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

Cricket paralysis virus (CrPV) was isolated in 1970 by Carl Reinganum (47), who observed that laboratory colonies of Australian field crickets (Teleogryllus oceanicus and T. commodus) contained some early-instar nymphs which developed a paralysis of the hind legs, became uncoordinated, and died. Electron microscopic sections of paralyzed insects revealed many virus-like particles in crystalline arrays reminiscent of those observed in picornavirus-infected cells. Although originally isolated from crickets, CrPV has a wide host range, infecting insects which belong to Diptera, Lepidoptera, Orthoptera, and Heteroptera species (8). Importantly, it also replicates in cultured cells from various insect species including Drosophila SL2 cells (37, 55). More recently, CrPV has been classified as a member of a group of insect picorna-like viruses which also includes Drosophila C virus (DCV) (24), Plautia stali intestine virus (PSIV) (53), himetobi P virus (HiPV) (40), and Rhopalosiphum padi virus (RhPV) (36). Insect picorna-like viruses share with mammalian picornaviruses many physical and morphological properties of the viral structural proteins (37, 56, 59), and the presence of a single plus-strand RNA genome with a genome-linked protein at the 5′ end (25) and polyadenosine residues at the 3′ end (9). In contrast to earlier reports (26), recent cloning and sequencing of several insect picorna-like viruses has revealed that the organization of these viral genomes differs from that of human picornaviruses (8, 28). Specifically, picornavirus RNA genomes consist of a single open reading frame (ORF) encoding a polyprotein which is posttranslationally processed to give rise to both structural (encoded in the N-terminal part of the polyprotein) and nonstructural viral proteins (49). In contrast, certain insect picorna-like virus genomes encode two distinct polyproteins. In these genomes, the polyprotein precursors to the nonstructural viral proteins are encoded by the upstream ORF (ORF1), while the structural protein precursors are encoded by the downstream ORF (ORF2) (24, 36, 40, 53). Furthermore, it has been noted that structural proteins accumulate in vast excess over nonstructural proteins in cells infected with such insect picorna-like viruses (37, 38). In contrast, cells infected with human picornaviruses produce equimolar amounts of structural and nonstructural proteins (49). The differential expression of the two insect virus polyproteins from a single mRNA has led to the suggestion that the two ORFs might be under independent translational control. By analogy to picornavirus polyproteins, which are known to be translated by an internal initiation mechanism, we speculated that both ORFs in picorna-like virus mRNAs might be translated by internal initiation. Consistent with this idea, ORF2 in the PSIV RNA genome is translated cap independently in the rabbit reticulocyte lysate (RRL) (52). Here we show that the CrPV RNA genome encodes two large, nonoverlapping ORFs with viral nonstructural and structural polyprotein precursors encoded by the upstream and downstream ORFs, respectively. Each ORF is preceded by an internal ribosome entry site (IRES), demonstrating that the CrPV RNA genome is a naturally occurring eukaryotic RNA that is functionally dicistronic. Mutational analysis of the downstream IRES has revealed that the first three nucleotides in the CrPV downstream ORF are CCU, which differs from the canonical AUG start codon at all three positions. This finding, together with the fact that complementarity of the CCU codon with an upstream sequence in the IRES must be maintained to preserve IRES function, suggests an unusual mechanism of translation initiation mediated by the intragenic region (IGR) of CrPV.