Stable expression of the reovirus mu 2 protein in mouse L cells complements the growth of a reovirus ts mutant with a defect in its M1 gene

Reovirus mu 2 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 mu 2 protein was detected by mu 2-specific antibody produced through immunization of rabbits with Trp-E-mu 2 fusion proteins expressed in Escherichia coli. The expression levels of mu 2 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. mu 2-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 mu 2 expression that were less stable and thus decreased on continued cell culture. The mu 2 protein expressed in transfectants was authentic as shown by peptide mapping comparison with mu 2 protein from reovirus-infected cells and that from in vitro transcription and translation of the M1 gene. It was further shown that the mu 2 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 mu 2 protein stably expressed by transfection is functionally equivalent to mu 2 protein expressed by reovirus. 1995 Academic Press, Inc.