Panavas Tadas, Nagy Peter D
Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA.
Virology. 2003 Sep 15;314(1):315-25. doi: 10.1016/s0042-6822(03)00436-7.
Defective interfering (DI) RNA associated with Tomato bushy stunt virus (TBSV), which is a plus-strand RNA virus, requires p33 and p92 proteins of TBSV or the related Cucumber necrosis virus (CNV), for replication in plants. To test if DI RNA can replicate in a model host, we coexpressed TBSV DI RNA and p33/p92 of CNV in yeast. We show evidence for replication of DI RNA in yeast, including (i) dependence on p33 and p92 for DI replication; (ii) presence of active CNV RNA-dependent RNA polymerase in isolated membrane-containing preparations; (iii) increasing amount of DI RNA(+) over time; (iv) accumulation of (-)stranded DI RNA; (v) presence of correct 5' and 3' ends in DI RNA; (vi) inhibition of replication by mutations in the replication enhancer; and (vii) evolution of DI RNA over time, as shown by sequence heterogeneity. We also produced evidence supporting the occurrence of DI RNA recombinants in yeast. In summary, development of yeast as a host for replication of TBSV DI RNA will facilitate studies on the roles of viral and host proteins in replication/recombination.
与番茄丛生矮化病毒（TBSV）相关的缺陷干扰（DI）RNA是一种正链RNA病毒，其在植物中的复制需要TBSV或相关黄瓜坏死病毒（CNV）的p33和p92蛋白。为了测试DI RNA是否能在模式宿主中复制，我们在酵母中共表达了TBSV DI RNA和CNV的p33/p92。我们展示了DI RNA在酵母中复制的证据，包括：（i）DI复制依赖p33和p92；（ii）在分离的含膜制剂中存在活性CNV RNA依赖性RNA聚合酶；（iii）随着时间的推移，DI RNA（+）的量增加；（iv）（-）链DI RNA的积累；（v）DI RNA中存在正确的5'和3'末端；（vi）复制增强子中的突变抑制复制；以及（vii）如序列异质性所示，DI RNA随时间进化。我们还提供了支持酵母中DI RNA重组体出现的证据。总之，将酵母开发为TBSV DI RNA复制的宿主将有助于研究病毒和宿主蛋白在复制/重组中的作用。
