A multidisciplinary approach to the identification of the protein-RNA connectome in double-stranded RNA virus capsids.
A multidisciplinary approach to the identification of the protein-RNA connectome in double-stranded RNA virus capsids.
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DOI：
10.1093/nar/gkad274
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发表时间：
2023-06-09
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
--
作者：
对于许多病毒来说，多节段双链RNA(DsRNA)病毒如何正确地将其基因组整合到衣壳中尚不清楚，包括呼肠孤病毒科成员蓝舌病毒(BTV)，其基因组由10个片段组成。为了解决这个问题，我们使用了RNA交联和多肽指纹分析(RCAP)来确定内衣壳蛋白VP3、病毒聚合酶VP1和封闭酶VP4的RNA结合位点。利用突变、反向遗传学、重组蛋白和体外组装的组合，我们验证了这些区域在病毒感染性中的重要性。此外，为了确定哪些RNA片段和序列与这些蛋白质相互作用，我们使用了病毒光激活核糖核苷交联(VPAR-CL)，这表明较大的RNA片段(S1-S4)和最小的片段(S10)与病毒蛋白的相互作用比其他较小的片段更多。此外，使用序列丰富分析，我们确定了一个由9个碱基组成的RNA基序，该基序被较大的片段共享。这一基序对病毒复制的重要性通过突变和病毒恢复得到了证实。我们进一步证明，这些方法可以应用于具有人类流行病影响的呼肠孤病毒科相关成员轮状病毒(RV)，为针对人类病原体的新干预策略提供了可能性。双链RNA病毒的分子连接体。
How multi-segmented double-stranded RNA (dsRNA) viruses correctly incorporate their genomes into their capsids remains unclear for many viruses, including Bluetongue virus (BTV), a Reoviridae member, with a genome of 10 segments. To address this, we used an RNA-cross-linking and peptide-fingerprinting assay (RCAP) to identify RNA binding sites of the inner capsid protein VP3, the viral polymerase VP1 and the capping enzyme VP4. Using a combination of mutagenesis, reverse genetics, recombinant proteins and in vitro assembly, we validated the importance of these regions in virus infectivity. Further, to identify which RNA segments and sequences interact with these proteins, we used viral photo-activatable ribonucleoside crosslinking (vPAR-CL) which revealed that the larger RNA segments (S1-S4) and the smallest segment (S10) have more interactions with viral proteins than the other smaller segments. Additionally, using a sequence enrichment analysis we identified an RNA motif of nine bases that is shared by the larger segments. The importance of this motif for virus replication was confirmed by mutagenesis followed by virus recovery. We further demonstrated that these approaches could be applied to a related Reoviridae member, rotavirus (RV), which has human epidemic impact, offering the possibility of novel intervention strategies for a human pathogen. The molecular connectome of double-stranded RNA viruses.
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