Analysis of SARS Coronavirus-Host Cell Interactions

描述（由申请人提供）：许多病毒在感染细胞中抑制宿主基因表达；病毒诱导的宿主基因表达抑制很可能有利于病毒复制并影响病毒的致病性。诱导宿主基因表达抑制的病毒蛋白往往是主要的病毒毒力因子。严重急性呼吸系统综合症（SARS）是最近出现的一种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。我们证明，编码于SCoV基因1 n端的SCoV nsp1蛋白可促进宿主mrna的降解，而非rnas，从而在表达细胞中导致强烈的宿主蛋白合成抑制。进一步分析表明，nsp1不促进SCoV mrna样mrna的降解，这表明在已知的病毒蛋白中，SCoV nsp1蛋白是第一个选择性地促进宿主mrna降解而不影响病毒mrna稳定性的病毒蛋白。对SCoV感染细胞和携带不再抑制宿主基因表达的突变nsp1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表达。我们假设SCoV nspl促进感染细胞中宿主mrna的降解，作为抑制各种“敌对”宿主细胞蛋白积累的一种手段；为了促进自身复制，SCoV可能抑制抗病毒蛋白，如I型干扰素和促凋亡蛋白，以防止细胞早期死亡，并且抑制参与先天免疫和获得性免疫的细胞蛋白可能使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降解可能有利于SCoV蛋白的高效翻译。我们怀疑nsp1可能是SCoV的主要毒力因子。目前的应用建议：利用体外系统揭示nsp1诱导宿主mRNA降解的机制；鉴定与nsp1相互作用的宿主蛋白，这可能为nsp1选择性地导致mRNA与rRNA的降解提供线索；阐明SCoV mRNA对nsp1诱导的mRNA失稳具有抗性的原因；并研究某些宿主mRNA物种是否也对nsp1诱导的宿主mRNA不稳定具有抗性。此外，我们将使用携带改变的nsp1的SCoV及其突变体，该突变体不再促进宿主mRNA降解，以确定nsp1在受感染细胞中抑制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.