The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5ʹ untranslated region (UTR) in conjunction with the first 120 nucleotides of the <i>gag</i> gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation <i>versus</i> packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

包装小鼠乳腺肿瘤病毒（mouse mammary tumor virus, MMTV）的基因组RNA（genomic RNA, gRNA），需要完整的5'非翻译区（5' untranslated region, UTR）与gag基因的前120个核苷酸协同作用。该区域包含多个回文（palindromic, pal）序列以及稳定的茎环（stem loop, SL）结构。其中，茎环4（stem loop 4, SL4）呈现分叉状结构，由三个茎段、两个顶端环与一个内部环构成。位于其中一个顶端环内的Pal II可介导gRNA二聚化，这一过程与病毒包装过程紧密相关。据此我们提出假说：分叉状的SL4结构可能是gRNA包装的核心决定元件。为验证该假说，我们对构成分叉状SL4结构的两个顶端环及其侧翼序列分别进行了单点突变。所有上述突变均对gRNA包装与病毒增殖传播产生了不利影响。随后，我们设计了单突变与补偿突变，以先破坏分叉状SL4结构，再重建该天然结构。通过生物信息学预测与RNA化学探针分析开展的结构-功能研究显示：破坏SL4分叉结构的突变会完全消除RNA包装与病毒增殖传播能力；而重建天然SL4结构的补偿突变，则可将RNA包装与增殖传播能力恢复至野生型水平。综上，本研究结果证实SL4是MMTV gRNA的核心包装决定元件。本研究结果还进一步表明，SL4可作为一种结构开关，不仅能够区分用于翻译的RNA与用于包装/二聚化的RNA，其所处的位置还使得在gRNA衣壳化过程中，可以区分剪接与未剪接的RNA。