Translation of the reovirus M1 gene initiates from the first AUG codon in both infected and transfected cells

Abstract

Reovirus μ2 protein can be expressed via the mouse phosphoglycerate kinase promoter to low levels in stably transfected L cells. To increase μ2 expression, the terminal regions of the M1 gene cDNA constructs were modified and the effect on μ2 expression was analyzed. The M1 gene has a single large open reading frame beginning at nucleotide 14 with another, in frame, AUG codon at nucleotide 161 reported to be used for translation initiation. Unexpectedly, deletions of the M1 5′ terminal sequence upstream of the reported translation initiation codon, AUG 161, resulted in loss of detection of μ2 expression. When expression was driven by the stronger T7 promoter in the presence of recombinant vaccinia virus expressing the T7 RNA polymerase, constructs with the M1 5′-terminal deletion produced a smaller protein product of ≈ 68 kDa, compared to ≈ 73 kDa for the protein produced from the full-length M1-containing constructs consistent with the loss of 49 amino acids. The amount of shorter μ2 product was increased by producing an improved ‘Kozak’ consensus sequence around the AUG codon at nucleotide 161 or by introducing an internal ribosome entry site at this location. Full-length M1 gene constructs produced a protein of the same size as the authentic μ2 protein from virus-infected cells. It was further shown that the ≈ 73 kDa product was expressed when the M1 gene was in different plasmid backgrounds and even when the M1 gene transcript was preceeded by a 1 kb gene. This study demonstrated that translation of the reovirus M1 gene initiates from the first AUG codon in both infected and transfected cells.