Synergism of the 3′-Untranslated Region and an Internal Ribosome Entry Site Differentially Enhances the Translation of a Plant Virus Coat Protein*

The use of internal ribosome entry sites (IRESs) is one of the unorthodox mechanisms exploited by viruses to initiate the translation of internal genes. Herein, we report a plant virus exploiting an IRES and its 3′-untranslated region (UTR) to express its internal genes, notably the 3′-proximal viral coat protein gene. Hibiscus chlorotic ringspot virus (HCRSV), a positive-strand non-polyadenylated RNA virus, was demonstrated to harbor a unique 100-nucleotide (nt) IRES, located 124 nt upstream of the coat protein gene, that could function in wheat germ extract, rabbit reticulocyte lysate, and mammalian cells. In comparison with other known IRESs of picornaviruses and eukaryotic mRNAs, this 100-nt IRES is distinctively short and simple. The IRES activity was tested in homologous and heterologous bicistronic constructs, and the expression of the 3′-proximal gene was enhanced when the 3′-UTR was present. When the IRES element was bisected, each half still possessed IRES activity and could initiate internal translation on its own. Site-directed mutagenesis and deletion analyses revealed that the primary sequence within the 5′ half was crucial for IRES activity, whereas the primary sequence of the second half and a GNRA motif were non-essential. To our knowledge, this is the first report describing a mechanism whereby an IRES, located in the 3′ portion of the virus genome, co-operates with the 3′-UTR to enhance gene expression differentially.