Discovery of frameshifting in Alphavirus 6K resolves a 20-year enigma.
Discovery of frameshifting in Alphavirus 6K resolves a 20-year enigma.
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DOI：
10.1186/1743-422x-5-108
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发表时间：
2008-09-26
期刊：
影响因子：
4.8
通讯作者：
Atkins JF
中科院分区：
文献类型：
--
作者：
Firth AE;Chung BY;Fleeton MN;Atkins JF
甲病毒属包括几种潜在致命的人类病毒。此外，诸如辛德毕斯病毒和塞姆利基森林病毒的物种是基因治疗、疫苗接种和癌症研究的重要载体，也是病毒体组装和结构分析的重要模型。基因组编码9种已知蛋白质，包括小的“6 K”蛋白质。6 K似乎参与包膜蛋白加工、膜透化、病毒体组装和病毒出芽。在蛋白质凝胶中，6 K作为双峰迁移-迄今为止，这一结果已被归因于不同程度的酰化。然而，尽管多年的研究，其作用仍然是相对知之甚少。我们报告了核糖体-1移码，估计效率为~10- 18%，发生在编码6 K的序列内的保守UUUUUA基序处，导致合成另一种蛋白质，称为TF（transFrame蛋白; ~8 kDa），其中C-末端氨基酸由-1框架编码。通过质谱法证实了塞姆利基森林病毒粒子中存在TF。TF和6 K的表达模式进行了研究，脉冲追踪标记，免疫沉淀和免疫荧光，使用野生型病毒和TF敲除突变体。我们发现，它主要是TF，被纳入病毒体，而不是6 K之前认为的。对负责UUUUUUA基序处的有效移码的3'刺激信号的研究揭示了不同甲病毒物种之间的信号的显著多样性。我们的结果为6 K双重体提供了令人惊讶的新解释，需要对甲病毒6 K蛋白的现有数据进行根本性的重新解释，并为未来进一步表征6 K和TF蛋白的进展开辟了道路。这些结果对甲病毒生物学、病毒粒子结构、病毒孔蛋白、核糖体移码和病毒和细胞生物中新型移码表达基因的生物信息学鉴定具有意义。
The genus Alphavirus includes several potentially lethal human viruses. Additionally, species such as Sindbis virus and Semliki Forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. The genome encodes nine known proteins, including the small '6K' protein. 6K appears to be involved in envelope protein processing, membrane permeabilization, virion assembly and virus budding. In protein gels, 6K migrates as a doublet – a result that, to date, has been attributed to differing degrees of acylation. Nonetheless, despite many years of research, its role is still relatively poorly understood. We report that ribosomal -1 frameshifting, with an estimated efficiency of ~10–18%, occurs at a conserved UUUUUUA motif within the sequence encoding 6K, resulting in the synthesis of an additional protein, termed TF (TransFrame protein; ~8 kDa), in which the C-terminal amino acids are encoded by the -1 frame. The presence of TF in the Semliki Forest virion was confirmed by mass spectrometry. The expression patterns of TF and 6K were studied by pulse-chase labelling, immunoprecipitation and immunofluorescence, using both wild-type virus and a TF knockout mutant. We show that it is predominantly TF that is incorporated into the virion, not 6K as previously believed. Investigation of the 3' stimulatory signals responsible for efficient frameshifting at the UUUUUUA motif revealed a remarkable diversity of signals between different alphavirus species. Our results provide a surprising new explanation for the 6K doublet, demand a fundamental reinterpretation of existing data on the alphavirus 6K protein, and open the way for future progress in the further characterization of the 6K and TF proteins. The results have implications for alphavirus biology, virion structure, viroporins, ribosomal frameshifting, and bioinformatic identification of novel frameshift-expressed genes, both in viruses and in cellular organisms.