Switch from cap- to factorless IRES-dependent 0 and +1 frame translation during cellular stress and dicistrovirus infection.
ABSTRACT: Internal ribosome entry sites (IRES) are utilized by a subset of cellular and viral mRNAs to initiate translation during cellular stress and virus infection when canonical cap-dependent translation is compromised. The intergenic region (IGR) IRES of the Dicistroviridae uses a streamlined mechanism in which it can directly recruit the ribosome in the absence of initiation factors and initiates translation using a non-AUG codon. A subset of IGR IRESs including that from the honey bee viruses can also direct translation of an overlapping +1 frame gene. In this study, we systematically examined cellular conditions that lead to IGR IRES-mediated 0 and +1 frame translation in Drosophila S2 cells. Towards this, a novel bicistronic reporter that exploits the 2A "stop-go" peptide was developed to allow the detection of IRES-mediated translation in vivo. Both 0 and +1 frame translation by the IGR IRES are stimulated under a number of cellular stresses and in S2 cells infected by cricket paralysis virus, demonstrating a switch from cap-dependent to IRES-dependent translation. The regulation of the IGR IRES mechanism ensures that both 0 frame viral structural proteins and +1 frame ORFx protein are optimally expressed during virus infection.
Project description:Cricket paralysis virus (CrPV) is a dicistrovirus. Its positive-sense single-stranded RNA genome contains two internal ribosomal entry sites (IRESs). The 5' untranslated region (5'UTR) IRES5'UTR mediates translation of non-structural proteins encoded by ORF1 whereas the well-known intergenic region (IGR) IRESIGR is required for translation of structural proteins from open reading frame 2 in the late phase of infection. Concerted action of both IRES is essential for host translation shut-off and viral translation. IRESIGR has been extensively studied, in contrast the IRES5'UTR remains largely unexplored. Here, we define the minimal IRES element required for efficient translation initiation in drosophila S2 cell-free extracts. We show that IRES5'UTR promotes direct recruitment of the ribosome on the cognate viral AUG start codon without any scanning step, using a Hepatitis-C virus-related translation initiation mechanism. Mass spectrometry analysis revealed that IRES5'UTR recruits eukaryotic initiation factor 3, confirming that it belongs to type III class of IRES elements. Using Selective 2'-hydroxyl acylation analyzed by primer extension and DMS probing, we established a secondary structure model of 5'UTR and of the minimal IRES5'UTR. The IRES5'UTR contains a pseudoknot structure that is essential for proper folding and ribosome recruitment. Overall, our results pave the way for studies addressing the synergy and interplay between the two IRES from CrPV.
Project description:The dicistrovirus intergenic internal ribosome entry site (IGR IRES) directly recruits the ribosome and initiates translation using a non-AUG codon. A subset of IGR IRESs initiates translation in either of two overlapping open reading frames (ORFs), resulting in expression of the 0 frame viral structural polyprotein and an overlapping +1 frame ORFx. A U-G base pair adjacent to the anticodon-like pseudoknot of the IRES directs +1 frame translation. Here, we show that the U-G base pair is not absolutely required for +1 frame translation. Extensive mutagenesis demonstrates that 0 and +1 frame translation can be uncoupled. Ribonucleic acid (RNA) structural probing analyses reveal that the mutant IRESs adopt distinct conformations. Toeprinting analysis suggests that the reading frame is selected at a step downstream of ribosome assembly. We propose a model whereby the IRES adopts conformations to occlude the 0 frame aminoacyl-tRNA thereby allowing delivery of the +1 frame aminoacyl-tRNA to the A site to initiate translation of ORFx. This study provides a new paradigm for programmed recoding mechanisms that increase the coding capacity of a viral genome.
