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

Wang, Qing S.; Jan, Eric

Similar to that observed with the CrPV IGR IRES, transfection of reporter RNAs containing the IAPV IGR IRES resulted in stimulation of FLuc translation in both the 0 and +1 frames during CrPV infection (Figure 7) . In CrPV-infected cells, both 0 and +1 frame IAPV IGR IRES translation was stimulated 2.5-5 fold as compared to that in mock-infected cells, whereas the capdependent RLuc expression was inhibited ( Figure 7A-7D) . The T2A-containing 0 frame reporter RNA showed a similar fold increase at 6.5 h.p.i. CrPV infection as that observed with the T2A-minus 0 frame reporter RNA (data not shown). As expected, mutations that disrupt PKI basepairing in the IAPV IRES abolished 0 and +1 frame FLuc expression in mock-and CrPVinfected cells ( Figure 7A-7B) . A time course following the expression of RLuc and FLuc showed that cap-dependent translation was inhibited early in infection and remained shutoff (,90% inhibition) throughout infection. In contrast, IGR IRESdependent 0 and +1 frame translation increased throughout the course of infection ( Figure 7C-7D) . Over the course of infection, the relative +1 to 0 frame translation did not appear to change significantly, suggesting that both 0 and +1 frame IAPV IRESmediated translation were similar during CrPV infection (Figure 7E ). We do note that at 3.5-4.5 h.p.i., the relative ratio of +1 to 0 frame translation decreases slightly, suggesting that 0 and +1 frame translation may be differentially regulated albeit to a minor extent. Similar to that observed with CrPV IRES translation, 0 frame IAPV IGR IRES translation was 1.1% and 11% of capdependent translation in mock-and CrPV-infected cells, indicating a switch from cap-dependent to IRES-dependent translation. In summary, these results demonstrate that IAPV IGR IRES translation is stimulated during CrPV infection.