Coronavirus defective-interfering RNA as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase.
Coronavirus defective-interfering RNA as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase.
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DOI：
10.1006/viro.1995.1155
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发表时间：
1995-04-01
期刊：
影响因子：
3.7
通讯作者：
Lai MM
中科院分区：
文献类型：
--
作者：
Liao CL;Zhang X;Lai MM
我们已经开发了一种表达载体系统，使用小鼠肝炎病毒（MHV）的缺陷干扰（DI）RNA，原型冠状病毒，在MHV感染的细胞中传递和表达外源基因。该载体含有MHV基因间序列以促进外源基因的表达。在这项研究中，我们使用这个载体引入一个血凝素酯酶（HE）蛋白，一个可选的MHV结构蛋白，到MHV感染的细胞。工程化的HE蛋白可以被有效地整合到不合成其自身HE蛋白的病毒粒子中，从而产生表达外源HE蛋白的假重组病毒。通过在羧基末端连接8个氨基酸的肽标签，可以使工程化的HE蛋白与天然蛋白区分开。来自产生HE的病毒培养物的工程化和天然HE蛋白都可以掺入病毒体中，从而产生表型混合的病毒颗粒。我们还表明，表达HE的DI RNA可以掺入病毒中，并且工程化的HE蛋白在感染的细胞中表达至少三个连续的病毒传代。此外，我们已经取得了两个突变体，其中部分的外部结构域的HE蛋白已被删除，研究的序列要求，稳定表达的HE和它的纳入MHV病毒粒子。虽然这两种突变的HE蛋白可以在MHV感染的细胞中表达，但它们未能被掺入病毒粒子中，这表明HE蛋白的胞外结构域对于其掺入病毒粒子中的重要性。该载体系统首次成功地将选定的冠状病毒蛋白掺入病毒体中，并证明了其作为研究冠状病毒分子生物学的表达载体的实用性。
We have developed an expression vector system using a defective-interfering (DI) RNA of mouse hepatitis virus (MHV), a prototype coronavirus, to deliver and express a foreign gene in MHV-infected cells. This vector contains an MHV intergenic sequence to promote the expression of foreign genes. In this study, we used this vector to introduce a hemagglutininesterase (HE) protein, an optional MHV structural protein, into the MHV-infected cells. The engineered HE protein could be efficiently incorporated into the virion which did not synthesize its own HE protein, thus generating a pseudorecombinant virus that expresses an exogenous HE protein. The engineered HE protein could be made distinguishable from the native protein by attaching an 8-amino-acid peptide tag at the carboxyl-terminus. Both the engineered and native HE proteins from the HE-producing virus train could be incorporated into the virion, thus generating phenotypically mixed virus parficles. We also showed that the HE-expressing DI RNA could be incorporated into viruses, and the engineered HE protein expressed in the infected cells for at least three serial virus passages. Furthermore, we have made two mutants, in which parts of the external domain of the HE protein have been deleted, to study the sequence requirements for the stable expression of HE and its incorporation into MHV virions. Although both of the mutant HE proteins could be expressed in the MHV-infected cells, they failed to be incorporated into virions, suggesting the importance of the extracellular domain of HE protein for its incorporation into virus particles. This vector system enabled the first successful incorporation of a selected coronaviral protein into virions and demonstrates its utility as an expression vector for studying the molecular biology of coronaviruses.