Project description:The dicistrovirus Cricket Paralysis virus contains a unique dicistronic RNA genome arrangement, encoding two main open reading frames that are driven by distinct internal ribosome entry sites (IRES). The intergenic region (IGR) IRES adopts an unusual structure that directly recruits the ribosome and drives translation of viral structural proteins in a factor-independent manner. While structural, biochemical, and biophysical approaches have provided mechanistic details into IGR IRES translation, these studies have been limited to in vitro systems and little is known about the behavior of these IRESs during infection. Here, we examined the role of previously characterized IGR IRES mutations on viral yield and translation in CrPV-infected Drosophila S2 cells. Using a recently generated infectious CrPV clone, introduction of a subset of mutations that are known to disrupt IRES activity failed to produce virus, demonstrating the physiological relevance of specific structural elements within the IRES for virus infection. However, a subset of mutations still led to virus production, thus revealing the key IRES-ribosome interactions for IGR IRES translation in infected cells, which highlights the importance of examining IRES activity in its physiological context. This is the first study to examine IGR IRES translation in its native context during virus infection.
Project description:The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2alpha phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2alpha by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2alpha phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2alpha phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5' untranslated region (5'UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5'UTR and IGR IRES.
Project description:Some viral and cellular messages use an alternative mechanism to initiate protein synthesis that involves internal recruitment of the ribosome to an internal ribosome entry site (IRES). The Dicistroviridae intergenic regions (IGR) have been studied as model IRESs to understand the mechanism of IRES-mediated translation. In this study, the in vivo activity of IGR IRESs were compared. Our analysis demonstrates that Class I and II IGR IRESs have comparable translation efficiency in yeast and that Class II is significantly more active in mammalian cells. Furthermore, while Class II IGR IRES activity was enhanced in yeast grown at a higher temperature, temperature did not affect IGR IRES activity in mammalian cells. This suggests that Class II IRESs may not function optimally with yeast ribosomes. Examination of chimeric IGR IRESs, established that the IRES strength and temperature sensitivity are mediated by the ribosome binding domain. In addition, the sequence of the first translated codon is also an important determinant of IRES activity. Our findings provide us with a comprehensive overview of IGR IRES activities and allow us to begin to understand the differences between Classes I and II IGR IRESs.
Project description:The Dicistroviridae is a growing virus family characterized by a dicistronic genome, wherein each open reading frame (ORF) is translated from an independent internal ribosome entry site (IRES). The 5' IRES that translates the first open reading frame (ORF1) is similar to the picornaviral IRESs. However the second IRES, referred to as the intergenic region (IGR) IRES, - translates ORF2 by and uses an unusual mechanism of initiating protein synthesis. It folds into a compact RNA structure that can bind directly to 40S ribosomal subunits and form 80S complexes to initiate translation in the absence of any initiation factors. Despite its unusual mechanism, the IGR IRES has proven to be an elegant model for elucidating initiation mechanisms employed by IRESs, as well as making it a powerful research tool with diverse applications.
Project description:Internal ribosome entry site (IRES) elements found in the 5' untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems.
Project description: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.
Project description:All viruses must successfully harness the host translational apparatus and divert it towards viral protein synthesis. Dicistroviruses use an unusual internal ribosome entry site (IRES) mechanism whereby the IRES adopts a three-pseudoknot structure that accesses the ribosome tRNA binding sites to directly recruit the ribosome and initiate translation from a non-AUG start site. A subset of dicistroviruses, including the honey bee Israeli acute paralysis virus (IAPV), encode an extra stem-loop (SLVI) 5' -adjacent to the IGR IRES. Previously, the function of this additional stem-loop is unknown. Here, we provide mechanistic and functional insights into the role of SLVI in IGR IRES translation and in virus infection. Biochemical analyses of a series of mutant IRESs demonstrated that SLVI does not function in ribosome recruitment but is required for proper ribosome positioning on the IRES to direct translation. Using a chimeric infectious clone derived from the related Cricket paralysis virus, we showed that the integrity of SLVI is important for optimal viral translation and viral yield. Based on structural models of ribosome-IGR IRES complexes, the SLVI is predicted to be in the vicinity of the ribosome E site. We propose that SLVI of IAPV IGR IRES functionally mimics interactions of an E-site tRNA with the ribosome to direct positioning of the tRNA-like domain of the IRES in the A site.IMPORTANCEViral internal ribosome entry sites are RNA elements and structures that allow some positive-sense monopartite RNA viruses to hijack the host ribosome to start viral protein synthesis. We demonstrate that a unique stem-loop structure is essential for optimal viral protein synthesis and for virus infection. Biochemical evidence shows that this viral stem-loop RNA structure impacts a fundamental property of the ribosome to start protein synthesis.
Project description:The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.