INTRACELLULAR PROCESSING OF THE N-TERMINAL ORF-1A PROTEINS OF THE CORONAVIRUS MHV-A59 REQUIRES MULTIPLE PROTEOLYTIC EVENTS
INTRACELLULAR PROCESSING OF THE N-TERMINAL ORF-1A PROTEINS OF THE CORONAVIRUS MHV-A59 REQUIRES MULTIPLE PROTEOLYTIC EVENTS
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DOI：
10.1016/0042-6822(92)90703-r
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发表时间：
1992-07-01
期刊：
影响因子：
3.7
通讯作者：
WEISS, SR
中科院分区：
文献类型：
--
作者：
DENISON, MR;ZOLTICK, PW;WEISS, SR
使用针对 ORF1a 前 6.5 kb 编码的融合蛋白的抗血清，在 MHV-A59 感染的 DBT 细胞中表征了 MHV-A59 ORF 1a 的几种多肽产物。其中包括先前鉴定的 N 端 ORF 1a 产物、p28，以及 290、240 和 50 kDa 多肽。 P28 始终被检测为不连续的条带，没有较大的前体，这表明 p28 在合成后立即快速裂解。 p28 被切割后，在 2 小时内蛋白质几乎没有降解。与基因组 RNA 体外翻译过程中获得的结果相反，p28 的细胞内裂解不受蛋白酶抑制剂亮肽素的抑制（Denison 和 Perlman，1986）。这些数据表明不同的蛋白酶活性可能导致p28在体外和体内的裂解。 290-kDa 蛋白质是源自大于 400 kDa 的前体的中间裂解产物。 290-kDa 产物随后被切割成 50 和 240 kDa 的二级产物。 290-kDa多肽的细胞内切割被亮抑肽酶抑制，其浓度不抑制p28的早期切割或290-kDa产物从其较大的多蛋白前体的切割。在氯化锌存在的情况下，检测到 >320 kDa 的产物，该产物似乎在其氨基末端掺入了 p28。这表明 ORF1a 蛋白的加工可能需要至少两种蛋白酶活性，其中一种可裂解 p28，对氯化锌敏感但对亮肽素具有抗性，另一种可裂解 290-kDa 前体并对两种抑制剂敏感。 290 kDa 和 240 kDa 蛋白质均应包含预测编码两种木瓜蛋白酶样蛋白酶活性的序列。
Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusionn roteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained duringin vitrotranslation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28in vitroandin vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of >320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities.