Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.
Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.
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DOI：
10.1128/jvi.01043-15
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发表时间：
2015-08
影响因子：
5.4
通讯作者：
Firth AE
中科院分区：
文献类型：
--
作者：
Finch LK;Ling R;Napthine S;Olspert A;Michiels T;Lardinois C;Bell S;Loughran G;Brierley I;Firth AE
泰勒氏鼠脑脊髓炎病毒（TMEV）是小核糖核酸病毒科（Picornaviridae）中杆状病毒属的成员，是一个正义单链RNA病毒家族。先前，我们证明了在相关的心脏病毒脑心肌炎病毒中，程序性-1核糖体移码（-1 PRF）发生在多蛋白开放阅读框（ORF）的2B编码区内的保守G_GUU_UUU序列上。在这里，我们发现-1 PRF发生在TMEV基因组翻译过程中的类似位点。此外，我们证明了预测的3′ RNA茎环结构在一个非规范的间距下游的移位位点是需要有效的移码在TMEV和移码也需要病毒感染。突变G_GUU_UUU移位位点以抑制移码导致具有降低的生长动力学和小空斑表型的减毒病毒。发现病毒背景下的移码非常有效，达到74 - 82%，据我们所知，这是迄今为止记录的任何病毒的最高移码效率。我们认为，TMEV中高效的−1 PRF提供了一种机制，可以逃避小核糖核酸病毒单聚蛋白表达策略中通常固有的等摩尔表达的限制。重要性许多病毒利用程序化的-1核糖体移码（-1 PRF）以确定的比例产生不同的蛋白质产物，或翻译重叠的ORF以增加编码能力。除了少数例外，-1 PRF发生在特定的“光滑”七核苷酸序列上，并由光滑位点下游5至9个核苷酸（nt）开始的RNA结构刺激。在这里，我们描述了一个在泰勒鼠脑脊髓炎病毒（TMEV）中不寻常的-1 PRF的例子，它非常有效（74%到82%的核糖体转移到替代阅读框架中），与其他-1 PRF的例子形成鲜明对比，它依赖于从滑动位点下游14 nt开始的茎环结构。此外，在转染细胞中基于TMEV的报告基因构建体中，有效的移码关键取决于病毒感染。我们认为，TMEV进化移码作为一种新的机制，删除核糖体的消息（“核糖体汇”）下调3′-编码的复制蛋白的合成。
Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed −1 ribosomal frameshift (−1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that −1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3′ RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient −1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses. IMPORTANCE Many viruses utilize programmed −1 ribosomal frameshifting (−1 PRF) to produce different protein products at a defined ratio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, −1 PRF occurs on specific “slippery” heptanucleotide sequences and is stimulated by RNA structure beginning 5 to 9 nucleotides (nt) downstream of the slippery site. Here we describe an unusual case of −1 PRF in Theiler's murine encephalomyelitis virus (TMEV) that is extraordinarily efficient (74 to 82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of −1 PRF, is dependent upon a stem-loop structure beginning 14 nt downstream of the slippery site. Furthermore, in TMEV-based reporter constructs in transfected cells, efficient frameshifting is critically dependent upon virus infection. We suggest that TMEV evolved frameshifting as a novel mechanism for removing ribosomes from the message (a “ribosome sink”) to downregulate synthesis of the 3′-encoded replication proteins.