DENGUE-2 VIRUS NS2B AND NS3 FORM A STABLE COMPLEX THAT CAN CLEAVE NS3 WITHIN THE HELICASE DOMAIN
DENGUE-2 VIRUS NS2B AND NS3 FORM A STABLE COMPLEX THAT CAN CLEAVE NS3 WITHIN THE HELICASE DOMAIN
复制标题
DOI：
10.1006/viro.1993.1198
复制
发表时间：
1993-04-01
期刊：
影响因子：
3.7
通讯作者：
STRAUSS, JH
中科院分区：
文献类型：
--
作者：
ARIAS, CF;PREUGSCHAT, F;STRAUSS, JH
黄病毒基因组RNA被翻译成一个大的多聚蛋白，该多聚蛋白被加工成结构蛋白和非结构蛋白。几种非结构蛋白的N-末端是通过由NS 2B和NS 3组成的双组分病毒蛋白酶在二元位点切割产生的。NS 3在其N-末端含有胰蛋白酶样丝氨酸蛋白酶结构域，而NS 2B在蛋白水解中的功能尚未确定。在非变性条件下，我们用NS 3特异性抗血清证明了NS 2B和NS 3在体外和体内形成复合物。NS 3的N端184个残基足以与NS 2B形成复合物。当NS 2B和NS 3从两种不同的mRNA翻译时，以及当NS 2B和NS 3从相同的mRNA翻译为多蛋白时，复合物有效地形成。嵌合复合物可以在黄热病NS 2B和嵌合黄热病-登革2型NS 3之间形成。使用抗NS 3抗血清，我们还发现，50 kDa的片段的NS 3，由N-末端约460个残基，是在感染的哺乳动物细胞中产生。该片段不在感染的蚊子细胞中产生，但将在蚊子细胞的Triton X-100裂解物中形成。NS 3裂解形成该片段由NS 3蛋白酶本身催化，蛋白水解需要NS 2B。NS 3的氨基酸序列的检查揭示了一个潜在的保守切割位点，类似于由NS 3/NS 2B蛋白酶切割的其他位点;该位点发生在保守的RNA解旋酶序列基序内。NS 3的这种替代加工形式的重要性及其在登革病毒复制周期中的作用仍有待确定。
Flavivirus genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. The N-termini of several nonstructural proteins are produced by cleavage at dibasic sites by a two-component viral proteinase consisting of NS2B and NS3. NS3 contains a trypsin-like serine proteinase domain at its N-terminus, whereas the function of NS2B in proteolysis is yet to be determined. We have used an NS3-specific antiserum, under nondenaturing conditions, to demonstrate that NS2B and NS3 form a complex bothin vitroandin vivo. The N-terminal 184 residues of NS3 are sufficient to form the complex with NS2B. The complex forms efficiently when the NS2B and NS3 are translated from two different mRNAs as well as when NS2B and NS3 are translated as a polyprotein from the same mRNA. A chimeric complex can be formed between yellow fever NS2B and a chimeric yellow fever-dengue 2 NS3. Using anti-NS3 antisera, we also found that a 50-kDa fragment of NS3, consisting of the N-terminal approximately 460 residues, is produced in infected mammalian cells. This fragment is not produced in infected mosquito cells, but will form in Triton X-100 lysates of mosquito cells. The cleavage of NS3 to form this fragment is catalyzed by the NS3 proteinase itself and proteolysis requires NS2B. Examination of the amino acid sequence of NS3 reveals a potential conserved cleavage site that resembles other sites cleaved by the NS3/NS2B proteinase; this site occurs within a conserved RNA helicase sequence motif. The importance of this alternatively processed form of NS3 and its role in the replication cycle of dengue virus remain to be determined.