Naturally Occurring Dicistronic Cricket Paralysis Virus RNA Is Regulated by Two Internal Ribosome Entry Sites

Abstract

Cricket paralysis virus is a member of a group of insect picorna-like viruses. Cloning and sequencing of the single plus-strand RNA genome revealed the presence of two nonoverlapping open reading frames, ORF1 and ORF2, that encode the nonstructural and structural proteins, respectively. We show that each ORF is preceded by one internal ribosome entry site (IRES). The intergenic IRES is located 6,024 nucleotides from the 5 end of the viral RNA and is more active than the IRES located at the 5 end of the RNA, providing a mechanistic explanation for the increased abundance of structural proteins relative to nonstructural proteins in infected cells. Mutational analysis of this intergenic-region IRES revealed that ORF2 begins with a noncognate CCU triplet. Complementarity of this CCU triplet with sequences in the IRES is important for IRES function, pointing to an involvement of RNA-RNA interactions in translation initiation. Thus, the cricket paralysis virus genome is an example of a naturally occurring, functionally dicistronic eukaryotic mRNA whose translation is controlled by two IRES elements located at the 5 end and in the middle of the mRNA. This finding argues that eukaryotic mRNAs can express multiple proteins not only by polyprotein processing, reinitiation and frameshifting but also by using multiple IRES elements.

Regulation of Internal Ribosomal Entry Site-mediated Translation by Phosphorylation of the Translation Initiation Factor eIF2α

“…Pim-1 is a serine-threonine kinase that functions with c-Myc in cellular transformation (22). This mRNA is translated in poliovirusinfected cells via an IRES element within its 5Ј-UTR (23). We show here that the activity of these IRESs is maintained but not increased by the cellular stresses that increase eIF2␣ phosphorylation.…”

“…pSVCAT/BiP/LUC encodes an mRNA with the 5Ј-UTR of the BiP mRNA as the ICS (21). Three bicistronic plasmids encoding renilla luciferase (RLUC) as the 5Ј-cistron and firefly luciferase (FLUC) as the 3Ј-cistron were used (23). The IGR construct (SV40/ T7⌬EMCV/Fluc-IGR/CrPVORF2) contains 207 bp from the intergenic region (IGR) of cricket paralysis virus (CrPV) in the ICS (23,24).…”

New insights into internal ribosome entry site elements relevant for viral gene expression

“…A fully divergent class of IRES elements are present in the genome of the family Dicistroviridae (Wilson et al, 2000b). This family of RNA viruses possesses a genome that is naturally dicistronic (Fig.…”

Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

“…1A) (5,6). IGR IRES RNAs bind directly to host cell ribosomes, initiate translation of the downstream message using a non-AUG codon, and induce translocation before a peptide bond is made (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Thus, the IGR IRESs are direct manipulators of the translation machinery, and they show how an RNA can drive its own translation using direct ribosome recruitment without protein intermediaries, reminiscent of an RNA world.…”