Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity.
Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity.
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DOI：
10.1186/1743-422x-2-63
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发表时间：
2005-08-16
期刊：
影响因子：
4.8
通讯作者：
Hruby DE
中科院分区：
文献类型：
--
作者：
Byrd CM;Hruby DE
通过使用瞬时表达测定和定向遗传学，牛痘病毒（VV）I7 L基因产物已被认为是病毒体组装期间发生病毒核心蛋白裂解所需的主要成熟蛋白酶。为了证实这一假设，并使生物化学检查的I7 L半胱氨酸蛋白酶，在体外裂解试验的开发。使用VV感染的细胞的提取物作为酶的来源，开发了反应条件，其允许准确和有效地切割外源添加的核心蛋白前体（P4 a、P4 b和P25 K）。切割反应以时间依赖性方式进行，并且当在25°C下孵育时是最佳的。I7 L介导的裂解不受金属蛋白酶、天冬氨酸蛋白酶或丝氨酸蛋白酶的选定抑制剂（分别为EDTA、胃酶抑素和PMSF）的影响，但对几种一般半胱氨酸蛋白酶抑制剂（E-64、EST、碘乙酸和NEM）以及I7 L活性位点抑制剂TTP-6171敏感[C. Byrd等人，J. Virol. 78：12147-12156（2004）]。最后，在抗体下拉实验中，可以证明单特异性α I7 L血清耗尽了酶活性，而针对VV金属蛋白酶的对照血清（包括α G1 L）则没有。综上所述，这些数据提供了生物化学证据，即I7 L是一种直接参与VV核心蛋白切割的半胱氨酸蛋白酶。此外，建立这种I7 L介导的体外裂解试验，使未来的研究，酶和辅因子的蛋白水解反应的要求，并促进抗病毒药物的开发，对这个重要的目标。
Through the use of transient expression assays and directed genetics, the vaccinia virus (VV) I7L gene product has been implicated as the major maturational proteinase required for viral core protein cleavage to occur during virion assembly. To confirm this hypothesis and to enable a biochemical examination of the I7L cysteine proteinase, an in vitro cleavage assay was developed. Using extracts of VV infected cells as the source of enzyme, reaction conditions were developed which allowed accurate and efficient cleavage of exogenously added core protein precursors (P4a, P4b and P25K). The cleavage reaction proceeded in a time-dependent manner and was optimal when incubated at 25°C. I7L-mediated cleavage was not affected by selected inhibitors of metalloproteinases, aspartic acid proteinases or serine proteinases (EDTA, pepstatin, and PMSF, respectively), but was sensitive to several general cysteine proteinase inhibitors (E-64, EST, Iodoacetic acid, and NEM) as well as the I7L active site inhibitor TTP-6171 [C. Byrd et al., J. Virol. 78:12147–12156 (2004)]. Finally, in antibody pull down experiments, it could be demonstrated that monospecific αI7L serum depleted the enzyme activity whereas control sera including αG1L, directed against the VV metalloproteinase, did not. Taken together, these data provide biochemical evidence that I7L is a cysteine proteinase which is directly involved in VV core protein cleavage. Furthermore, establishment of this I7L-mediated in vitro cleavage assay should enable future studies into the enzymology and co-factor requirements of the proteolysis reaction, and facilitate antiviral drug development against this essential target.