Departamento de Virología y Microbiología, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain.
PLoS One. 2010 May 20;5(5):e10735. doi: 10.1371/journal.pone.0010735.
Success of a viral infection requires that each infected cell delivers a sufficient number of infectious particles to allow new rounds of infection. In picornaviruses, viral replication is initiated by the viral polymerase and a viral-coded protein, termed VPg, that primes RNA synthesis. Foot-and-mouth disease virus (FMDV) is exceptional among picornaviruses in that its genome encodes 3 copies of VPg. Why FMDV encodes three VPgs is unknown.
we have constructed four mutant FMDVS that encode only one VPG: either VPg(1), VPg(3), or two chimeric versions containing part of VPg(1) and VPg(3). All mutants, except that encoding only VPg(1), were replication-competent. Unexpectedly, despite being replication-competent, the mutants did not form plaques on BHK-21 cell monolayers. The one-VPg mutant FMDVs released lower amounts of encapsidated viral RNA to the extracellular environment than wild type FMDV, suggesting that deficient plaque formation was associated with insufficient release of infectious progeny. Mutant FMDVs subjected to serial passages in BHK-21 cells regained plaque-forming capacity without modification of the number of copies of VPg. Substitutions in non-structural proteins 2C, 3A and VPg were associated with restoration of plaque formation. Specifically, replacement R55W in 2C was repeatedly found in several mutant viruses that had regained competence in plaque development. The effect of R55W in 2C was to mediate an increase in the extracellular viral RNA release without a detectable increase of total viral RNA that correlated with an enhanced capacity to alter and detach BHK-21 cells from the monolayer, the first stage of cell killing.
The results link the VPg copies in the FMDV genome with the cytopathology capacity of the virus, and have unveiled yet another function of 2C: modulation of picornavirus cell-to-cell transmission. Implications for picornaviruses pathogenesis are discussed.
病毒感染的成功需要每个感染的细胞都能提供足够数量的传染性颗粒，以允许新的感染周期。在小核糖核酸病毒中，病毒复制是由病毒聚合酶和一种称为 VPg 的病毒编码蛋白启动的，VPg 可以启动 RNA 合成。口蹄疫病毒（FMDV）在小核糖核酸病毒中是一个例外，因为它的基因组编码了 3 个 VPg。为什么 FMDV 编码 3 个 VPg 是未知的。
我们构建了四个只编码一个 VPg 的突变 FMDVS：VPg(1)、VPg(3)或两个包含 VPg(1)和 VPg(3)部分的嵌合版本。除了只编码 VPg(1)的突变体外，所有突变体都是复制有效的。出乎意料的是，尽管具有复制能力，但突变体在 BHK-21 细胞单层上不能形成噬菌斑。一个 VPg 的突变 FMDVs 释放到细胞外环境中的包裹病毒 RNA 的量低于野生型 FMDV，这表明缺陷性噬菌斑形成与传染性子代的释放不足有关。在 BHK-21 细胞中连续传代的突变 FMDVs 在不改变 VPg 拷贝数的情况下恢复了形成噬菌斑的能力。非结构蛋白 2C、3A 和 VPg 中的替换与恢复噬菌斑形成有关。具体来说，在几个恢复形成噬菌斑能力的突变病毒中，2C 中的 R55W 替换被反复发现。2C 中的 R55W 的作用是介导细胞外病毒 RNA 释放的增加，而不会导致总病毒 RNA 的可检测增加，这与增强改变和从单层上分离 BHK-21 细胞的能力相关，这是细胞杀伤的第一阶段。
结果将 FMDV 基因组中的 VPg 拷贝数与病毒的细胞病变能力联系起来，并揭示了 2C 的另一个功能：调节小核糖核酸病毒的细胞间传播。讨论了对小核糖核酸病毒发病机制的影响。
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