Establishment of a cell line expressing human parvovirus B19 non-structural protein from an inducible promoter.
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.
Leruez-Ville
M
M
Service de Microbiologie, Unité de Virologie, Hôpital Saint-Louis, CNRS UPR9051, Paris, France.
Vassias
I
I
Pallier
C
C
Cecille
A
A
Hazan
U
U
Morinet
F
F
D015153:Blotting, Western; D002460:Cell Line; D046508:Culture Techniques / Q000379:methods; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D016732:Parvovirus B19, Human / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000096:biosynthesis*
D017931:DNA Primers; D017361:Viral Nonstructural Proteins