Leruez-Ville M, Vassias I, Pallier C, Cecille A, Hazan U, Morinet F
Service de Microbiologie, Unité de Virologie, Hôpital Saint-Louis, CNRS UPR9051, Paris, France.
J Gen Virol. 1997 Jan;78 ( Pt 1):215-9. doi: 10.1099/0022-1317-78-1-215.
Human parvovirus B19 non-structural (NS) protein is supposed to play a major role in B19 replication and transcription, and therefore in B19 pathogenicity. Constitutive expression of NS protein in stable cell lines has failed so far, presumably because of its cytotoxicity. To avoid this cytotoxic effect, we have cloned the NS gene in an Epstein-Barr virus episomal vector under the control of a steroid inducible promoter (5xGRE) and transfected this construction into HeLa cells. We obtained stable cell lines inducibly expressing high level of NS protein, with 50% of the cells demonstrating specific nucleo-cytoplasmic staining. In Western blot analysis, three B19 NS proteins (72, 68 and 60 kDa) were found but a unique NS transcript was detected by Northern blotting. The NS protein expressed in HeLa cell lines was demonstrated to be functional as it trans-activates the B19 P6 promoter. These cell lines might be major tools for further study and characterization of B19 NS protein.
人细小病毒B19非结构（NS）蛋白被认为在B19的复制和转录中起主要作用，因此在B19的致病性中也起主要作用。迄今为止，NS蛋白在稳定细胞系中的组成型表达均告失败，推测是由于其细胞毒性所致。为避免这种细胞毒性作用，我们将NS基因克隆到一个在类固醇诱导型启动子（5xGRE）控制下的Epstein-Barr病毒附加型载体中，并将此构建体转染到HeLa细胞中。我们获得了可诱导表达高水平NS蛋白的稳定细胞系，其中50%的细胞呈现特异性核质染色。在蛋白质印迹分析中，发现了三种B19 NS蛋白（72、68和60 kDa），但通过Northern印迹检测到一个独特的NS转录本。在HeLa细胞系中表达的NS蛋白被证明具有功能，因为它能反式激活B19 P6启动子。这些细胞系可能是进一步研究和鉴定B19 NS蛋白的主要工具。
