Torrent C, Bordet T, Darlix J L
LaboRetro INSERM, Unité de virologie humaine U412, Ecole Normale Supérieure de Lyon, France.
J Mol Biol. 1994 Jul 29;240(5):434-44. doi: 10.1006/jmbi.1994.1459.
A sequence of the rat retrotransposon virus-like 30 S RNA (VL30) located next to the 5' end of the Harvey murine sarcoma virus (HaMSV) genome was recently found to form stable dimeric RNA in vitro and to direct the efficient packaging of VL30-derived recombinant RNAs into MuLV virions. To study the structure-function relationships of the rat VL30 dimerization-encapsidation signal (E/DLS), we have performed biochemical and genetic studies of rat VL30 RNA dimerization in vitro. The results show that temperature and specific cation/RNA interactions are important for VL30 dimerization in vitro. VL30 RNA dimerization is optimal at 55 degrees C and Li+ dramatically enhances the stability of VL30 dimeric RNA. In addition, a genetic analysis of VL30 RNA dimerization reveals that a 5' G-rich sequence is critical for dimer formation and that a UGUCUUGUC repeat contributes to VL30 dimer stability. Interestingly enough, substitution of an A for a G in the 5' G-rich sequence is sufficient to abolish VL30 RNA dimerization in vitro. Taken together, these biochemical and genetic data indicate that dimerization of VL30 RNA involves non-canonical base-pairings and possible purine-purine interactions. Nucleocapsid protein NCp10 of murine leukemia virus (MuLV), a gag-encoded protein that is tightly associated with genomic RNA in the virion core, has been shown to have nucleic acid binding and annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to the retrotransposon VL30 RNA and to activate its dimerization. Moreover, mutations in the 5' G-rich sequence of the VL30 dimerization sequence impaired NCp10 binding to RNA. Recombinant MLV-VL30 vectors with mutations in the VL30 dimerization sequence were constructed. Results obtained in vivo clearly show that the mutations that had a deleterious effect on the packaging of MLV-VL30 retroviral vector in vivo were those that impaired VL30 RNA dimerization and interactions with NCp10 in vitro, even the single mutation in the 5' G-rich region. Therefore, these findings suggest that packaging of VL30 RNA into MuLV virions requires specific interactions between RNA dimerization sequences and viral NC protein molecules.
最近发现，位于哈维鼠肉瘤病毒（HaMSV）基因组5'端旁边的大鼠逆转录转座子病毒样30S RNA（VL30）序列在体外能形成稳定的二聚体RNA，并能将VL30衍生的重组RNA有效包装到鼠白血病病毒（MuLV）病毒粒子中。为了研究大鼠VL30二聚化-包装信号（E/DLS）的结构-功能关系，我们对大鼠VL30 RNA在体外的二聚化进行了生化和遗传学研究。结果表明，温度和特定的阳离子/RNA相互作用对VL30在体外的二聚化很重要。VL30 RNA二聚化在55℃时最适宜，Li+能显著增强VL30二聚体RNA的稳定性。此外，对VL30 RNA二聚化的遗传学分析表明，5'富含G的序列对二聚体形成至关重要，UGUCUUGUC重复序列有助于VL30二聚体的稳定性。有趣的是，在5'富含G的序列中用A取代G足以在体外消除VL30 RNA二聚化。综上所述，这些生化和遗传学数据表明，VL30 RNA二聚化涉及非经典碱基配对和可能的嘌呤-嘌呤相互作用。鼠白血病病毒（MuLV）的核衣壳蛋白NCp10是一种gag编码蛋白，与病毒粒子核心中的基因组RNA紧密相关，已被证明具有核酸结合和退火活性。在此我们报告，病毒NCp10蛋白能够紧密结合到退火活性上。在此我们报告，病毒NCp10蛋白能够紧密结合逆转录转座子VL30 RNA并激活其二聚化。此外，VL30二聚化序列5'富含G的序列中的突变会损害NCp10与RNA的结合。构建了VL30二聚化序列有突变的重组MLV-VL30载体。体内实验结果清楚地表明，对MLV-VL30逆转录病毒载体在体内包装有有害影响的突变是那些在体外损害VL30 RNA二聚化及与NCp10相互作用的突变，即使是5'富含G区域的单个突变。因此，这些发现表明，将VL30 RNA包装到MuLV病毒粒子中需要RNA二聚化序列与病毒NC蛋白分子之间的特定相互作用。
