Molecular anatomy of plant viral genome replication machinery for the development of cell-free viral replication system.

1.烟草花叶病毒复制复合体的分子解剖：烟草花叶病毒RNA依赖的RNA聚合酶(RdRp)与宿主蛋白激酶(NtPERK1)、蛋白磷酸酶2C(NtPP2C-2)、精氨酸脱羧酶(ADC)和环指蛋白(PHF15)特异地相互作用。此外，伸长因子eEF1a还与TMV RdRp和TMV RNA的3‘-UTR直接结合，SbCMV复制复合体的分子解剖：大豆褪绿斑驳病毒(SbCMV)的ORFⅡ产物显示了病毒致病所必需的自身相互作用。3.ASGV复制复合体的分子解剖：苹果茎沟病毒(ASGV)通过移码策略表达其蛋白，可变区参与了其致病。4.TVX复制复合体的分子解剖表明，马铃薯X病毒属于郁金香病毒X属(TVX)5.小麦花叶病毒复制复合体的分子解剖及建立无细胞复制系统的试验：土传小麦花叶病毒在17℃时的复制效率高于15℃或20℃。P19富含半胱氨酸的蛋白质是一种RNA沉默抑制因子，它促进了感染后期转基因细胞中RNA的积累，以及病毒RNA在细胞之间的移动。以小麦胚芽提取液为模板，以T7启动子为模板，以RNA1为模板，体外翻译SBWMV RNA1。证实在24℃时，p152基因的UGA终止密码子的翻译通读可以在体内发生。

This study was intended to analyze the molecular structure of viral replication complexes (VRC) of 5 different plant viruses for developing the cell-free viral replication systems.1.Molecular anatomy of TMV replication complex :Tobacco mosaic virus RNA dependent RNA polymerase (RdRp) was shown to interact specifically with the host protein kinase (NtPERK1), protein phosphatase 2C (NtPP2C-2), arginine decarboxylase (ADC) and ring-finger protein (PHF15) those might regulate the viral replication. Also elongation factor eEF1A was shown to bind directly with TMV RdRp as well as 3'-UTR of TMV RNA, which indicated that eEF1A is one of the essential factors of VRC.2.Molecular anatomy of SbCMV replication complex :ORF II product of Soybean chlorotic mottle virus (SbCMV) showed the self-interaction that was indispensable to viral pathogenicity.3.Molecular anatomy of ASGV replication complex :Apple stem grooving virus (ASGV) was revealed to express its proteins through the frameshift strategy and the variable region was shown to be involved in its pathogenicity.4.Molecular anatomy of TVX replication complex :Potato virus X belonging to the same genus of Tulip virus X (TVX) showed that the C-terminus region of the replicase and 5'untranslated region were responsible for different types of symptoms.5.Molecular anatomy of SBWMV replication complex and trial for developing cell-free replication system :Replication of Soil-borne wheat mosaic virus (SBWMV) was more efficient at 17℃ than at 15℃ or 20℃. The p19 cysteine-rich protein, an RNA silencing suppressor, facilitated RNA accumulation in transfected cells late in infection as well as cell-to-cell movement of viral RNA. SBWMV RNA1 was translated in vitro in transcription-coupled wheat germ extracts using RNA1 cDNA with the upstream T7 promoter as a template. It was confirmed that translational readthrough at the UGA termination codon of the p152 gene could occur as in vivo at 24℃.