Flores Ricardo, Gas Marída-Eugenia, Molina Diego, Hernández Carmen, Darós José-Antonio
Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas, Valencia, Spain.
Methods Mol Biol. 2008;451:167-83. doi: 10.1007/978-1-59745-102-4_12.
Viroids, as a consequence of not encoding any protein, are extremely dependent on their hosts. Replication of these minimal genomes, composed exclusively by a circular RNA of 246-401 nt, occurs in the nucleus (family Pospiviroidae) or in the chloroplast (family Avsunviroidae) by an RNA-based rolling-circle mechanism with three steps: (1) synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases recruited and redirected to transcribe RNA templates, (2) cleavage to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3) circularization through an RNA ligase or autocatalytically. This consistent but still fragmentary picture has emerged from a combination of studies with in vitro systems (analysis of RNA preparations from infected plants, transcription assays with nuclear and chloroplastic fractions, characterization of enzymes and ribozymes mediating cleavage and ligation of viroid strands, dissection of 5' terminal groups of viroid strands, and in situ hybridization and microscopy of subcellular fractions and tissues), and in vivo systems (tissue infiltration studies, protoplasts, studies in planta and use of transgenic plants expressing viroid RNAs).
由于不编码任何蛋白质，类病毒极其依赖其宿主。这些仅由246 - 401个核苷酸的环状RNA组成的最小基因组，通过基于RNA的滚环机制在细胞核（马铃薯纺锤块茎类病毒科）或叶绿体（鳄梨日斑类病毒科）中进行复制，该机制有三个步骤：（1）由被招募并重新定向以转录RNA模板的宿主DNA依赖性RNA聚合酶催化合成比单位链更长的链；（2）切割成单位长度，在鳄梨日斑类病毒科中由锤头状核酶介导；（3）通过RNA连接酶或自动催化进行环化。这一连贯但仍不完整的情况是通过体外系统（对感染植物的RNA制剂进行分析、对细胞核和叶绿体部分进行转录测定、对介导类病毒链切割和连接的酶和核酶进行表征、剖析类病毒链的5'末端基团以及对亚细胞部分和组织进行原位杂交和显微镜观察）和体内系统（组织浸润研究、原生质体、植物体内研究以及使用表达类病毒RNA的转基因植物）的研究相结合而得出的。
