a Department of Microbiology & Immunology College of Medicine and Health Sciences , United Arab Emirates University , Al Ain , United Arab Emirates (UAE).
b CNRS, Architecture et Réactivité de l'ARN, UPR , Université de Strasbourg , Strasbourg , France.
RNA Biol. 2019 May;16(5):612-625. doi: 10.1080/15476286.2019.1572424. Epub 2019 Feb 17.
The Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging signal is a highly-structured element with several stem-loops held together by two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences. These LRIs play a critical role in maintaining the structure of the 5´ end of the MPMV gRNA. Thus, one could hypothesize that the overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, Gag SL1, and Gag SL2) comprising of sequences from the distal parts of the 5´untranslated region (5' UTR) to ~ 120 nucleotides into gag, excluding gag sequences involved in forming the U5-Gag LRIs. To provide functional evidence for the biological significance of these stem loops during gRNA encapsidation, these structural motifs were mutated and their effects on MPMV RNA packaging and propagation were tested in a single round trans-complementation assay. The mutant RNA structures were further studied by high throughput SHAPE (hSHAPE) assay. Our results reveal that sequences involved in forming these three stem loops do not play crucial roles at an individual level during MPMV gRNA packaging or propagation. Further structure-function analysis indicates that the U5-Gag LRIs have a more important architectural role in stabilizing the higher order structure of the 5´ UTR than the three stem loops which have a more secondary and perhaps indirect role in stabilizing the overall RNA secondary structure of the region. Our work provides a better understanding of the molecular interactions that take place during MPMV gRNA packaging.
猴痘病毒（MPMV）基因组 RNA（gRNA）的包装信号是一个高度结构化的元件，由 U5 和 gag 互补序列之间的两个系统发育上保守的长程相互作用（LRIs）保持在一起。这些 LRIs 在维持 MPMV gRNA 5' 端的结构中起着关键作用。因此，人们可以假设该区域的整体 RNA 二级结构进一步由三个其他茎环（SL3、Gag SL1 和 Gag SL2）构成，这些茎环包含来自 5' 非翻译区（5'UTR）远端的序列~120 个核苷酸进入 gag，不包括参与形成 U5-Gag LRIs 的 gag 序列。为了提供这些茎环在 gRNA 包装过程中具有生物学意义的功能证据，我们对这些结构基序进行了突变，并在单次轮互补测定中测试了它们对 MPMV RNA 包装和传播的影响。通过高通量 SHAPE（hSHAPE）测定进一步研究了突变 RNA 结构。我们的结果表明，形成这些三个茎环的序列在 MPMV gRNA 包装或传播过程中单独水平上并不起关键作用。进一步的结构功能分析表明，U5-Gag LRIs 在稳定 5'UTR 的高级结构方面比三个茎环具有更重要的结构作用，这三个茎环在稳定该区域的整体 RNA 二级结构方面具有更次要的、也许是间接的作用。我们的工作提供了对 MPMV gRNA 包装过程中发生的分子相互作用的更好理解。
