Yamshchikov V F, Compans R W
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322.
J Virol. 1995 Apr;69(4):1995-2003. doi: 10.1128/JVI.69.4.1995-2003.1995.
One of the late processing events in the flavivirus replication cycle involves cleavage of the intracellular form of the flavivirus capsid protein (Cint) to the mature virion form (Cvir) lacking the carboxy-terminal stretch of hydrophobic amino acids which serves as a signal peptide for the downstream prM protein. This cleavage event was hypothesized to be effected by a viral protease and to be associated with virion formation. We have proposed a model of flavivirus virion formation in which processing of the C-prM precursor at the upstream signalase site is upregulated by interaction of the NS2B part of the protease with the prM signal peptide or with an adjacent carboxy-terminal region of the capsid protein in the precursor, and processing of Cint by the NS2B-NS3 protease follows the signalase cleavage. Recently, an alternative hypothesis was proposed which suggests a reverse order of these two cleavage events, namely, that cleavage of the C-prM precursor by the NS2B-NS3 protease at the Cint-->Cvir dibasic cleavage site is a prerequisite for the subsequent signalase cleavage of the prM signal peptide. To distinguish between these alternative models, we prepared a series of expression cassettes carrying mutations at the Cint-->Cvir dibasic cleavage site and investigated the effects of these mutations on signalase processing of C-prM and on formation and secretion of prM-E heterodimers. For certain mutated C-prM precursors, namely, for those with Lys-->Gly disruption of the dibasic site, efficient formation of prM was observed upon expression from larger cassettes encoding the viral protease, despite the absence of processing at the Cint-->Cvir cleavage site. Surprisingly, formation and secretion of prM-E heterodimers accompanied by late cleavage of prM was also observed for these cassettes, with an efficiency comparable to that of the wild-type expression cassette. These observations contradict the model in which cleavage of the C-prM precursor at the Cint-->Cvir dibasic site is a prerequisite for signalase cleavage.
黄病毒复制周期中的晚期加工事件之一涉及将黄病毒衣壳蛋白的细胞内形式（Cint）切割成成熟病毒体形式（Cvir），后者缺乏作为下游prM蛋白信号肽的羧基末端疏水氨基酸延伸段。据推测，这种切割事件由病毒蛋白酶介导，并与病毒体形成有关。我们提出了一个黄病毒病毒体形成模型，其中上游信号肽酶位点处C-prM前体的加工通过蛋白酶的NS2B部分与前体中prM信号肽或衣壳蛋白相邻羧基末端区域的相互作用而上调，并且NS2B-NS3蛋白酶对Cint的加工跟随信号肽酶切割。最近，有人提出了另一种假说，认为这两个切割事件的顺序相反，即NS2B-NS3蛋白酶在Cint→Cvir双碱性切割位点对C-prM前体的切割是随后prM信号肽进行信号肽酶切割的先决条件。为了区分这些替代模型，我们制备了一系列在Cint→Cvir双碱性切割位点携带突变的表达盒，并研究了这些突变对C-prM的信号肽酶加工以及对prM-E异二聚体形成和分泌的影响。对于某些突变的C-prM前体，即那些双碱性位点的赖氨酸被甘氨酸取代的突变体，尽管在Cint→Cvir切割位点没有加工，但从编码病毒蛋白酶的较大表达盒中表达时，仍观察到prM的有效形成。令人惊讶的是，对于这些表达盒，还观察到了prM-E异二聚体的形成和分泌以及伴随的prM晚期切割，其效率与野生型表达盒相当。这些观察结果与C-prM前体在Cint→Cvir双碱性位点的切割是信号肽酶切割的先决条件这一模型相矛盾。
