Biosynthesis of Reovirus-Specified Polypeptides: Identification of Regions of the Bicistronic Reovirus S1 mRNA That Affect the Efficiency of Translation in Animal Cells

Abstract

The reovirus S1 gene cDNA was systematically altered by site-directed mutagenesis in an attempt to identify regions important in determining the relative efficiency of translation of the two open reading frames of the bicistronic S1 mRNA. The synthesis of the minor capsid protein σ1 encoded by ORF1, extending from AUG14 to UGA1424, and the synthesis of the nonstructural protein σ1NS encoded by ORF2, extending from AUG75 to UAG432, were examined in transfected COS cells. Deletion of the 5′-untranslated region upstream of ORF1 AUG14 did not significantly affect either the relative amount, or the ratio, of σ1 and σ1NS synthesized. Creation of a strong context ORF1 initiation site by substitution of 5′-untranslated region nucleotides flanking AUG 14 likewise did not affect σ1 synthesis, but σ1 NS synthesis from ORF2 was decreased about twofold relative to wild-type S1 mRNA. The amount of σ1NS synthesis was increased less than twofold either by elimination of the ORF1 AUG14 initiation codon or by termination of σl synthesis shortly after initiation from AUG14. No σ1 synthesis was detected when the ORF1 AUG14 was mutated to a UUG14 codon. When ribosomes which initiated translation at AUG14 were frameshifted at the next codon after AUG14, elongation occurred with comparable efficiency in the σ1NS ORF2 and in σ1 ORF1. No σ1NS synthesis was detected when the ORF2 AUG75 was mutated to an CUG75 codon, and this mutation did not affect the amount of σ1 synthesis. err NS synthesis was not affected by truncation of the 3′-untranslated region or premature termination of σl synthesis shortly after the ORF2 UAG432. However, truncation of the ORF2 5′-untranslated region at either 6 or 36 nt following the ORF1 AUG14 significantly increased the efficiency of σ1NS synthesis. These results indicate that the region of the S1 mRNA immediately downstream of the σ1 ORF1 initiation codon AUG14 but well upstream of the σ1NS ORF2 initiation codon AUG75 plays a major role in determining the relative efficiency of synthesis of σ1 and σ1NS from the reovirus bicistronic s1 mRNA.