Sun Xiaoping, Simon Anne E
Department of Cell Biology and Molecular Genetics, Microbiology Building, University of Maryland College Park, College Park, MD 20742, USA.
Virology. 2006 Aug 15;352(1):39-51. doi: 10.1016/j.virol.2006.03.051. Epub 2006 Jun 6.
Turnip crinkle virus (TCV) (family Tombusviridae, genus Carmovirus) is a positive-sense RNA virus containing a 4054-base genome. Previous results indicated that insertion of Hairpin 4 (H4) into a TCV-associated satellite RNA enhanced replication 6-fold in vivo (Nagy, P., Pogany, J., Simon, A. E., 1999. EMBO J. 18:5653-5665). A detailed structural and functional analysis of H4 has now been performed to investigate its role in TCV replication. RNA structural probing of H4 in full-length TCV supported the sequence forming hairpin structures in both orientations in vitro. Deletion and mutational analyses determined that H4 is important for efficient accumulation of TCV in protoplasts, with a 98% reduction of genomic RNA levels when H4 was deleted. In vitro transcription using p88 [the TCV RNA-dependent RNA polymerase] demonstrated that H4 in its plus-sense orientation [H4(+)] caused a nearly 2-fold increase in RNA synthesis from a core hairpin promoter located on TCV plus-strands. H4 in its minus-sense orientation [H4(-)] stimulated RNA synthesis by 100-fold from a linear minus-strand promoter. Gel mobility shift assays indicated that p88 binds H4(+) and H4(-) with equal affinity, which was substantially greater than the binding affinity to the core promoters. These results support roles for H4(+) and H4(-) in TCV replication by enhancing syntheses of both strands through attracting the RdRp to the template.
芜菁皱缩病毒（TCV）（番茄丛矮病毒科， Carmovirus属）是一种正义RNA病毒，其基因组包含4054个碱基。先前的结果表明，将发夹4（H4）插入与TCV相关的卫星RNA中可在体内使复制增强6倍（Nagy, P., Pogany, J., Simon, A. E., 1999. EMBO J. 18:5653 - 5665）。现在已经对H4进行了详细的结构和功能分析，以研究其在TCV复制中的作用。对全长TCV中的H4进行RNA结构探测，支持该序列在体外以两种方向形成发夹结构。缺失和突变分析确定，H4对于TCV在原生质体中的有效积累很重要，当H4缺失时，基因组RNA水平降低98%。使用p88[TCV RNA依赖性RNA聚合酶]进行的体外转录表明，正链方向的H4[H4(+)]使位于TCV正链上的核心发夹启动子的RNA合成增加了近2倍。负链方向的H4[H4(-)]使线性负链启动子的RNA合成增加了100倍。凝胶迁移率变动分析表明，p88以相等的亲和力结合H4(+)和H4(-)，这大大高于对核心启动子的结合亲和力。这些结果支持H4(+)和H4(-)通过将RdRp吸引到模板上增强两条链的合成，从而在TCV复制中发挥作用。
