Enzymatic characterization of hepatitis C virus NS3/4A complexes expressed in mammalian cells by using the herpes simplex virus amplicon system
Enzymatic characterization of hepatitis C virus NS3/4A complexes expressed in mammalian cells by using the herpes simplex virus amplicon system
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DOI：
10.1128/jvi.70.7.4261-4268.1996
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发表时间：
1996-07-01
影响因子：
5.4
通讯作者：
Kwong, AD
中科院分区：
文献类型：
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作者：
Hong, Z;Ferrari, E;Kwong, AD
丙型肝炎病毒（HCV）NS 3蛋白具有三种酶活性：N-末端丝氨酸蛋白酶活性、C-末端RNA刺激的NTR酶活性和RNA解旋酶活性。为了表征它们，在哺乳动物细胞中用HSV扩增子缺陷型病毒表达全长NS 3（631）/4A和三种C末端截短的蛋白酶（NS 3（201）/4A、NS 3（181）/4A和NS 3（155）/4A），我们的研究结果表明，所有在哺乳动物细胞中产生的NS 3/4A蛋白，（除了NS 3（155）/4A）在处理顺式和反式切割位点中是有活性的。温度优化研究表明，该蛋白酶在4至25摄氏度的温度范围内更具活性，在42摄氏度时完全失活。RNA刺激的ATP酶活性用部分纯化的NS 3（631）/4A级分表征，并且在37至42 ℃具有更高的最适温度。洗涤剂对NS 3蛋白酶和RNA刺激的ATP酶的影响相似。非离子型去污剂如Triton X-100、Nonidet P-40和Tween 20不影响活性，而阴离子型去污剂如十二烷基硫酸钠和脱氧胆酸则具有抑制作用。两性离子去污剂如3-[（3-胆酰胺丙基）-二甲基-铵基]-1-丙磺酸盐（CHAPS）在0.5%（8 mM）的浓度下抑制蛋白酶活性，这对ATP酶活性没有影响。最后，在NS 3（631）/4A级分中证明了RNA解旋活性，但在类似纯化的NS 3（181）/4A和NS 3（201）/4A级分中没有。NS 3（631）/4A解旋具有3'而非5'单链突出端的RNA双链体，表明NS 3 RNA解旋酶以3 '至5'方向起作用。
The hepatitis C virus (HCV) NS3 protein possesses three enzymatic activities: an N-terminal serine protease activity, a C-terminal RNA-stimulated NTPase activity, and an RNA helicase activity. To characterize them, the full-length NS3(631)/4A and three C-terminal truncated proteases (NS3(201)/4A, NS3(181)/4A, and NS3(155)/4A) were expressed in mammalian cells with HSV amplicon-defective viruses, Our results revealed that all of the NS3/4A proteins produced in mammalian cells (except NS3(155)/4A) are active in processing both cis and trans cleavage sites. Temperature optimization studies revealed that the protease is more active at temperatures ranging from 4 to 25 degrees C and is completely inactive at 42 degrees C. The RNA-stimulated ATPase activity was characterized with a partially purified NS3(631)/4A fraction and has a higher optimal temperature at 37 to 42 degrees C. The effects of detergents on both NS3 protease and RNA-stimulated ATPase were similar. Nonionic detergents such as Triton X-100, Nonidet P-40 and Tween 20 did not affect the activities, while anionic detergents such as sodium dodecyl sulfate and deoxycholic acid were inhibitory. Zwitterionic detergent such as 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) inhibited protease activity at a concentration of 0.5% (8 mM), which had no effect on ATPase activity, Finally, RNA-unwinding activity was demonstrated in the NS3(631)/4A fraction but not in the similarly purified NS3(181)/4A and NS3(201)/4A fractions. NS3(631)/4A unwinds RNA duplexes with 3' but not 5' single-stranded overhangs, suggesting that the NS3 RNA helicase functions in a 3'-to-5' direction.