Forng R Y, Frey T K
Department of Biology, Georgia State University, Atlanta 30302-4010.
Virology. 1995 Feb 1;206(2):843-53. doi: 10.1006/viro.1995.1007.
Five segments of the rubella virus (RUB) nonstructural protein open reading frame (NSP-ORF) were cloned into pATH (trpE) bacterial fusion protein expression plasmid vectors. Antisera raised in rabbits against these fusion proteins were used to identify RUB nonstructural polypeptides in lysates from RUB-infected Vero cells and from BHK cells transfected with pTM3/nsRUB, a vector from which the RUB NSP-ORF is expressed. In both systems, three polypeptides were immunoprecipitated. A 200-kDa polypeptide (P200) was immunoprecipitated by all of the antisera and therefore is the primary translation product of the ORF. A 150-kDa polypeptide (P150) was immunoprecipitated by antisera against fusion proteins containing N-terminal regions of the ORF, and a 90-kDa polypeptide (P90) was immunoprecipitated by sera against fusion proteins containing C-terminal regions of the ORF. The order of these polypeptides within the NSP-ORF is thus NH2-P150-P90-COOH. It was shown in a previous study that a protease within P200 catalyzes cleavage of P200 (L. D. Marr et al., Virology 198, 586-592, 1994). When hypertonic block was used to synchronize initiation of translation in RUB-infected cells, P200 was detected within 10 min following release of the block, while P150 and P90 were not detected until 20 min, indicating that translation of the precursor is completed before proteolytic cleavage occurs. Pulse-chase experiments showed that cleavage of the P200 precursor was complete within 90 min of synthesis. P150 was stable for 24 hr following processing, while turnover of P90 was detected within 90 min. In immunofluorescence experiments on RUB-infected cells, antisera that recognized P150 stained a perinuclear focus, a thread-like cytoplasmic structure, and the nuclear membrane.
将风疹病毒（RUB）非结构蛋白开放阅读框（NSP - ORF）的五个片段克隆到pATH（trpE）细菌融合蛋白表达质粒载体中。用针对这些融合蛋白在兔体内产生的抗血清来鉴定来自RUB感染的Vero细胞裂解物以及用pTM3/nsRUB转染的BHK细胞裂解物中的RUB非结构多肽，pTM3/nsRUB是一个表达RUB NSP - ORF的载体。在这两个系统中，三种多肽被免疫沉淀。一种200 kDa的多肽（P200）被所有抗血清免疫沉淀，因此是该开放阅读框的主要翻译产物。一种150 kDa的多肽（P150）被针对包含开放阅读框N端区域的融合蛋白的抗血清免疫沉淀，一种90 kDa的多肽（P90）被针对包含开放阅读框C端区域的融合蛋白的血清免疫沉淀。因此，这些多肽在NSP - ORF内的顺序是NH2 - P150 - P90 - COOH。在先前的一项研究中表明，P200内的一种蛋白酶催化P200的切割（L.D. Marr等人，《病毒学》198，586 - 592，1994）。当使用高渗阻滞来同步RUB感染细胞中的翻译起始时，在解除阻滞10分钟内检测到P200，而直到20分钟才检测到P150和P90，这表明前体的翻译在蛋白水解切割发生之前完成。脉冲追踪实验表明，P200前体的切割在合成后90分钟内完成。加工后P150稳定24小时，而在90分钟内检测到P90的周转。在对RUB感染细胞的免疫荧光实验中，识别P150的抗血清染色出一个核周聚焦区、一种丝状细胞质结构和核膜。
