Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging

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 <i>gag</i> 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 (5ʹ UTR) to ~ 120 nucleotides into <i>gag</i>, excluding <i>gag</i> 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 <i>trans</i>-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.

梅森-菲策猴病毒（Mason-Pfizer monkey virus, MPMV）的基因组RNA（genomic RNA, gRNA）包装信号是一类高度结构化的元件，由多个茎环结构组成，这些结构通过U5与gag互补序列之间的两处系统发育保守的长程相互作用（long-range interactions, LRIs）维系。此类长程相互作用在维持MPMV gRNA 5'端结构中发挥关键作用。据此可提出假说：该区域的整体RNA二级结构还可通过另外三个茎环（SL3、Gag SL1及Gag SL2）进一步在结构上得到稳固，这三个茎环的序列覆盖范围从5'非翻译区（5ʹ untranslated region, 5ʹ UTR）远端延伸至gag基因内约120个核苷酸的区域，但不包含参与形成U5-Gag长程相互作用的gag序列。
为了为这些茎环在gRNA衣壳化过程中的生物学意义提供功能证据，我们对这些结构基序进行了突变，并通过单轮反式互补试验检测了其对MPMV RNA包装与增殖的影响。此外，我们还通过高通量SHAPE（high throughput SHAPE, hSHAPE）检测进一步研究了突变后的RNA结构。
研究结果显示，构成这三个茎环的序列在MPMV gRNA包装或增殖过程中，单独发挥的作用并不关键。进一步的结构-功能分析表明，相较于这三个茎环，U5-Gag长程相互作用在稳定5' UTR的高级结构中发挥了更为重要的结构维系作用，而这三个茎环仅在稳定该区域整体RNA二级结构中扮演次要且可能间接的角色。本研究增进了我们对MPMV gRNA包装过程中发生的分子相互作用的理解。