Zoll J, van Kuppeveld F J, Galama J M, Melchers W J
Department of Medical Microbiology, University of Nijmegen, The Netherlands.
J Gen Virol. 1998 Jan;79 ( Pt 1):17-25. doi: 10.1099/0022-1317-79-1-17.
To examine the functional requirements of mengovirus 2A for virus reproduction, a series of mutants with overlapping deletions within the 2A region of mengovirus, and two chimeric constructs in which 2A is replaced either by Theiler's murine encephalomyelitis virus (TMEV) 2A or by coxsackie B3 virus (CBV3) 2Apro were generated. In vitro polyprotein synthesis showed that in both deletion mutants and the TMEV 2A chimeric construct, viral 3C protease (3Cpro)-mediated cleavage at the VP1-2A junction was disturbed, which resulted in decreased formation of mature capsid proteins and accumulation of the P1-2A precursor. 2Apro-mediated processing of the chimeric VP1-2Apro junction was highly efficient. Although the resulting L-P1 precursor was cleaved at the L-VP4 junction, further processing of the P1 precursor was abrogated. Two deletion mutant viruses and a TMEV 2A chimeric virus were obtained after transfection. The CBV 2Apro construct did not result in viable virus. Deletion mutant virus production was less than 3% compared to wild-type virus production, whereas chimeric virus production was reduced to 25%. Although inhibition of host-cell translation was identical in wild-type and mutant virus-infected cells, viral protein and RNA synthesis were reduced in cells infected with mutant virus, independently of the impaired P1-2A processing. It is concluded that mengovirus 2A may play a functional role in either virus translation or replication, and that the functional aspects of mengovirus and TMEV 2A cannot be exchanged. The results also confirm that the processing cascade of L-P1-2A occurs sequentially and is probably regulated by subsequent conformational transitions of the cleavage products after each proteolytic event. The sequential release of L and 2A may be essential in the context of their function in virus replication.
为研究脑心肌炎病毒2A对病毒复制的功能需求，构建了一系列脑心肌炎病毒2A区域内有重叠缺失的突变体，以及两个嵌合构建体，其中2A分别被泰勒氏鼠脑脊髓炎病毒（TMEV）2A或柯萨奇B3病毒（CBV3）2Apro取代。体外多蛋白合成显示，在缺失突变体和TMEV 2A嵌合构建体中，病毒3C蛋白酶（3Cpro）介导的VP1-2A连接处的切割均受到干扰，导致成熟衣壳蛋白形成减少和P1-2A前体积累。2Apro介导的嵌合VP1-2Apro连接处的加工效率很高。虽然产生的L-P1前体在L-VP4连接处被切割，但P1前体的进一步加工被消除。转染后获得了两种缺失突变病毒和一种TMEV 2A嵌合病毒。CBV 2Apro构建体未产生有活力的病毒。与野生型病毒产生相比，缺失突变病毒产生量不到3%，而嵌合病毒产生量降至25%。虽然野生型和突变病毒感染的细胞中宿主细胞翻译抑制情况相同，但感染突变病毒的细胞中病毒蛋白和RNA合成减少，与受损的P1-2A加工无关。结论是脑心肌炎病毒2A可能在病毒翻译或复制中发挥功能作用，且脑心肌炎病毒和TMEV 2A的功能方面不能相互交换。结果还证实L-P1-2A的加工级联是顺序发生的，可能受每次蛋白水解事件后切割产物后续构象转变的调节。L和2A的顺序释放可能在它们在病毒复制中的功能背景下至关重要。
