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

Wang, Qing S.; Jan, Eric

Infection by dicistroviruses leads to a shutoff of host protein synthesis concomitant with preferential viral translation [27] [28] [29] . To date, the mechanisms underlying the translational shutoff during dicistrovirus infection is not completely understood. Previously, we showed that eIF2a is phosphorylated and eIF4E-eIF4G interactions are compromised during infection [27] . Both mechanisms likely contribute to the inhibition of host translation, which in turn leads to preferential IRES-dependent translation of the viral nonstructural and structural proteins during infection [27] . Furthermore, the structural proteins are expressed in molar excess of the nonstructural proteins, suggesting that IGR IRES translation is stimulated during infection [27] [28] [29] . As IGR IRES exemplifies one of the more unusual mechanisms for translational initiation and for ribosome recruitment, an outstanding question is why dicistroviruses may have evolved this type of mechanism. A probable explanation may be due to, as discovered during poliovirus infection [6, 30] , the availability of cellular and viral factors including many initiation factors during dicistroviruses infection. It is therefore predicted that IGR IRES, which can assemble ribosome without factors, would be translated under many situations when cap-dependent translation is compromised. However, other alternative explanations exist such as the possibility that the replicating viral RNA simply outcompetes the host mRNAs for the translational machinery. Here, we have systematically explored IGR IRES-dependent translation in insect cells under dicistrovirus infection as well as during cellular stresses that compromise different initiation factors. Toward this, we established a transfection approach using capped dicistronic reporter RNAs to monitor both cap-and IGR IRES-mediated translation. We also developed T2A-containing bisictronic constructs to maximize detection of reporter luciferase activities. Using these assays, we determined whether 0 and +1 frame IGR IRES-mediated translation are differentially regulated in vivo.