Internal initiation of translation in retroviral vectors carrying picornavirus 5' nontranslated regions

Abstract

Previous work has shown that picornavirus 5' nontranslated regions (NTRs) can initiate internal translation of downstream coding regions both in vitro and in transient in vivo assays. We have used 5' NTR sequences from encephalomyocarditis virus and poliovirus to construct retroviral vectors that are designed to express two proteins from a single mRNA. Inclusion of 5' NTR sequences did not adversely affect vector titer. Protein expression was studied with stable cell lines generated by vector infection of mouse NIH 3T3 cells and human and canine skin fibroblasts. Expression of a coding region in the downstream position was at levels from 25 to 100% of the same coding region in the upstream position. Expression of downstream coding regions in control vectors that did not contain the 5' NTR sequences was very low, in agreement with the predictions of the scanning model for eukaryotic translation. These experiments demonstrate coordinate expression of two coding regions from a single mRNA in stable cell lines and provide further support for the model of internal translation initiation by sequences in the 5' NTRs of picornaviruses.