Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
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DOI：
10.1128/jvi.78.24.13600-13612.2004
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发表时间：
2004-12-01
影响因子：
5.4
通讯作者：
Baker, SC
中科院分区：
文献类型：
--
作者：
Harcourt, BH;Jukneliene, D;Baker, SC
严重急性呼吸综合征（SARS）相关冠状病毒的基因1编码复制酶多聚蛋白，预计这些多聚蛋白会被两种病毒蛋白酶，即木瓜蛋白酶样蛋白酶（PLpro）和3C样蛋白酶（3CLpro）加工成16种非结构蛋白（nsp1至nsp16）。在此，我们鉴定了SARS冠状病毒氨基末端复制酶产物nsp1、nsp2和nsp3，并描述了表征产生这些产物所需蛋白酶活性的反式切割实验。我们制备了针对谷胱甘肽S - 转移酶 - 复制酶融合蛋白的多克隆抗血清，并利用该抗血清在脉冲追踪实验中检测复制酶中间体和产物。我们发现nsp1（p20）能从复制酶多聚蛋白中快速被加工出来。相比之下，nsp2/3位点的加工效率较低，因为检测到了一种约300 kDa的中间体（NSP2 - 3），但最终还是产生了nsp2（p71）和nsp3（p213）。我们发现SARS冠状病毒复制酶产物在感染细胞的细胞质中感染后4小时即可被检测到，并且nsp1至nsp3与新合成的病毒RNA在点状的核周位点共定位，这与它们在病毒RNA合成中所预期的作用相符。为了确定PLpro是否负责加工这些产物，我们克隆并表达了PLpro结构域以及预期的底物，并建立了PLpro反式切割实验。我们发现PLpro结构域足以加工预期的nsp1/2和nsp2/3位点。有趣的是，加工预期的nsp3/4位点需要表达包含下游疏水结构域的PLpro延伸区域。我们发现疏水结构域插入到膜中，并且腔内结构域在天冬酰胺残基2249和2252处被糖基化。因此，疏水结构域可能将复制复合物锚定到细胞内膜上。这些研究表明，PLpro能够在三个预期的切割位点进行反式切割，并且它需要与膜结合才能加工nsp3/4切割位点。
Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a approximate to300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro, can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.