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 t he same phosphoglycerate kinase promoter. Translation of neo was initi ated with the cap-independent translation initiation element from ence phalomyocarditis 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 l evels of mu 2 proteins of serotype 1 (T1) and serotype 3 (T3) were dif ferent and varied in different mouse cell lines with T1 being expresse d more efficiently than T3. mu 2-expressing L929 cell lines generated with the dicistronic constructs were highly stable. Inclusion of the t ransforming fragment of bovine papillomavirus in the dicistronic const ruct lead to higher levels of mu 2 expression that were less stable an d 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 th at 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 transfecti on is functionally equivalent to mu 2 protein expressed by reovirus. 1 995 Academic Press, Inc.