Max Planck Institute of Biochemistry, Munich, Germany.
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Elife. 2023 Jan 26;12:e68670. doi: 10.7554/eLife.68670.
Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that support segmented genome assembly and replication via a poorly understood mechanism. Here, we analysed the RV transcriptome by combining DNA-barcoded smFISH of rotavirus-infected cells. Rotavirus RNA stoichiometry in viroplasms appears to be distinct from the cytoplasmic transcript distribution, with the largest transcript being the most enriched in viroplasms, suggesting a selective RNA enrichment mechanism. While all 11 types of transcripts accumulate in viroplasms, their stoichiometry significantly varied between individual viroplasms. Accumulation of transcripts requires the presence of 3' untranslated terminal regions and viroplasmic localisation of the viral polymerase VP1, consistent with the observed lack of polyadenylated transcripts in viroplasms. Our observations reveal similarities between viroplasms and other cytoplasmic RNP granules and identify viroplasmic proteins as drivers of viral RNA assembly during viroplasm formation.
轮状病毒转录 11 种不同的 RNA，这些 RNA 必须在复制之前共同包装，才能形成具有感染性的病毒粒子。在感染过程中，非翻译的轮状病毒转录本在细胞质蛋白-RNA 颗粒中积累，这些颗粒被称为 viroplasms，通过一种尚未完全了解的机制支持分段基因组的组装和复制。在这里，我们通过结合感染轮状病毒的细胞的 DNA 条形码 smFISH 分析了 RV 转录组。viroplasms 中的轮状病毒 RNA 计量似乎与细胞质转录本分布不同，最大的转录本在 viroplasms 中富集最多，表明存在一种选择性的 RNA 富集机制。虽然所有 11 种类型的转录本都在 viroplasms 中积累，但它们在单个 viroplasms 中的计量存在显著差异。转录本的积累需要 3'非翻译末端区域的存在和病毒聚合酶 VP1 的 viroplasmic 定位，这与在 viroplasms 中观察到的缺乏多聚腺苷酸化转录本一致。我们的观察结果揭示了 viroplasms 与其他细胞质 RNP 颗粒之间的相似性，并确定了 viroplasmic 蛋白是 viroplasm 形成过程中病毒 RNA 组装的驱动因素。
