Guo Rong, Lin Wai, Zhang Jiuchun, Simon Anne E, Kushner David B
Department of Cell Biology and Molecular Genetics, University of Maryland-College Park, Maryland 20742, USA.
J Virol. 2009 Jan;83(2):927-39. doi: 10.1128/JVI.02060-08. Epub 2008 Nov 12.
Satellite RNAs usually lack substantial homology with their helper viruses. The 356-nucleotide satC of Turnip crinkle virus (TCV) is unusual in that its 3'-half shares high sequence similarity with the TCV 3' end. Computer modeling, structure probing, and/or compensatory mutagenesis identified four hairpins and three pseudoknots in this TCV region that participate in replication and/or translation. Two hairpins and two pseudoknots have been confirmed as important for satC replication. One portion of the related 3' end of satC that remains poorly characterized corresponds to juxtaposed TCV hairpins H4a and H4b and pseudoknot psi(3), which are required for the TCV-specific requirement of translation (V. A. Stupina et al., RNA 14:2379-2393, 2008). Replacement of satC H4a with randomized sequence and scoring for fitness in plants by in vivo genetic selection (SELEX) resulted in winning sequences that contain an H4a-like stem-loop, which can have additional upstream sequence composing a portion of the stem. SELEX of the combined H4a and H4b region in satC generated three distinct groups of winning sequences. One group models into two stem-loops similar to H4a and H4b of TCV. However, the selected sequences in the other two groups model into single hairpins. Evolution of these single-hairpin SELEX winners in plants resulted in satC that can accumulate to wild-type (wt) levels in protoplasts but remain less fit in planta when competed against wt satC. These data indicate that two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts.
卫星RNA通常与其辅助病毒缺乏显著的同源性。芜菁皱缩病毒（TCV）的356个核苷酸的satC则不同寻常，因为其3'端的后半部分与TCV的3'端具有高度的序列相似性。通过计算机建模、结构探测和/或补偿性诱变，在这个TCV区域鉴定出了四个发夹结构和三个假结结构，它们参与复制和/或翻译。已证实两个发夹结构和两个假结结构对satC的复制很重要。satC相关3'端中一个特征尚不明确的部分对应于并列的TCV发夹结构H4a和H4b以及假结结构psi(3)，它们是TCV特异性翻译所必需的（V. A. Stupina等人，《RNA》14:2379 - 2393，2008年）。用随机序列替换satC的H4a，并通过体内遗传选择（SELEX）在植物中对适应性进行评分，得到的获胜序列包含一个类似H4a的茎环结构，该结构可能有额外的上游序列构成茎的一部分。对satC中H4a和H4b组合区域进行SELEX产生了三组不同的获胜序列。一组模拟成两个类似于TCV的H4a和H4b的茎环结构。然而，另外两组中所选序列模拟成单个发夹结构。这些单发卡SELEX获胜者在植物中的进化产生了satC，其在原生质体中可以积累到野生型（wt）水平，但在与wt satC竞争时在植物体内的适应性仍然较低。这些数据表明，H4a和H4b区域中两种高度不同的RNA构象可以介导satC在原生质体中的适应性。
