Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans

Author:

Hardy W R1,Strauss J H1

Affiliation:

1. Division of Biology, California Institute of Technology, Pasadena 91125.

Abstract

The processing of the Sindbis virus nonstructural polyprotein translated in vitro has been studied. When Sindbis virus genomic RNA was translated in a reticulocyte lysate, polyprotein P123 was cleaved efficiently to produce nsP1, nsP2, and nsP3. Inhibition of this processing by anti-nsP2 antibodies, but not by antibodies specific for nsP1, nsP3, or nsP4, suggested that the viral proteinase was present in nsP2. To localize the proteolytic activity more precisely, deletions were made in a full-length cDNA clone of Sindbis virus, and RNA was transcribed from these constructs with SP6 RNA polymerase and translated in vitro. Although virtually all of the nsP1, nsP3, and nsP4 sequences could be deleted without affecting processing, deletions in the N-terminal half of nsP2 led to aberrant processing, and deletions in the C-terminal half abolished proteolysis. However, inactive polyproteins containing the nsP2 deletions could be processed by exogenously supplied proteins translated from virion RNA, demonstrating that cleavage was virus specific and not due to a protease present in the reticulocyte lysate and that the deleted polyproteins still served as substrates for the enzyme. From these results and from experiments in which processing was studied at increasingly higher dilution, we have concluded the following: (i) the viral nonstructural proteinase is located in the C-terminal half of nsP2; (ii) in the P123 precursor the cleavage between nsP2 and nsP3 occurs efficiently as a bimolecular reaction (in trans) to remove nsP3, while the bond between nsP1 and nsP2 is cleaved inefficiently, but detectably, in trans, but no autoproteolysis of P123 was detected; (iii) once nsP3 has been removed, the bond between nsP1 and nsP2 in the P12 precursor is cleaved efficiently by autoproteolysis (in cis). This mode of processing leads to a slow rate of cleavage, particularly early in infection, suggesting that the polyproteins might play roles in virus RNA replication distinct from those of the cleaved products. A hypothesis is presented that the proteinase is a thiol protease related to papain.

Publisher

American Society for Microbiology

Subject

Virology,Insect Science,Immunology,Microbiology

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3