The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus
The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus
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DOI：
10.1006/viro.1996.0165
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发表时间：
1996-04-01
期刊：
影响因子：
3.7
通讯作者：
Spaan, WJM
中科院分区：
文献类型：
--
作者：
Bos, ECW;Luytjes, W;Spaan, WJM
我们研究了在牛痘-T7聚合酶重组病毒感染的L细胞中感染性DI颗粒的产生和释放，所述L细胞用表达合成DI RNA MIDI-HD和鼠冠状病毒MHV-A59的四种结构蛋白（M、N、S和E）的五种不同质粒转染。DI cDNA含有丁型肝炎核酶序列，以在转染的细胞中产生确定的3'端。在转染细胞的EM研究中，在囊泡中观察到病毒样颗粒（VLP）。通过释放到培养上清液中的蛋白质的免疫沉淀来研究颗粒释放到培养基中。颗粒释放不依赖于S或N，但需要M和E。E和M的共表达足以释放颗粒。结构蛋白和MIDI-HD RNA的共表达导致感染性DI颗粒的产生和释放。通过将辅助病毒MHV-A59加入到含有VLP的培养基中并使用该混合物感染新的L细胞来测定DI-颗粒的亲和性。分离几个后续未稀释传代的细胞内RNA以检测MIDI-HD RNA。MIDI-HD RNA的传代依赖于转染细胞中MHV-A59结构蛋白的表达。在不存在E或M的情况下，MIDI-HD RNA不能传代至新鲜L细胞。因此，我们开发了一个系统，在其中我们可以产生冠状病毒样颗粒和检测其感染性的方法。(C)出版社：Academic Press，Inc.
We have studied the production and release of infectious DI-particles in vaccinia-T7-polymerase recombinant virus-infected L cells that were transfected with five different plasmids expressing the synthetic DI RNA MIDI-HD and the four structural proteins (M, N, S, and E) of the murine coronavirus MHV-A59. The DI cDNA contains the hepatitis delta ribozyme sequences to generate in the transfected cells a defined 3' end. In EM studies of transfected cells virus-like particles (VLP) were observed in vesicles. Release of the particles into the medium was studied by immunoprecipitations of proteins released into the culture supernatant Particle release was independent of S or N, but required M and E. Coexpression of E and M was sufficient for particle release. Coexpression of the structural proteins and the MIDI-HD RNA resulted in the production and release of infectious DI-particles. Infectivity of the DI-particles was determined by adding helper virus MHV-A59 to the medium containing the VLPs and using this mixture to infect new L cells. Intracellular RNA of several subsequent undiluted passages was isolated to detect the MIDI-HD RNA. Passage of the MIDI-HD RNA was dependent on the expression of the structural proteins of MHV-A59 in the transfected cells. In the absence of either E or M, MIDI-HD RNA could not be passaged to fresh L cells. We have thus developed a system in which we can produce coronavirus-like particles and an assay to test their infectivity. (C) 1996 Academic Press, Inc.