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

Wang, Qing S.; Jan, Eric

We next compared translation from a panel of dicistrovirus IGR IRESs including the honey bee viruses ABPV and KBV and the fire ant SINV-1 virus. Similar to the cloning strategy for the IAPV and CrPV IGR IRESs, we subcloned sequences adjacent to the IRES to ensure optimal translational activity (see Materials and Methods for details). In S2 cells, the fire ant dicistrovirus SINV-1 IGR IRES showed the highest translational activity, approximately 2-fold higher than that from the IAPV, ABPV and KBV IGR IRESs. As a comparison, the CrPV IGR IRES had the lowest translational activity under basal conditions ( Figure 3B ). We also found that IAPV IGR IRES-dependent translation can be readily detected in another Drosophila cell line, Kc167 cells ( Figure S3, A) . In summary, we have established a general protocol to monitor IGR IRES-mediated translation in Drosophila cells. We next investigated whether +1 frame translation directed by the IAPV IGR IRES can be monitored using the T2A-containing reporter construct in Drosophila S2 cells. Compared to a reporter construct which does not contain an IRES (empty) or a construct containing a mutant IRES (DPKI), +1 frame translation of FLuc using the wild-type IAPV IGR IRES-dependent FLuc translation was detected above background ( Figure 4A, 4C) . Previously, we showed that mutation of G6618 to U, which disrupts the U6562/ G6618 base pairing adjacent to PKI, abolishes +1 frame ORFx translation [25] . The mutant G6618U IRES (G/U) resulted in loss of +1 frame FLuc translation both in vitro and in S2 cells to a level similar to the empty reporter construct ( Figure 4A, 4B, lane 4 and 4C) . A similar result was observed using the reporter construct with a stop codon in place of the +1 frame GCG starting codon ( Figure 4A, 4B, lane 3 and 4C) . Note that insertion of the stop codon in the +1 frame did not affect 0 frame *sORF2 expression ( Figure 4B, lane 3) , confirming that +1 frame translation was measured ( Figure 4B, 4C) . Thus, we report a T2A-containing bicistronic reporter construct that faithfully monitors IAPV IGR IRES-dependent +1 frame translation in vivo.