EXPRESSION AND ANALYSIS OF THE HUMAN CYTOMEGALOVIRUS-UL80-ENCODED PROTEASE - IDENTIFICATION OF AUTOPROTEOLYTIC SITES
EXPRESSION AND ANALYSIS OF THE HUMAN CYTOMEGALOVIRUS-UL80-ENCODED PROTEASE - IDENTIFICATION OF AUTOPROTEOLYTIC SITES
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DOI：
10.1128/jvi.67.1.497-506.1993
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发表时间：
1993-01-01
影响因子：
5.4
通讯作者：
GLUZMAN, Y
中科院分区：
文献类型：
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作者：
BAUM, EZ;BEBERNITZ, GA;GLUZMAN, Y
人巨细胞病毒的45-kDa装配蛋白由UL 8 O开放阅读框（ORF）的C-末端部分编码。对于单纯疱疹病毒，DNA的包装伴随着其装配蛋白前体在其C末端附近的位点被由相同ORF的N末端区域编码的蛋白酶切割（F. Liu和B. Roizman，J. Virol. 65：5149-5156，1991）。通过与单纯疱疹病毒类比，我们研究了蛋白酶是否包含在人巨细胞病毒UL 80 ORF的N-末端部分。在噬菌体T7启动子的控制下，在大肠杆菌中表达整个UL 80 ORF。UL 80应该编码85 kDa的蛋白。相反，野生型构建体产生一组分子量为50、30、16、13和5 kDa的蛋白质。相反，当突变体UL 80缺失前14个氨基酸时，它仅产生85-kDa蛋白。这些结果表明，UL 80多聚蛋白进行自蛋白水解。我们通过缺失分析和N-末端测序证明30-kDa蛋白是蛋白酶，并且它起源于UL 80的N末端。UL 80多聚蛋白在以下三个位点被切割：（i）在装配蛋白结构域的C末端，（ii）在30-和50-kDa蛋白质之间，和（iii）在30-kDa蛋白酶本身内，这产生16-和13-kDa蛋白质，并且可能是使蛋白酶变性的机制。
The 45-kDa assembly protein of human cytomegalovirus is encoded by the C-terminal portion of the UL8O open reading frame (ORF). For herpes simplex virus, packaging of DNA is accompanied by cleavage of its assembly protein precursor at a site near its C terminus, by a protease encoded by the N-terminal region of the same ORF (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). By analogy with herpes simplex virus, we investigated whether a protease is contained within the N-terminal portion of the human cytomegalovirus UL80 ORF. The entire UL8O ORF was expressed in Escherichia coli, under the control of the phage T7 promoter. UL80 should encode a protein of 85 kDa. Instead, the wild-type construct produces a set of proteins with molecular masses of 50, 30, 16, 13, and 5 kDa. In contrast, when mutant UL80 is deleted of the first 14 amino acids, it produces only an 85-kDa protein. These results suggest that the UL80 polyprotein undergoes autoproteolysis. We demonstrate by deletional analysis and by N-terminal sequencing that the 30-kDa protein is the protease and that it originates from the N terminus of UL80. The UL8O polyprotein is cleaved at the following three sites: (i) at the C terminus of the assembly protein domain, (ii) between the 30- and 50-kDa proteins, and (iii) within the 30-kDa protease itself, which yields the 16- and 13-kDa proteins and may be a mechanism to inactivate the protease.