Analysis of SARS Coronavirus-Host Cell Interactions

描述（由申请方提供）：许多病毒抑制感染细胞中的宿主基因表达;病毒诱导的宿主基因表达抑制最有可能有利于病毒复制并影响病毒致病性。诱导宿主基因表达抑制的病毒蛋白通常是主要的病毒毒力因子。严重急性呼吸综合征（SARS）是近年来出现的一种与肺炎相关的人类疾病。新型SARS冠状病毒（SCoV）是SARS的病原体。我们证明了在SCoV基因1的N-末端编码的SCoV nsp 1蛋白促进宿主mRNA的降解，但不促进rRNA的降解，导致表达细胞中宿主蛋白质合成的强烈抑制。进一步的分析表明，nsp 1不促进SCoV mRNA样mRNA的降解，证明SCoV nsp 1蛋白是任何已知病毒蛋白中第一个选择性促进宿主mRNA降解而不影响病毒mRNA稳定性的病毒蛋白。在SCoV感染的细胞中和用携带不再抑制宿主基因表达的突变的nsp 1的SCoV感染的细胞中宿主mRNA积累的分析强烈地表明，nsp 1促进了感染细胞中宿主mRNA的降解。因此，SCoV似乎使用一种新的策略来抑制宿主基因表达。我们假设SCoV nsp 1促进宿主mRNA在感染细胞中的降解，作为抑制各种“敌对”宿主细胞蛋白积累的手段;为了促进自身的复制，SCoV可以抑制抗病毒蛋白，如I型干扰素，和促凋亡蛋白，以防止早期细胞死亡，并且抑制参与先天性和获得性免疫的细胞蛋白质可以使病毒绕过宿主免疫识别，和/或诱导反应。nsp 1诱导的宿主mRNA降解也可能有利于SCoV蛋白的有效翻译。我们怀疑nsp 1可能是一个主要的SCoV毒力因子。本申请提出：利用体外系统揭示nsp 1诱导宿主mRNA降解的机制;鉴定与nsp 1相互作用的宿主蛋白质，这可能产生关于nsp 1选择性导致mRNA与rRNA降解的线索;澄清为什么SCoV mRNA对nsp 1诱导的mRNA不稳定具有抗性;并检查一些宿主mRNA种类是否也对nsp 1诱导的宿主mRNA不稳定具有抗性。此外，我们将使用SCoV及其突变体携带改变的nsp 1，不再促进宿主mRNA降解，以建立nsp 1在抑制感染细胞中宿主基因表达中的作用。

DESCRIPTION (provided by applicant): Many viruses suppress host gene expression in infected cells; virus-induced host gene expression inhibition is most probably beneficial for virus replication and affects viral pathogenicity. Viral proteins that induce suppression of host gene expression are often major viral virulence factors. Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. A novel SARS coronavirus (SCoV) is the etiological agent of SARS. We demonstrated that SCoV nsp1 protein, which is encoded at the N-terminus of the SCoV gene 1, promotes degradation of host mRNAs, but not rRNAs, leading to strong host protein synthesis inhibition in expressing cells. Further analysis suggested that nsp1 did not promote degradation of SCoV mRNA-like mRNAs, demonstrating that SCoV nsp1 protein was the first viral protein among any known viral proteins that selectively promotes degradation of host mRNAs without affecting the stabilities of viral mRNAs. Analysis of host mRNA accumulation in SCoV-infected cells and in cells infected with SCoV carrying a mutated nsp1 that no longer suppressed host gene expression strongly suggested that nsp1 promoted host mRNA degradation in infected cells. Thus, SCoV appears to use a novel strategy to suppress host gene expression. We hypothesized that SCoV nspl promotes degradation of host mRNAs in infected cells as a means to suppress accumulation of various "hostile" host- cell proteins; to promote its own replication, SCoV may suppress antiviral proteins, like type I interferon, and proapoptotic proteins to prevent early cell death, and suppression of cellular proteins involved in both innate and acquired immunities may allow the virus to circumvent host immune recognition, and/or induction responses. Also the nsp1-induced host mRNA degradation may be beneficial for efficient translation of SCoV proteins. We suspect that nsp1 may be a major SCoV virulence factor. The present application proposes: to uncover the mechanisms of nsp1-induced host mRNA degradation using in vitro systems; to identify host proteins that interact with nsp1, which might yield clues about nsp1 selectively leading to degradation of mRNA vs. rRNA; to clarify why SCoV mRNAs were resistant to the nsp1-induced mRNA destabilization; and to examine whether some host mRNA species are also resistant to nsp1-induced host mRNA destabilization. Furthermore, we will use SCoV and its mutant carrying an altered nsp1 that no longer promotes host mRNA degradation to establish the role of nsp1 in suppressing host gene expression in infected cells.