Investigations on the Tobacco Necrosis Virus D p60 Replicase Protein

Tobacco Necrosis Virus D (TNV-D), in the genus Betanecrovirus (family Tombusviridae), possesses a single-stranded, positive-sense RNA genome containing six open reading frames (ORFs). Two 5'-proximal ORFs (1 and 2) encode overlapping polypeptides of 22 and 82 kDa (p22 and p82, respectively) which are both required for replication. The p22 auxiliary protein contains no replication motifs but the C-terminal region, downstream of a leaky stop codon, encodes a 60 kDa polypeptide (p60) which contains conserved RNA-dependent RNA polymerase (RdRP) motifs. Here we have expressed and purified recombinant p60 and show that in vitro it binds and efficiently synthesises both TNV-D RNA and Satellite tobacco necrosis virus C RNA. Alanine scanning mutagenesis of conserved amino acids in characteristic motifs in p60 revealed that some mutations significantly reduced RNA synthesis but mutating the second asparagine residue in the conserved GDD box was lethal. The effects of mutating identical amino acids in p60 on virus replication in vivo were examined in Nicotiana benthamiana plants following infection with RNA transcribed from wild type (wt) and mutant constructs. In inoculated leaves the behaviour of the mutants paralleled the in vitro data but systemic infection was precluded in all but one mutant which had reverted to wt. This study is the first to demonstrate the nucleic acid-binding and synthetic capabilities of a betanecrovirus polymerase.

烟草坏死病毒D（Tobacco Necrosis Virus D, TNV-D）隶属于番茄丛矮病毒科（Tombusviridae）下的贝塔坏死病毒属（Betanecrovirus），其基因组为单股正链RNA，包含6个开放阅读框（ORFs）。两个位于5'近端的开放阅读框（ORF1和ORF2）分别编码22 kDa和82 kDa的重叠多肽（分别为p22与p82），二者均为病毒复制所必需。p22辅助蛋白不具备复制基序，但在渗漏终止密码子下游的C端区域，可编码一个60 kDa的多肽（p60），该多肽包含保守的RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase, RdRP）基序。本研究对重组p60蛋白进行了表达与纯化，并证实其在体外可结合并高效合成烟草坏死病毒D RNA以及卫星烟草坏死病毒C RNA。对p60特征基序内的保守氨基酸进行丙氨酸扫描诱变后发现，部分突变可显著降低RNA合成效率，而突变保守GDD基序中的第二个天冬酰胺残基则会导致其功能完全丧失。本研究还利用野生型（wt）及突变体构建物转录得到的RNA接种本氏烟（Nicotiana benthamiana），探究了p60中相同氨基酸突变对病毒体内复制的影响。结果显示，接种叶片中突变体的表现与体外实验结果一致，但除一株恢复为野生型的突变体外，其余所有突变体均无法实现系统侵染。本研究首次证实了贝塔坏死病毒属聚合酶具备核酸结合与合成能力。