Polynucleotide modulation of the protease, nucleoside triphosphatase, and helicase activities of a hepatitis C virus NS3-NS4A complex isolated from transfected COS cells
Polynucleotide modulation of the protease, nucleoside triphosphatase, and helicase activities of a hepatitis C virus NS3-NS4A complex isolated from transfected COS cells
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DOI：
10.1128/jvi.71.5.3767-3775.1997
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发表时间：
1997-05-01
影响因子：
5.4
通讯作者：
Thomson, JA
中科院分区：
文献类型：
--
作者：
Morgenstern, KA;Landro, JA;Thomson, JA
丙型肝炎病毒（HCV）非结构蛋白3（NS 3）是一种70 kDa的多功能酶，具有三种已知的催化活性，分离在两个有点独立的结构域。丝氨酸蛋白酶的基本机制位于蛋白质的N-末端三分之一，核苷三磷酸酶（NTH）和解旋酶活性位于剩余的C-末端区域。NS 4A是在病毒多蛋白中紧邻NS 3下游表达的54个残基的蛋白质，并且NS 4A中疏水残基的中心延伸形成NS 3丝氨酸蛋白酶结构域的完整结构组分。没有证据表明NS 3的两个结构域通过体内蛋白水解加工而分离。这可能反映了病毒基本复制成分的经济包装，但这也可能意味着这两个结构域之间存在功能上的相互依赖。在本研究中，在瞬时转染的COS细胞中表达和自动加工后分离全长NS 3-NS 4A复合物。该蛋白质用于检查多核苷酸对NTR酶、解旋酶和蛋白酶活性的影响。与先前报道的单独表达的NS 3解旋酶结构域的行为不同，完整的NS 3-NS 4A复合物在pH 7.5和8.5之间表现出最佳的NTR活性。所有三种NS 3-NS 4A活性都受到多核苷酸的调节，其中poly（U）具有最显著的效果。这些发现表明，NS 3内的结构域可能会影响彼此的活性，并且HCV基因组元件的相互作用可能会调节这种复杂的HCV复制酶组分的酶活性。
The hepatitis C virus (HCV) nonstructural 3 protein (NS3) is a 70-kDa multifunctional enzyme with three known catalytic activities segregated in two somewhat independent domains. The essential machinery of a serine protease is localized in the N-terminal one-third of the protein, and nucleoside triphosphatase (NTPase) and helicase activities reside in the remaining C-terminal region. NS4A is a 54-residue protein expressed immediately downstream of NS3 in the viral polyprotein, and a central stretch of hydrophobic residues in NS4A form an integral structural component of the NS3 serine protease domain. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic processing in vivo. This may reflect economical packaging of essential viral replicative components, but it could also mean that there is functional interdependence between the two domains. In this study, a full-length NS3-NS4A complex was isolated after expression and autoprocessing in transiently transfected COS cells. The protein was used to examine the effects of polynucleotides on the NTPase, helicase, and protease activities. Unlike the previously reported behavior of a separately expressed NS3 helicase domain, the full NS3-NS4A complex demonstrated optimal NTPase activity between pH 7.5 and 8.5. All three NS3-NS4A activities were modulated by polynucleotides, with poly(U) having the most remarkable effect. These findings suggest that the domains within NS3 may influence the activity of one another and that the interplay of HCV genomic elements may regulate the enzyme activities of this complex HCV replicase component.