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 examined whether 0 and +1 frame IAPV IGR IRESmediated translation is regulated differentially during dicistrovirus infection. The ideal experiment would be to monitor IAPV IGR IRES translation during honey bee dicistrovirus infection, however, to date, an appropriate honey bee cell line which can be infected with a pure honey bee dicistrovirus has not been established. As a result, we used S2 cells infected with a related dicistrovirus member, CrPV, as a model system. IRES-containing bicistronic reporter RNAs were transfected into S2 cells 30 minutes after CrPV infection and then harvested at either 6 hours or at the different time points post transfection. CrPV infection in S2 cells results in a rapid shutdown of host protein synthesis [27] . Similar to these findings, comparing to mock-infected cells, capdependent RLuc translation was significantly down regulated by 80-90% as early as 1.5-2.5 hours of infection and by 6.5 hours post infection ( Figure 6A,6B) . Note in Figure 6B , the RLuc and FLuc activities are normalized to that at 6 hours after transfection in mock-infected cells. In contrast, FLuc expression driven by the CrPV IGR IRES was significantly enhanced at all times post infection. At 6.5 hours post infection, CrPV IGR IRES-mediated translation was stimulated approximately 2.5-3 fold as compared to that during mock-infection ( Figure 6A ). As expected, the mutant DPKI IRES did not result in significant FLuc expression in mock-or CrPV-infected S2 cells ( Figure 6A ). We next examined the relative CrPV IGR IRES (FLuc) to cap-dependent (RLuc) translation ratio in mock-and CrPV-infected cells. In mock-infected cells, CrPV IGR IRES translation is 0.8% of capdependent translation, indicating that IRES translation is weak in vivo. Under virus infection, CrPV IGR IRES translation was 16% of cap-dependent translation, which is in part due to the 80-90% shutoff of overall cap-dependent translation and the stimulation of IRES translation. These results demonstrate that there is a switch from cap-dependent to IRES-dependent translation during CrPV infection.