NMD restricts KSHV reactivation by targeting key viral mRNAs as well as cellular transcripts in pathways important for viral gene expression and that the virus antagonizes NMD through viral-encoded mechanisms. To test this hypothesis, we propose an integrated series of experiments aimed at determining the interactions between NMD and KSHV. In Aim 1, we will investigate how NMD-dependent regulation of the UPR pathway regulates the KSHV lifecycle. In Aim 2, we will investigate a class of KSHV mRNAs that escape NMD despite harboring sequence features that should render them NMD-susceptible. In Aim 3, we will determine the mechanism by which KSHV-encoded proteins inhibit NMD. Completion of these studies is expected to determine how NMD restricts the KSHV lifecycle as well as the mechanisms the virus employs to antagonize it. These will represent fundamental new insights into how DNA virus infection is regulated by cell-intrinsic mechanisms and can be harnessed for the development of new therapeutic strategies.