Switch from Cap- to Factorless IRES-Dependent 0 and +1 Frame Translation during Cellular Stress and Dicistrovirus Infection

Wang, Qing S.; Jan, Eric

For all constructs, expression of FLuc is IRES-dependent and RLuc is cap-dependent. Sequences upstream and downstream of the IRES were also included as we and others have shown that inclusion of these sequences enhances IRES translation [19, 25, 31, 33] . Specifically, the downstream region of the CrPV (nucleotides 6217-6372) and IAPV ORF2 (called short ORF2 or sORF2, nucleotides 6618-6908), which includes the overlapping IAPV ORFx gene, were cloned in frame with the FLuc gene ( Figure S1A , S1B). The starting codons of the IAPV and CrPV structural proteins are glycine GGC and alanine GCU codons, respectively. To monitor IAPV IGR IRES-mediated +1 frame translation, we created mutations within the ORFx sequence such that ORFx is fused in frame with FLuc ( Figure 2B ). In generating these constructs, a concern was that inclusion of viral protein sequences in frame with the reporter FLuc ORF may affect the luciferase enzymatic activity, thereby underestimating the actual IRES activity [31] . To circumvent these issues, we developed a novel bicistronic reporter construct which exploits the properties of the 2A 'self-cleaving' peptide ( Figure 2A ) [31] . The 2A peptide stalls translating ribosome by interacting with the exit channel leading to codon-independent translational termination [34] . Ribosomal pausing results in nascent peptide cleavage between the two final amino acids of the peptide, glycine and proline. However, instead of ribosome dissociating from the mRNA, the ribosome continues translation starting from the proline codon. The 2A peptide, termed 'stop-go' or 'stop-carry' translational recoding mechanism, is used by several positive strand RNA viruses to produce separate proteins during translation of the polyprotein. For our studies, we chose the T2A peptide from the insect virus, Thosea asigna, which has been shown to be one of the most efficient and highly active in insect cells [35] [36] [37] . We subcloned the T2A peptide directly upstream and in frame of the FLuc ORF, thereby creating T2A-constructs that monitor both IAPV IGR IRES-mediated 0 ( Figure S1 , B) and +1 frame ( Figure 2B ) translation. We first tested whether there were differences in IGR IRESmediated 0 frame translation between T2A-less and T2Acontaining bicistronic reporter constructs ( Figure S1 ). We previously showed that IGR IRES translation can be assayed by incubating bicistronic reporter constructs in a Sf21 translation extract system and monitoring protein synthesis by either incorporation of radioactive [ 35 S]-methionine or quantitation of luciferase enzymatic activities [25, 31] . As shown previously, a bicistronic reporter construct (minus T2A) containing the IAPV IGR IRES resulted in expression of three radiolabelled proteins, RLuc (scanning-dependent) and IGR IRES-dependent 0 frame sORF-FLuc and +1 frame ORFx ( Figure S1 , C lane 2) [31] . Mutations that disrupt PKI basepairing (DPKI, CC6615-6GG) ( Figure S1 , A) abolished expression of 0 and +1 frame proteins, indicating that IRES translation was being measured (Figure S1, C lane 1). In the case of the T2A-containing bicistronic construct (plus T2A), the T2A 'self-cleaving' activity led to the expression of two 0 frame translation products, sORF2-T2A and the FLuc protein with a proline at the N-terminus (proline-FLuc, P-FLuc) (Figure S1, C lane 3). In addition, because sequences downstream of the IRES were included, +1 frame ORFx protein was also synthesized (Figure S1, C lane 3). The mutant