ROLES OF NONSTRUCTURAL POLYPROTEINS AND CLEAVAGE PRODUCTS IN REGULATING SINDBIS VIRUS-RNA REPLICATION AND TRANSCRIPTION
ROLES OF NONSTRUCTURAL POLYPROTEINS AND CLEAVAGE PRODUCTS IN REGULATING SINDBIS VIRUS-RNA REPLICATION AND TRANSCRIPTION
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DOI：
10.1128/jvi.67.4.1916-1926.1993
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发表时间：
1993-04-01
影响因子：
5.4
通讯作者：
RICE, CM
中科院分区：
文献类型：
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作者：
LEMM, JA;RICE, CM
使用牛痘病毒表达辛德毕斯病毒（SIN）的非结构蛋白（nsPs）和模板RNA，我们以前表明，所有三种病毒RNA的合成只发生在整个非结构编码区或多蛋白前体P123和P34的表达。在这份报告中，牛痘病毒系统被用来表达切割缺陷的多聚蛋白和nsP 4蛋白含有各种N-末端延伸，直接检查的P123和P34多聚蛋白在RNA复制中的作用。在P34和P123多聚蛋白的共表达过程中发生复制和亚基因组mRNA转录，其中切割在1/2和2/3位点中的一个或两个位点被阻断。然而，对于所有切割缺陷的P123多聚蛋白，亚基因组RNA与基因组RNA的比率降低，表明1/2和2/3切割对于有效的亚基因组RNA转录都是必需的。这些研究表明，未切割的P123多聚蛋白可以作为病毒复制酶的一个组成部分，能够合成正链和负链RNA。相反，切割缺陷的P34不能在RNA复制中起作用，即使在由nsP 4提供负链RNA的互补实验中也是如此。切割位点未改变的P34多聚蛋白只能在活性nsP 2蛋白酶存在下在RNA复制中起作用。虽然nsP 4，假定的RNA聚合酶，能够合成只有负链RNA在与P123共表达，只有22个上游残基添加到nsP 4允许复制和转录的亚基因组RNA发生。这些数据表明，nsP 3和nsP 4聚合酶的保守结构域不需要存在于P3/4多聚蛋白中以形成功能性正链复制酶-转录酶，并表明活性nsP 2蛋白酶和可切割的3/4位点的存在与所有病毒特异性RNA种类的合成相关。
Using vaccinia virus to express Sindbis virus (SIN) nonstructural proteins (nsPs) and template RNAs, we showed previously that synthesis of all three viral RNAs occurred only during expression of either the entire nonstructural coding region or the polyprotein precursors P123 and P34. In this report, the vaccinia virus system was used to express cleavage-defective polyproteins and nsP4 proteins containing various N-terminal extensions to directly examine the roles of the P123 and P34 polyproteins in RNA replication. Replication and subgenomic mRNA transcription occurred during coexpression of P34 and P123 polyproteins in which cleavage was blocked at either or both of the 1/2 and 2/3 sites. For all cleavage-defective P123 polyproteins, however, the ratio of subgenomic to genomic RNA was decreased, suggesting that both the 1/2 and 2/3 cleavages are required for efficient subgenomic RNA transcription. These studies indicate that the uncleaved P123 polyprotein can function as a component of the viral replicase capable of synthesizing both plus- and minus-strand RNAs. In contrast, cleavage-defective P34 was unable to function in RNA replication, even in complementation experiments in which minus-strand RNAs were provided by nsP4. A P34 polyprotein whose cleavage site was not altered could only function in RNA replication in the presence of an active nsP2 protease. Although nsP4, the putative RNA polymerase, was capable of synthesizing only minus-strand RNAs during coexpression with P123, the addition of only 22 upstream residues to nsP4 allowed both replication and transcription of subgenomic RNA to occur. These data show that the conserved domains of both nsP3 and the nsP4 polymerase do not need to be present in a P34 polyprotein to form a functional plus-strand replicase-transcriptase and suggest that the presence of an active nsP2 protease and a cleavable 3/4 site correlates with synthesis of all virus-specific RNA species.