title: Reverse genetics system for Chandipura virus: tagging the viral matrix protein with green fluorescent protein.
abstract: Chandipura virus (CV; genus Vesiculovirus, family Rhabdoviridae) is an emerging arbovirus, responsible for a number of outbreaks of severe viral encephalitis affecting children in India. A reverse genetics system has been constructed which allows recovery of infectious recombinant CV (rCV) entirely from cDNA. This system was used to construct a virus, rCVE, which has an additional transcription unit encoding green fluorescent protein (EGFP) between the 3rd and 4th CV genes. This virus grew to titres comparable to the parental rCV and stably expressed EGFP in infected cells for at least 4 passages. A second virus, rCVGM, was constructed in which the CV matrix (M) coding region was replaced with the coding region for an EGFP-M fusion protein. Compared to rCV and rCVE, rCVGM was attenuated, giving a small plaque phenotype and lower yield, although it did express the EGFP-M protein in infected cells. Passage of rCVGM resulted in viruses with a standard plaque phenotype which no longer expressed EGFP. Analysis of two of these viruses showed that most or all of the EGFP ORF was deleted. The EGFP-M fusion protein showed cleavage to EGFP-sized and M-sized products, both in rCVGM-infected cells and when expressed from a plasmid. The EGFP-M fusion protein was not detected in virus particles, suggesting it was incompatible with virus assembly, and particles of rCVGM likely contained the M-sized cleavage product in its place.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. tony@southbank3.wanadoo.co.uk
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Marriott
authors.forename: Crystal A
authors.initials: CA
authors.lastname: Hornsey
mesh_terms: D005818:Genetic Engineering / Q000379:methods; D005827:Genetics, Microbial / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D018116:Vesiculovirus / Q000235:genetics* / Q000254:growth & development; D014763:Viral Matrix Proteins / Q000235:genetics; D010948:Viral Plaque Assay; D014773:Virology / Q000379:methods*
chemical_list: D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins