Abstract
Internal ribosome entry is a key mechanism for viral protein synthesis in a subset of RNA viruses. Cricket paralysis virus (CrPV), a member of Dicistroviridae, has a positive-sense single strand RNA genome that contains two internal ribosome entry sites (IRES), a 5′untranslated region (5′UTR) and intergenic region (IGR) IRES, that direct translation of open reading frames (ORF) encoding the viral non-structural and structural proteins, respectively. The regulation of and the significance of the CrPV IRESs during infection are not fully understood. In this study, using a series of biochemical assays including radioactive-pulse labelling, reporter RNA assays and ribosome profiling, we demonstrate that while 5′UTR IRES translational activity is constant throughout infection, IGR IRES translation is delayed and then stimulated two to three hours post infection. The delay in IGR IRES translation is not affected by inhibiting global translation prematurely via treatment with Pateamine A. Using a CrPV replicon that uncouples viral translation and replication, we show that the increase in IGR IRES translation is dependent on expression of non-structural proteins and is greatly stimulated when replication is active. Temporal regulation by distinct IRESs within the CrPV genome is an effective viral strategy to ensure optimal timing and expression of viral proteins to facilitate infection.
Highlights
Viral protein synthesis is an essential process in all viral life cycles
It is well established that viral structural proteins are expressed in molar excess over the viral non-structural proteins during Cricket paralysis virus (CrPV) infection [7,8,9], we carefully re-examined viral protein synthesis in CrPV-infected S2 cells by metabolic [35 S]-Met/Cys pulse-labelling at different times after infection
Using CrPV(Fluc) replicons to decouple viral translation from viral replication, we showed that the increase in intergenic region (IGR) internal ribosome entry sites (IRES) translation is partly dependent on viral non-structural proteins (Figure 7)
Summary
Viral protein synthesis is an essential process in all viral life cycles. As such, viruses have adapted diverse strategies to recruit the host ribosome and the translational machinery. Numerous positive strand RNA viruses use a strategy whereby infection leads to an inhibition of host translation concomitant with an increase in viral protein synthesis [1]. One of the best-studied examples of this switch from host to viral protein synthesis is during poliovirus infection: the translation factors eIF4G and poly A binding protein are cleaved by virally-encoded proteases thereby shutting off cap-dependent translation [2,3,4]. The poliovirus internal ribosome entry site, IRES, recruits the ribosome using a subset of translation factors and IRES trans-acting factors (ITAFs) to direct cap-independent viral protein synthesis [5]. The strategies that underlie the switch from host to viral protein synthesis such as the signalling pathways that target translation factors and the mechanisms that direct viral protein expression are fundamental to virus infection
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