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仙台病毒缺陷干扰颗粒核衣壳在稳定辅助细胞系中的长期复制

Long-term replication of Sendai virus defective interfering particle nucleocapsids in stable helper cell lines.

作者信息

Willenbrink W, Neubert W J

机构信息

Abteilung für Virusforschung, Max-Planck-Institut für Biochemie, Martinsried, Germany.

出版信息

J Virol. 1994 Dec;68(12):8413-7. doi: 10.1128/JVI.68.12.8413-8417.1994.

DOI:10.1128/JVI.68.12.8413-8417.1994
PMID:7966637
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC237314/
Abstract

An essential prerequisite for generating a stable helper cell line, which constitutively expresses functional Sendai virus RNA-dependent RNA polymerase, is the expression of all three Sendai virus nucleocapsid (NC) proteins, NP, P, and L, simulataneously. Generating a stable helper cell line was accomplished by cotransfecting cell line 293 with all three corresponding viral genes under the control of cytomegalovirus promoter-enhancer elements. Cotransfection with a dominant selectable marker enabled selection for stably transfected cells. The levels of the expressed P and NP proteins reached up to 1/10th and 1/20th of the protein levels in Sendai virus-infected cells, respectively. The Sendai virus polymerase activity of the coexpressed proteins was demonstrated by an in vivo polymerase assay. The cell clone H29 gave the strongest signal and produced DI genomes continuously for at least 3 months. This result demonstrates that it is possible to stably express adequate levels of all three viral NC proteins to form Sendai virus polymerase activity, thereby performing the replication and encapsidation of viral RNA, essential prerequisites for a helper cell line to be competent in producing recombinant viruses.

摘要

产生稳定表达功能性仙台病毒RNA依赖的RNA聚合酶的辅助细胞系的一个必要前提条件是同时表达所有三种仙台病毒核衣壳（NC）蛋白，即NP、P和L。通过在巨细胞病毒启动子-增强子元件的控制下将所有三个相应的病毒基因共转染到293细胞系中来产生稳定的辅助细胞系。与显性选择标记共转染能够选择稳定转染的细胞。所表达的P和NP蛋白水平分别达到仙台病毒感染细胞中蛋白水平的1/10和1/20。通过体内聚合酶测定证实了共表达蛋白的仙台病毒聚合酶活性。细胞克隆H29给出了最强的信号，并连续至少3个月产生DI基因组。这一结果表明，有可能稳定表达足够水平的所有三种病毒NC蛋白以形成仙台病毒聚合酶活性，从而进行病毒RNA的复制和衣壳化，这是辅助细胞系能够产生重组病毒的必要前提条件。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a2f/237314/ee4d42d0a0c7/jvirol00021-0762-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a2f/237314/2ba2e3753899/jvirol00021-0760-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a2f/237314/3de9fe8c2583/jvirol00021-0761-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a2f/237314/1fb272c4be65/jvirol00021-0762-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a2f/237314/ee4d42d0a0c7/jvirol00021-0762-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a2f/237314/2ba2e3753899/jvirol00021-0760-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a2f/237314/3de9fe8c2583/jvirol00021-0761-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a2f/237314/1fb272c4be65/jvirol00021-0762-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a2f/237314/ee4d42d0a0c7/jvirol00021-0762-b.jpg

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