title: The densovirus of Junonia coenia (Jc DNV) as an insect cell expression vector.
abstract: An infectious genome of the Junonia coenia densovirus (Jc DNV) has been recently cloned and sequenced. We investigated the ability of this cloned genome to be used as expression vector by inserting the lacZ gene of Escherichia coli as fusion gene in the major open reading frame (ORF 1) of the viral sequence. The resulting recombinant plasmid designated pBRJlac Z was transfected into insect SPC-SL 52 cells and the expression of beta-galactosidase (beta-gal) was detected qualitatively or quantitatively by using Xgal or ONPG as chromogenic substrates. Western blot analysis revealed that beta-gal was expressed as chimeric capsid-beta-gal polypeptides. This provided evidence that ORF1 codes for structural polypeptides which share a common C-terminal sequence. Construction of plasmids with alterations or deletions in ORF2, 3 or 4, allowed us to implicate nonstructural (NS) functions in viral DNA replication. Deletions in inverted terminal repeats or in NS functions did not abolish expression of capsid polypeptides but reduced it dramatically. Encapsidation of Jlac Z recombinant genome was achieved by trans-complementation with plasmids bearing intact structural and nonstructural functions. Detection of a beta-gal activity in SPC-SL 52 cells following several subcultures post-transfection suggests that Jlac Z recombinant genome could be maintained in an integrative or episomal state.
authors.affiliation: Station de Recherches de Pathologie Comparée, INRA-UA CNRS, Saint Christol-lez-Alès, France.
authors.forename: C
authors.initials: C
authors.lastname: Giraud
authors.forename: G
authors.initials: G
authors.lastname: Devauchelle
authors.forename: M
authors.initials: M
authors.lastname: Bergoin
mesh_terms: D000818:Animals; D002213:Capsid / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004274:DNA, Recombinant; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D005822:Genetic Vectors; D007313:Insecta / Q000166:cytology; D010321:Parvoviridae / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014758:Viral Core Proteins / Q000235:genetics; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins; D001616:beta-Galactosidase