Liu Ying, Franco David, Paul Aniko V, Wimmer Eckard
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
J Virol. 2007 Jun;81(11):5669-84. doi: 10.1128/JVI.02350-06. Epub 2007 Mar 14.
Poliovirus (PV) VPg is a genome-linked protein that is essential for the initiation of viral RNA replication. It has been well established that RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine (Y3) in VPg by the viral RNA polymerase 3D(pol), but it is not yet known whether the substrate for uridylylation in vivo is the free peptide itself or one of its precursors. The aim of this study was to use complementation analyses to obtain information about the true in vivo substrate for uridylylation by 3D(pol). Previously, it was shown that a VPg mutant, in which tyrosine 3 and threonine 4 were replaced by phenylalanine and alanine (3F4A), respectively, was nonviable. We have now tested whether wild-type forms of proteins 3B, 3BC, 3BCD, 3AB, 3ABC, and P3 provided either in trans or in cis could rescue the replication defect of the VPg(3F4A) mutations in the PV polyprotein. Our results showed that proteins 3B, 3BC, 3BCD, and P3 were unable to complement the RNA replication defect in dicistronic PV or dicistronic luciferase replicons in vivo. However, cotranslation of the P3 precursor protein allowed rescue of RNA replication of the VPg(3F4A) mutant in an in vitro cell-free translation-RNA replication system, but only poor complementation was observed when 3BC, 3AB, 3BCD, or 3ABC proteins were cotranslated in the same assay. Interestingly, only protein 3AB but not 3B and 3BC, when provided in cis by insertion of a wild-type 3AB coding sequence between the P2 and P3 domains of the polyprotein, supported the replication of the mutated genome in vivo. Elimination of cleavage between 3A and 3B in the complementing 3AB protein, however, led to a complete lack of RNA replication. Our results suggest that (i) VPg has to be delivered to the replication complex in the form of a large protein precursor (P3) to be fully functional in replication; (ii) the replication complex formed during PV replication in vivo is essentially inaccessible to proteins provided in trans, even if the complementing protein is translated from a different cistron of the same RNA genome; (iii) 3AB is the most likely precursor of VPg; and (iv) Y3 of VPg has an essential function in RNA replication in the context of both VPg and 3AB.
脊髓灰质炎病毒（PV）的基因组连接蛋白VPg对于病毒RNA复制的起始至关重要。目前已经明确，当一分子UMP通过病毒RNA聚合酶3D（pol）共价连接到VPg中酪氨酸（Y3）的羟基上时，RNA复制开始，但尚不清楚体内尿苷酸化的底物是游离肽本身还是其前体之一。本研究的目的是通过互补分析获取有关3D（pol）体内真正尿苷酸化底物的信息。此前研究表明，一种VPg突变体，其中酪氨酸3和苏氨酸4分别被苯丙氨酸和丙氨酸取代（3F4A），无法存活。我们现在测试了以反式或顺式提供的蛋白质3B、3BC、3BCD、3AB、3ABC和P3是否能够挽救PV多聚蛋白中VPg（3F4A）突变的复制缺陷。我们的结果表明，蛋白质3B、3BC、3BCD和P3在体内无法互补双顺反子PV或双顺反子荧光素酶复制子中的RNA复制缺陷。然而，P3前体蛋白的共翻译能够在体外无细胞翻译-RNA复制系统中挽救VPg（3F4A）突变体的RNA复制，但在相同实验中共翻译3BC、3AB、3BCD或3ABC蛋白时，仅观察到较差的互补作用。有趣的是，当通过在多聚蛋白的P2和P3结构域之间插入野生型3AB编码序列以顺式提供时，只有蛋白质3AB而不是3B和3BC能够支持体内突变基因组的复制。然而，互补的3AB蛋白中3A和3B之间切割的消除导致RNA复制完全缺失。我们的结果表明：（i）VPg必须以大蛋白前体（P3）的形式传递到复制复合体中才能在复制中完全发挥功能；（ii）即使互补蛋白是从同一RNA基因组的不同顺反子翻译而来，体内PV复制过程中形成的复制复合体基本上无法接受反式提供的蛋白质；（iii）3AB是VPg最可能的前体；（iv）在VPg和3AB的背景下，VPg的Y3在RNA复制中具有重要功能。
