Denison M R, Sims A C, Gibson C A, Lu X T
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Adv Exp Med Biol. 1998;440:121-7. doi: 10.1007/978-1-4615-5331-1_16.
The 3C-like proteinase of mouse hepatitis virus (MHV-3CLpro) is predicted to cleave at least 10 sites in the gene 1 polyprotein, resulting in processing of proteinase, polymerase and helicase proteins from the polyprotein. We have used E. coli expressed recombinant 3CLpro (r3CLpro) to define cleavage sites in carboxy-terminal region of the ORF 1a polyprotein. Polypeptides containing one or more putative 3CLpro cleavage site were translated in vitro from subcloned regions of gene 1, and the polypeptides were incubated with r3CLpro. Analysis of the cleavage products confirmed several putative cleavage sites, as well as identifying cleavage sites not previously predicted by analysis of the MHV sequence. Antibodies directed against a portion of the ORF 1a polyprotein were used to probe virus infected cells, and detected proteins that correspond to the cleavage sites used by 3CLpro in vitro. These results suggest that MHV 3CLpro cleaves at least 7 sites in the ORF 1a polyprotein, and that the specificity of 3CLpro for cleavage site dipeptides may be broader than previously predicted.
小鼠肝炎病毒的3C样蛋白酶（MHV - 3CLpro）预计会在基因1多聚蛋白中切割至少10个位点，从而从多聚蛋白中加工出蛋白酶、聚合酶和解旋酶蛋白。我们使用大肠杆菌表达的重组3CLpro（r3CLpro）来确定ORF 1a多聚蛋白羧基末端区域的切割位点。含有一个或多个假定的3CLpro切割位点的多肽从基因1的亚克隆区域体外翻译而来，并将这些多肽与r3CLpro一起孵育。对切割产物的分析证实了几个假定的切割位点，同时还鉴定出了先前通过MHV序列分析未预测到的切割位点。针对ORF 1a多聚蛋白一部分的抗体用于探测病毒感染细胞，并检测到了与3CLpro在体外使用的切割位点相对应的蛋白质。这些结果表明，MHV 3CLpro在ORF 1a多聚蛋白中切割至少7个位点，并且3CLpro对切割位点二肽的特异性可能比先前预测的更广泛。
