Tzeng Wen-Pin, Frey Teryl K
Department of Biology, Georgia State University, PO Box 4010, Atlanta, GA 30302-4010, USA.
Virology. 2005 Jul 5;337(2):327-34. doi: 10.1016/j.virol.2005.04.019.
The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA.
表达绿色荧光蛋白报告基因的风疹病毒（RUB）复制子（RUBrep/GFP）合成的亚基因组（SG）RNA与基因组RNA的比例，显著高于风疹病毒（RUB）合成的这些RNA种类的比例（RUBrep/GFP为4.3，而RUB为1.3 - 1.4）。据推测，病毒RNA合成的这种调节是由病毒结构蛋白基因之一引起的，并且发现将衣壳（C）蛋白基因作为与GFP的读码框内融合引入复制子中，会导致基因组RNA产量增加（降低SG/基因组RNA比例），证实了这一推测，并表明C基因是负责调节作用的部分。C基因的N端三分之一是发挥该作用所必需的。在相关的甲病毒辛德毕斯病毒的复制子中未观察到类似现象。有趣的是，当RUBrep/GFP与表达C基因的其他RUBrep或质粒构建体共转染时，未观察到调节作用，这表明调节仅在顺式提供C基因时才会发生。阻止C蛋白翻译的突变未能调节RNA合成，表明C蛋白是负责调节的部分；与此结论一致，当在C基因的5'端引入同义密码子突变以改变C基因序列而不改变C蛋白的氨基酸序列时，RNA合成的调节得以维持。这些结果表明，在病毒复制复合物附近翻译的C蛋白，可能来自新合成的SG RNA，参与调节病毒RNA的复制。
