Attachment of ribosomal complexes and retrograde scanning during initiation on the Halastavi árva virus IRES.
Attachment of ribosomal complexes and retrograde scanning during initiation on the Halastavi árva virus IRES.
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DOI：
10.1093/nar/gkw016
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发表时间：
2016-03-18
影响因子：
14.9
通讯作者：
Hellen CU
中科院分区：
文献类型：
--
作者：
Abaeva IS;Pestova TV;Hellen CU
Halastavi árva 病毒 (HalV) 具有正义 RNA 基因组，具有 827 nt 长的 5' UTR 和分隔两个开放阅读框的基因间区域。虽然编码的蛋白质与双顺反子病毒多蛋白最同源，但其 5'UTR 是不同的。在这里，我们报道HalV 5' UTR包含由长单链区域分隔的小茎环结构域和围绕起始密码子AUG828的大的富含A的非结构化区域，并且具有跨界内部核糖体进入位点（IRES）活性。与大多数病毒 IRES 相比，它不依赖于结构完整性以及结构化元件与翻译成分的特定相互作用，而是由 AUG828 侧翼的非结构化区域决定。 eIF2、eIF3、eIF1 和 eIF1A 在 AUG828 促进有效的 48S 起始复合物形成，在省略 eIF1 和 eIF1A 时，48S 起始复合物的形成减少约 5 倍。引发涉及43S预引发复合物在AUG828处或其下游的短窗口内直接附着。 40S 和 eIF3 足以进行初始结合。附着后，43S 复合物进行逆行扫描，强烈依赖于 eIF1 和 eIF1A。 eIF4A/eIF4G 仅在低温下或在突变体上刺激起始，其中 AUG828 周围的区域已被异源序列取代。然而，他们强烈促进了 AUG872 处的起始，产生富含脯氨酸的寡肽。
Halastavi árva virus (HalV) has a positive-sense RNA genome, with an 827 nt-long 5′ UTR and an intergenic region separating two open reading frames. Whereas the encoded proteins are most homologous to Dicistrovirus polyproteins, its 5′ UTR is distinct. Here, we report that the HalV 5′ UTR comprises small stem-loop domains separated by long single-stranded areas and a large A-rich unstructured region surrounding the initiation codon AUG828, and possesses cross-kingdom internal ribosome entry site (IRES) activity. In contrast to most viral IRESs, it does not depend on structural integrity and specific interaction of a structured element with a translational component, and is instead determined by the unstructured region flanking AUG828. eIF2, eIF3, eIF1 and eIF1A promote efficient 48S initiation complex formation at AUG828, which is reduced ∼5-fold on omission of eIF1 and eIF1A. Initiation involves direct attachment of 43S preinitiation complexes within a short window at or immediately downstream of AUG828. 40S and eIF3 are sufficient for initial binding. After attachment, 43S complexes undergo retrograde scanning, strongly dependent on eIF1 and eIF1A. eIF4A/eIF4G stimulated initiation only at low temperatures or on mutants, in which areas surrounding AUG828 had been replaced by heterologous sequences. However, they strongly promoted initiation at AUG872, yielding a proline-rich oligopeptide.