Kusov Y, Gauss-Müller V
Institute for Medical Microbiology and Hygiene, Medical University of Lübeck, Lübeck, Germany.
J Virol. 1999 Dec;73(12):9867-78. doi: 10.1128/JVI.73.12.9867-9878.1999.
The orchestrated liberation of viral proteins by 3C(pro)-mediated proteolysis is pivotal for gene expression by picornaviruses. Proteolytic processing is regulated either by the amino acid sequence at the cleavage site of the substrate or by cofactors covalently or noncovalently linked to the viral proteinase. To determine the role of the amino acid sequence at cleavage sites 3A/3B and 3B/3C that are essential for the liberation of 3C(pro) from its precursors and to assess the function of the stable processing intermediates 3AB and 3ABC, we studied the effect of cleavage site mutations on hepatitis A virus (HAV) polyprotein processing, particle formation, and replication. Using the recombinant vaccinia virus system, we showed that the normally retarded cleavage at the 3A/3B junction can be improved by altering the amino acid sequence at the scissile bond such that it matches the preferred HAV 3C cleavage sites. In contrast to the processing products of the wild-type polyprotein, 3ABC was no longer detectable in the mutant. VP0 and VP3 were generated less efficiently, implying that processing of the structural protein precursor P1-2A depends on the presence of stable 3ABC and/or 3AB. In addition, cleavage of 2BC was impaired in 3AB/3ABC-deficient mutants. Formation of HAV particles was not affected in mutants with blocked 3A/3B and/or 3B/3C cleavage sites. However, 3ABC-deficient mutants produced small numbers of HAV particles, which could be augmented by coexpressing 3AB or 3ABC. The hydrophobic domain of 3A that has been proposed to mediate membrane anchorage of the replication complex was crucial for restoration of defective particle formation. In vitro transcripts of the various cleavage site mutants were unable to initiate an infectious cycle, and no progeny viruses were obtained even after blind passages. Taken together, the data suggest that accumulation of uncleaved HAV 3AB and/or 3ABC is pivotal for both viral replication and efficient particle formation.
由3C（蛋白酶）介导的蛋白水解作用精心安排病毒蛋白的释放，这对于小核糖核酸病毒的基因表达至关重要。蛋白水解加工过程受底物切割位点的氨基酸序列调控，或者受与病毒蛋白酶共价或非共价连接的辅因子调控。为了确定3A/3B和3B/3C切割位点的氨基酸序列在从其前体释放3C（蛋白酶）过程中的作用，并评估稳定加工中间体3AB和3ABC的功能，我们研究了切割位点突变对甲型肝炎病毒（HAV）多聚蛋白加工、颗粒形成和复制的影响。使用重组痘苗病毒系统，我们发现通过改变可裂解键处的氨基酸序列使其与甲型肝炎病毒3C的优选切割位点匹配，可以改善3A/3B连接处通常延迟的切割。与野生型多聚蛋白的加工产物不同，在突变体中不再能检测到3ABC。VP0和VP3的产生效率较低，这意味着结构蛋白前体P1-2A的加工依赖于稳定的3ABC和/或3AB的存在。此外，在缺乏3AB/3ABC的突变体中，2BC的切割受到损害。3A/3B和/或3B/3C切割位点被阻断的突变体中，甲型肝炎病毒颗粒的形成不受影响。然而，缺乏3ABC的突变体产生少量甲型肝炎病毒颗粒，通过共表达3AB或3ABC可以增加颗粒数量。已提出介导复制复合物膜锚定的3A疏水结构域对于恢复有缺陷的颗粒形成至关重要。各种切割位点突变体的体外转录本无法启动感染周期，即使经过盲目传代也未获得子代病毒。综上所述，数据表明未切割的甲型肝炎病毒3AB和/或3ABC的积累对于病毒复制和有效颗粒形成均至关重要。
