Stable expression of the reovirus mu2 protein in mouse L cells complements the growth of a reovirus ts mutant with a defect in its M1 gene.
Reovirus mu2 protein was constitutively expressed in mammalian cells transfected with dicistronic constructs in which the reovirus M1 gene and the selectable neomycin-resistant gene (neo) were both driven by the same phosphoglycerate kinase promoter. Translation of neo was initiated with the cap-independent translation initiation element from encephalomyocarditis virus. Expression of mu2 protein was detected by mu2-specific antibody produced through immunization of rabbits with Trp-E-mu2 fusion proteins expressed in Escherichia coli. The expression levels of mu2 proteins of serotype 1 (T1) and serotype 3 (T3) were different and varied in different mouse cell lines with T1 being expressed more efficiently than T3. mu2 expressing L929 cell lines generated with the dicistronic constructs were highly stable. Inclusion of the transforming fragment of bovine papillomavirus in the dicistronic construct lead to higher levels of mu2 expression that were less stable and thus decreased on continued cell culture. The mu2 protein expressed in transfectants was authentic as shown by peptide mapping comparison with mu2 protein from reovirus-infected cells and that from in vitro transcription and translation of the M1 gene. It was further shown that the mu2 protein expressed in a stable L929 cell line complemented the growth of the reovirus tsH11.2 mutant with a defect in its M1 gene. It is concluded that the mu2 protein stably expressed by transfection is functionally equivalent to mu2 protein expressed by reovirus.
Zou
S
S
Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ontario, Canada.
Brown
E G
EG
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D005814:Genes, Viral; D005816:Genetic Complementation Test; D007739:L Cells; D051379:Mice; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012087:Reoviridae / Q000235:genetics*; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000914:Antibodies, Viral; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins