Replicating reoviruses with a transgene replacing the codons for the head domain of the viral spike
Replicating reoviruses with a transgene replacing the codons for the head domain of the viral spike
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DOI：
10.1038/gt.2014.126
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发表时间：
2015-03-01
期刊：
影响因子：
5.1
通讯作者：
Hoeben, R. C.
中科院分区：
文献类型：
--
作者：
van den Wollenberg, D. J. M.;Dautzenberg, I. J. C.;Hoeben, R. C.
修饰呼肠孤病毒基因组的能力有助于产生新的治疗性呼肠孤病毒。我们描述了一种用于产生携带异源转基因的可复制呼肠孤病毒的方法。该策略是基于扩大嗜性呼肠孤病毒突变体Jin-3，其可以独立于呼肠孤病毒受体连接粘附分子A（JAM-A）感染细胞。Jin-3在Si片段中具有突变，导致刺突蛋白σ 1的尾部中的G196 R取代。jin-3尾编码Si区段的使用允许用外源序列的多达522个核苷酸替换JAM-A结合头部结构域的密码子，而不超过野生型Si区段的大小。我们将猪捷申病毒-1 2A元件的密码子与编码荧光蛋白iLOV的密码子融合。通过共转染所有呼肠孤病毒区段的表达质粒来产生复制rS 1His-2A-iLOV呼肠孤病毒。这些呼肠孤病毒含有S1 His-2A-iLOV区段，而不存在野生型S1区段。密度梯度离心证实了σ 1-尾片段与衣壳的关联。暴露于rS 1His-2A-iLOV呼肠孤病毒的JAM-A阳性和阴性细胞均表现出iLOV荧光，证实了jin-3衍生的扩增嗜性表型。这些数据证明了产生携带异源转基因的去头复制型T3 D呼肠孤病毒的可行性。
The capacity to modify the reovirus genome facilitates generation of new therapeutic reoviruses. We describe a method for generating replication-competent reoviruses carrying a heterologous transgene. The strategy is based on the expanded-tropism reovirus mutant jin-3, which can infect cells independent of the reovirus receptor junction-adhesion molecule A (JAM-A). Jin-3 harbors a mutation in the Si segment, resulting in a G196R substitution in the tail of the spike protein sigma 1. The use of the jin-3 tail-encoding Si segment allows replacing the codons for the JAM-A-binding head domain by up to 522 nucleotides of foreign sequences, without exceeding the size of the wild-type Si segment. We inserted the codons for the porcine teschovirus-1 2A element fused with those encoding the fluorescent protein iLOV. Replicating rS1His-2A-iLOV reoviruses were generated by co-transfection of expression plasmids for all reovirus segments. These reoviruses contain the S1His-2A-iLOV segment in the absence of the wild-type Si segment. Density-gradient centrifugation confirmed the association of the sigma 1-tail fragment with the capsid. Both JAM-A-positive and -negative cells exposed to the rS1His-2A-iLOV reoviruses exhibited iLOV fluorescence, confirming the jin-3-derived expanded-tropism phenotype. These data demonstrated the feasibility of generating decapitated replication-competent T3D reoviruses carrying a heterologous transgene.