Stable expression of the reovirus μ2 protein in mouse L cells complements the growth of a reovirus ts mutant with a defect in its M1 gene

Abstract

Reovirus μ2 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 μ2 protein was detected by μ2-specific antibody produced through immunization of rabbits with Trp-E-μ2 fusion proteins expressed in Escherichia coli. The expression levels of μ2 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. μ2-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 μ2 expression that were less stable and thus decreased on continued cell culture. The μ2 protein expressed in transfectants was authentic as shown by peptide mapping comparison with μ2 protein from reovirus-infected cells and that from in vitro transcription and translation of the M1 gene. It was further shown that the μ2 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 μ2 protein stably expressed by transfection is functionally equivalent to μ2 protein expressed by reovirus.