FIG. 1.

The ORF 3 protein is produced by cell-free translation of ORF 3 and ORFs 2+3 transcripts. (A) Analysis of proteins produced by translation of synthetic mRNAs using RRLs in the absence (−) or presence (+) of canine pancreatic microsomal membranes. The translated proteins were labeled using Tran³⁵S-label. An aliquot (10 μl/25 μl) of the translation reaction mixture was analyzed on an SDS–15% polyacrylamide gel. (B) Immunoprecipitation of the ORF 3 protein from cell-free translation reaction mixtures using the ORF 3 C-terminal peptide antiserum. The remaining aliquot of the translation reaction mixtures in panel A was immunoprecipitated in RIPA buffer, and the entire sample was analyzed by autoradiography of an SDS–15% polyacrylamide gel. Lane M contains ¹⁴C-methylated protein molecular weight markers (Amersham); lane ORFs 2+3 contains the entire 3′ end of the genome beginning with the ORF 2 coding sequences; lane G10 contains rotavirus gene 10; lane Vector contains pGem7Zf(+) vector with no insert. The ORF 2, ORF 3, G10, and glycosylated G10 (gG10) protein products are designated.