Analysis of SARS Coronavirus-Host Cell Interactions

描述(申请人提供)：许多病毒抑制宿主基因在感染细胞中的表达；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的致病性。引起宿主基因表达抑制的病毒蛋白通常是病毒的主要毒力因素。严重急性呼吸综合征(SARS)是一种新近出现的与肺炎相关的人类疾病。一种新型SARS冠状病毒(SCOV)是SARS的病原体。我们证明了编码在SCOV基因1 N端的SCOV NSP1蛋白可以促进宿主mRNAs的降解，但不能促进rRNAs的降解，导致表达细胞的宿主蛋白合成受到强烈的抑制。进一步的分析表明，NSP1不能促进SCOV类mRNAs的降解，表明Socv NSP1蛋白是已知病毒蛋白中第一个选择性地促进宿主mRNAs降解而不影响病毒mRNAs稳定性的病毒蛋白。对SCOV感染细胞和携带突变的NSP1不再抑制宿主基因表达的SCOV感染细胞中宿主mRNA积累的分析强烈表明，NSP1促进了感染细胞中宿主mRNA的降解。因此，SCOV似乎使用了一种新的策略来抑制宿主基因的表达。我们推测，SCOV NSPL促进感染细胞中宿主mRNAs的降解，以此作为一种手段来抑制各种“敌对”宿主细胞蛋白的积累；为了促进自身复制，SCOV可能会抑制抗病毒蛋白，如I型干扰素和促凋亡蛋白，以防止细胞早期死亡，而抑制参与先天性免疫和获得性免疫的细胞蛋白可能允许病毒绕过宿主免疫识别和/或诱导反应。此外，NSP1诱导的宿主mRNA的降解可能有利于SCOV蛋白的高效翻译。我们怀疑NSP1可能是SCOV毒力的一个主要因素。目前的应用建议：利用体外系统揭示NSP1诱导的宿主mRNA降解的机制；鉴定与NSP1相互作用的宿主蛋白，这可能提供关于NSP1选择性地导致mRNA与rRNA降解的线索；澄清为什么cov mRNAs抵抗nsp1诱导的mRNA失稳；以及检查是否一些宿主mRNA物种也抵抗nsp1诱导的宿主mRNA失稳。此外，我们将使用SCOV及其突变体携带不再促进宿主mRNA降解的改变的NSP1来确定NSP1在抑制感染细胞中宿主基因表达的作用。

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.