Orr D C, Long A C, Kay J, Dunn B M, Cameron J M
Department of Virology, Glaxo Group Research Limited, Greenford, Middlesex, U.K.
J Gen Virol. 1989 Nov;70 ( Pt 11):2931-42. doi: 10.1099/0022-1317-70-11-2931.
The 3C proteins of several picornaviruses, including poliovirus, foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV), have been demonstrated to be cysteine-type proteinases, involved in the processing of the respective polyproteins expressed by the monocistronic RNA genome. Nucleotide sequencing data have indicated that the human rhinovirus 14 (HRV-14) RNA genome encodes a homologous 3C protein. The HRV-14 3C protein was purified to homogeneity from Escherichia coli expressing the cloned 3C genomic fragment. The enzyme was assayed against peptides corresponding to those residues, predicted (by nucleotide sequencing data) to occur at authentic cleavage sites within the polyprotein. The peptides representing the 1B/1C, 2A/2B, 2C/3A, 3A/3B, 3B/3C and 3C/3D cleavage sites, where proteolysis was predicted to occur at a Gln-Gly junction, were all cleaved by the 3C proteinase. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase. By contrast, peptides corresponding to the predicted 2B/2C and 1C/1D cleavage sites, where the processing was presumed to occur at a Gln-Ala or Glu-Gly bond respectively, were not cleaved by the 3C proteinase. The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys----Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV-14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile. The HRV-14 3C proteinase probably plays an important role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy.
几种微小核糖核酸病毒的3C蛋白，包括脊髓灰质炎病毒、口蹄疫病毒（FMDV）和脑心肌炎病毒（EMCV），已被证明是半胱氨酸型蛋白酶，参与由单顺反子RNA基因组表达的各自多聚蛋白的加工过程。核苷酸测序数据表明，人鼻病毒14型（HRV-14）RNA基因组编码一种同源3C蛋白。从表达克隆的3C基因组片段的大肠杆菌中纯化出了纯的HRV-14 3C蛋白。针对与那些（通过核苷酸测序数据预测）在多聚蛋白内真实切割位点出现的残基相对应的肽段对该酶进行了测定。代表1B/1C、2A/2B、2C/3A、3A/3B、3B/3C和3C/3D切割位点的肽段，预计蛋白水解发生在谷氨酰胺-甘氨酸连接处，均被3C蛋白酶切割。通过反相快速蛋白质液相色谱和氨基酸分析表明，水解作用特异性地发生在每个肽段的谷氨酰胺-甘氨酸键处。这些方便底物的可得性有助于对3C蛋白酶进行进一步表征。相比之下，与预测的2B/2C和1C/1D切割位点相对应的肽段，推测加工过程分别发生在谷氨酰胺-丙氨酸或谷氨酸-甘氨酸键处，未被3C蛋白酶切割。如果在该蛋白中引入半胱氨酸→丝氨酸（146）突变，HRV-14 3C蛋白酶水解合成肽段的能力会受到抑制。使用已知蛋白酶抑制剂的研究证实了以下结论：HRV-14 3C蛋白似乎是一种半胱氨酸蛋白酶，第146位的半胱氨酸残基可能是活性位点亲核试剂。HRV-14 3C蛋白酶可能在病毒复制中发挥类似于脊髓灰质炎病毒3C蛋白酶的重要作用，因此是抗病毒化疗的一个潜在靶点。
