Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression
Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression
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DOI：
10.1073/pnas.1220107110
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发表时间：
2013-05-14
影响因子：
11.1
通讯作者：
Nibert, Max L.
中科院分区：
文献类型：
--
作者：
Demidenko, Aleksander A.;Blattman, Joseph N.;Nibert, Max L.
我们测试了一种工程重组哺乳动物呼肠孤病毒（rMRV）以表达外源多肽的策略。一个重要特征是这些 rMRV 被设计为自主繁殖，因此可以作为潜在的疫苗载体在动物中进行测试。迄今为止，该策略已应用于 10 个 MRV 基因组片段中的三个：S3、M1 和 L1。为了设计修饰片段，复制每个片段中必需的长 ORF 的 5' 或 3' 区域，然后在重复之间插入外源序列。内部重复和外源插入片段与天然蛋白质编码序列位于读框内，但通过读框内的“2A样”序列元件与它们分开，该元件指定共翻译“停止/继续”事件，从必需的MRV蛋白中释放外源多肽。该设计保留了 MRV 基因组片段的末端区域，该区域在 RNA 包装、分类、复制、转录和/或翻译中具有重要活性，并在有限程度上改变了编码的 MRV 蛋白。通过对内部重复序列和外源插入序列进行摆动诱变，可以更有效地恢复具有较长插入序列的 rMRV，这可能通过同源重组和 RNA 结构的相应减少而有所帮助。通过检测 Gag 特异性 T 细胞反应，证实了由 L1 修饰的 rMRV 在小鼠中表达的猿猴免疫缺陷病毒 Gag 蛋白的 300 个氨基酸部分的免疫原性。该工程策略进一步用于绘制 MRV 基因组片段 S3 所必需的最小 5' 末端区域的图谱。
We tested a strategy for engineering recombinant mammalian reoviruses (rMRVs) to express exogenous polypeptides. One important feature is that these rMRVs are designed to propagate autonomously and can therefore be tested in animals as potential vaccine vectors. The strategy has been applied so far to three of the 10 MRV genome segments: S3, M1, and L1. To engineer the modified segments, a 5' or 3' region of the essential, long ORF in each was duplicated, and then exogenous sequences were inserted between the repeats. The inner repeat and exogenous insert were positioned in frame with the native protein-encoding sequences but were separated from them by an in-frame "2A-like" sequence element that specifies a cotranslational "stop/continue" event releasing the exogenous polypeptide from the essential MRV protein. This design preserves a terminal region of the MRV genome segment with essential activities in RNA packaging, assortment, replication, transcription, and/or translation and alters the encoded MRV protein to a limited degree. Recovery of rMRVs with longer inserts was made more efficient by wobble-mutagenizing both the inner repeat and the exogenous insert, which possibly helped via respective reductions in homologous recombination and RNA structure. Immunogenicity of a 300-aa portion of the simian immunodeficiency virus Gag protein expressed in mice by an L1-modified rMRV was confirmed by detection of Gag-specific T-cell responses. The engineering strategy was further used for mapping the minimal 5'-terminal region essential to MRV genome segment S3.