Dalmay T, Szittya G, Burgyán J
Agricultural Biotechnology Center, Plant Science Institute, Gödöllö, Hungary.
Virology. 1995 Mar 10;207(2):510-7. doi: 10.1006/viro.1995.1111.
Inoculation of Nicotiana clevelandii and N. benthamiana plants with in vitro transcripts of both genomic and defective interfering (DI) RNAs of cymbidium ringspot tombusvirus resulted in a rapid accumulation of new DI-like RNA species which were demonstrated by cloning and sequencing to be head-to-tail dimers of unit length DI RNAs. The junction regions of dimers were represented by sequences derived precisely from the 5' and 3' termini of DI RNAs. Only infection with DI RNAs of smaller size (DI-2 and DI-3, 402 and 482 nt, respectively) produced detectable amount of dimers; in contrast, infection with the largest DI RNA (DI-13, 679 nt) was unable to accumulate dimers during viral infection. Analysis of mutant DI RNAs containing deletions or insertions revealed that the size of the monomer molecule is a major factor in the accumulation of dimers. Monomeric DI RNAs were formed in both plants and protoplasts inoculated with in vitro-transcribed dimers. No heterodimers were found in plants inoculated simultaneously with DI-2 and DI-3 RNA molecules, which may indicate that replicase is not released from the template during synthesis of dimer molecules. However, the occurrence of a recombinant DI RNA dimer molecule derived from the two DI RNAs suggests that simultaneous infection of the same cells with two DI RNAs did indeed take place and that absence of heterodimers did not depend on compartmentalization.
用大花蕙兰环斑番茄病毒的基因组RNA和缺陷干扰（DI）RNA的体外转录本接种克利夫兰烟草和本氏烟草植株，会导致新的类DI RNA物种迅速积累，通过克隆和测序证明这些物种是单位长度DI RNA的头对头二聚体。二聚体的连接区域由精确来源于DI RNA 5'和3'末端的序列代表。只有用较小尺寸的DI RNA（DI-2和DI-3，分别为402和482个核苷酸）感染才会产生可检测量的二聚体；相反，用最大的DI RNA（DI-13，679个核苷酸）感染在病毒感染期间无法积累二聚体。对含有缺失或插入的突变DI RNA的分析表明，单体分子的大小是二聚体积累的主要因素。在接种体外转录二聚体的植物和原生质体中均形成了单体DI RNA。在同时接种DI-2和DI-3 RNA分子的植物中未发现异源二聚体，这可能表明在二聚体分子合成过程中复制酶未从模板上释放。然而，源自两种DI RNA的重组DI RNA二聚体分子的出现表明，同一细胞确实同时被两种DI RNA感染，并且异源二聚体的缺失并不取决于区室化。
