Falgout B, Pethel M, Zhang Y M, Lai C J
Molecular Viral Biology Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J Virol. 1991 May;65(5):2467-75. doi: 10.1128/JVI.65.5.2467-2475.1991.
The cleavages at the junctions of the flavivirus nonstructural (NS) proteins NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 share an amino acid sequence motif and are presumably catalyzed by a virus-encoded protease. We constructed recombinant vaccinia viruses expressing various portions of the NS region of the dengue virus type 4 polyprotein. By analyzing immune precipitates of 35S-labeled lysates of recombinant virus-infected cells, we could monitor the NS2A/NS2B, NS2B/NS3, and NS3/NS4A cleavages. A polyprotein composed of NS2A, NS2B, and the N-terminal 184 amino acids of NS3 was cleaved at the NS2A/NS2B and NS2B/NS3 junctions, whereas a similar polyprotein containing only the first 77 amino acids of NS3 was not cleaved. This finding is consistent with the proposal that the N-terminal 180 amino acids of NS3 constitute a protease domain. Polyproteins containing NS2A and NS3 with large in-frame deletions of NS2B were not cleaved at the NS2A/NS2B or NS2B/NS3 junctions. Coinfection with a recombinant expressing NS2B complemented these NS2B deletions for NS2B/NS3 cleavage and probably also for NS2A/NS2B cleavage. Thus, NS2B is also required for the NS2A/NS2B and NS2B/NS3 cleavages and can act in trans. Other experiments showed that NS2B was needed, apparently in cis, for NS3/NS4A cleavage and for a series of internal cleavages in NS3. Indirect evidence that NS3 can also act in trans was obtained. Models are discussed for a two-component protease activity requiring both NS2B and NS3.
黄病毒非结构（NS）蛋白NS2A/NS2B、NS2B/NS3、NS3/NS4A和NS4B/NS5连接处的切割具有一个氨基酸序列基序，推测由病毒编码的蛋白酶催化。我们构建了表达登革病毒4型多聚蛋白NS区各部分的重组痘苗病毒。通过分析重组病毒感染细胞的35S标记裂解物的免疫沉淀物，我们可以监测NS2A/NS2B、NS2B/NS3和NS3/NS4A的切割情况。由NS2A、NS2B和NS3的N端184个氨基酸组成的多聚蛋白在NS2A/NS2B和NS2B/NS3连接处被切割，而仅包含NS3前77个氨基酸的类似多聚蛋白未被切割。这一发现与NS3的N端180个氨基酸构成蛋白酶结构域的提议一致。含有NS2A和NS3且NS2B有大片框内缺失的多聚蛋白在NS2A/NS2B或NS2B/NS3连接处未被切割。与表达NS2B的重组体共感染可补充这些NS2B缺失，以进行NS2B/NS3切割，可能也进行NS2A/NS2B切割。因此，NS2B对于NS2A/NS2B和NS2B/NS3切割也是必需的，并且可以反式作用。其他实验表明，NS2B显然顺式作用于NS3/NS4A切割以及NS3中的一系列内部切割。获得了NS3也可反式作用的间接证据。讨论了需要NS2B和NS3两者的双组分蛋白酶活性模型。
