Ansardi D C, Morrow C D
Department of Microbiology, University of Alabama at Birmingham 35294-0007.
J Virol. 1993 Dec;67(12):7284-97. doi: 10.1128/JVI.67.12.7284-7297.1993.
Assembly of poliovirus virions requires proteolytic cleavage of the P1 capsid precursor polyprotein between two separate glutamine-glycine (QG) amino acid pairs by the viral protease 3CD. In this study, we have investigated the effects on P1 polyprotein processing and subsequent assembly of processed capsid proteins caused by substitution of the glycine residue at the individual QG cleavage sites with valine (QG-->QV). P1 cDNAs encoding the valine substitutions were created by site-directed mutagenesis and were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses which expressed the mutant P1 precursors. The recombinant vaccinia virus-expressed mutant P1 polyproteins were analyzed for proteolytic processing defects in cells coinfected with a recombinant vaccinia virus (VVP3) that expresses the poliovirus 3CD protease and for processing and assembly defects by using a trans complementation system in which P1-expressing recombinant vaccinia viruses provide capsid precursor to a defective poliovirus genome that does not express functional capsid proteins (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The QV-substituted precursors were proteolytically processed at the altered sites both in cells coinfected with VVP3 and in cells coinfected with defective poliovirus, although the kinetics of cleavage at the altered sites were slower than those of cleavage at the wild-type QG site in the precursor. Completely processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor containing a valine at the amino terminus of VP3 (VP3-G001V) were unstable and failed to assemble stable subviral structures in cells coinfected with defective poliovirus. In contrast, capsid proteins derived from the P1 precursor with a valine substitution at the amino terminus of VP1 (VP1-G001V) assembled empty capsid particles but were deficient in assembling RNA-containing virions. The assembly characteristics of the VP1-G001V mutant were compared with those of a previously described VP3-VP1 cleavage site mutant (K. Kirkegaard and B. Nelsen, J. Virol. 64:185-194, 1990) which contained a deletion of the first four amino-terminal residues of VP1 (VP1-delta 1-4) and which was reconstructed for our studies into the recombinant vaccinia virus system. Complete proteolytic processing of the VP1-delta 1-4 precursor also occurred more slowly than complete cleavage of the wild-type precursor, and formation of virions was delayed; however, capsid proteins derived from the VP1-G001V mutant assembled RNA-containing virions less efficiently than those derived from the VP1-delta 1-4 precursor.(ABSTRACT TRUNCATED AT 400 WORDS)
脊髓灰质炎病毒病毒粒子的组装需要病毒蛋白酶3CD在两个分开的谷氨酰胺 - 甘氨酸（QG）氨基酸对之间对P1衣壳前体多聚蛋白进行蛋白水解切割。在本研究中，我们研究了将各个QG切割位点处的甘氨酸残基替换为缬氨酸（QG→QV）对P1多聚蛋白加工以及随后加工的衣壳蛋白组装的影响。通过定点诱变创建了编码缬氨酸替换的P1 cDNA，并将其重组到野生型痘苗病毒中，以产生表达突变P1前体的重组痘苗病毒。分析了重组痘苗病毒表达的突变P1多聚蛋白在与表达脊髓灰质炎病毒3CD蛋白酶的重组痘苗病毒（VVP3）共感染的细胞中的蛋白水解加工缺陷，以及通过使用反式互补系统分析其加工和组装缺陷，在该系统中，表达P1的重组痘苗病毒为不表达功能性衣壳蛋白的缺陷脊髓灰质炎病毒基因组提供衣壳前体（D.C.安萨尔迪、D.C.波特和C.D.莫罗，《病毒学杂志》67:3684 - 3690，1993）。在与VVP3共感染的细胞以及与缺陷脊髓灰质炎病毒共感染的细胞中，QV取代的前体在改变的位点进行了蛋白水解加工，尽管改变位点处的切割动力学比前体中野生型QG位点处的切割动力学慢。源自VP3氨基末端含有缬氨酸的突变前体（VP3 - G001V）的完全加工的衣壳蛋白VP0、VP3和VP1不稳定，并且在与缺陷脊髓灰质炎病毒共感染的细胞中未能组装成稳定的亚病毒结构。相比之下，源自VP1氨基末端缬氨酸取代的P1前体（VP1 - G001V）组装成空衣壳颗粒，但在组装含RNA病毒粒子方面存在缺陷。将VP1 - G001V突变体的组装特性与先前描述的VP3 - VP1切割位点突变体（K.柯克加德和B.尼尔森，《病毒学杂志》64:185 - 194，1990）进行了比较，该突变体包含VP1的前四个氨基末端残基的缺失（VP1 - δ1 - 4），并且为我们的研究重建到重组痘苗病毒系统中。VP1 - δ1 - 4前体的完全蛋白水解加工也比野生型前体的完全切割更慢，并且病毒粒子的形成延迟；然而，源自VP1 - G001V突变体的衣壳蛋白组装含RNA病毒粒子的效率低于源自VP1 - δ1 - 4前体的衣壳蛋白。（摘要截短至400字）
