Eric Jan

Viruses have evolved a variety of mechanisms to subvert cellular processes, specifically at the point of translation, a highly regulated process that relies on recognition of the 5’-cap. One subversive mechanism that many RNA viruses use is the internal ribosome entry site (IRES), a RNA secondary structure that can bypass the necessity of the 5’-cap, translation factors, and even AUG start codon. The most streamlined IRESs are intergenic region (IGR) IRESs, derived from dicistroviruses, where it resides between two main viral open reading frames (ORFs).Interestingly, a subset of dicistroviruses have been identified to contain a +1-frame open reading frame (ORFx) that overlaps the open reading frame 2 (ORF2). Recent studies have indicated some possible mechanisms for +1 frame translation and the role of the resulting protein, but much of it remains elusive. In chapter 2, I evaluated dataset of dicistro-like viruses for predicted +1 open reading frame ORFx. Further, I examined the mechanism of the +1-frame ORFx translation from black queen cell virus (BQCV) IGR IRES, which has previously exemplified high ratios of translation. I determined that BQCV +1-frame translation is IGR IRES-dependent and driven by the downstream sequence. As well, I provide evidence that the BQCV IGR IRES-mediated +1-frame translation is dependent on translation of the 0-frame protein. I propose a model wherein translation begins in the 0-frame and frameshifts downstream into +1-frame.Recently, metagenomic screens of environmental samples have identified previously unknown dicistroviruses and dicistro-like viruses that contain an IGR IRES. Some of these genomes have uncommon structural and organizational characteristics. Specifically, some reside in coding regions of the first open reading frame (ORF1). In chapter 3, two novel predicted IGR IRESs, DS1 and DS2, are evaluated for functionality in vitro on their ability to bind to ribosomes and translation. Further, these are evaluated on how genome arrangement affects translation and translational regulation of the first and second open reading frames. Lastly, these IGR IRESs are analyzed for important structural motifs, providing insights on the similarity of these mechanisms to previously studied IGR IRESs.

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Internal ribosome entry sites (IRES) are structured RNA elements that recruit ribosomes using a subset of translation factors in a 5' cap-independent manner. The dicistrovirus IRES uses the most streamlined mechanism: the IRES recruits ribosomes directly without the use of initiation factors and directs translation from a non-AUG codon. The IRES adopts three pseudoknots, stem-loop, and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits. Analysis of dicistrovirus-like genomes identified via metagenomic studies predicts potentially novel atypical IRES RNA structures. Specifically, the Bemisia-associated dicistrovirus 2 (BaDV-2) IRES contains an unusual RNA structure that lacks conserved elements and altered structures that do not conform to the typical dicistrovirus IRES mechanism. Moreover, the BaDV-2 genome contains a predicted -1 frameshifting signal (FSS) with an RNA stem-loop structure that would direct the translation of the RNA-dependent RNA polymerase (RdRp) motif. In this study, using a bicistronic reporter, we demonstrate that BaDV-2 IGR supports internal ribosome entry activity in in vitro insect Sf-21 and rabbit reticulocyte translation extracts. Using deletion and mutagenesis analyses, we showed that the BaDV-2 IRES activity is within a minimal 140 nucleotide element containing a predicted stem loop. Moreover, the IRES is sensitive to eIF2 and eIF4A inhibitors, NSC1198983 and hippuristanol, respectively, indicating that the BaDV-2 IRES is factor-dependent, unlike typical factor-less dicistrovirus IRESs. Preliminary data shows that a chimeric dicistrovirus infectious clone does not support virus infection in Drosophila cells. Finally, we show that the predicted stem-loop structure -1 frameshifting signal can direct programmed frameshifting in vitro using a luciferase-based bicistronic reporter. In summary, we have provided evidence of the first -1 frameshifting signal and a novel IRES mechanism in this viral family, thus highlighting the diversity of viral strategies to direct viral protein synthesis.

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Death and dysfunction of insulin producing pancreatic β-cells is a major feature of diabetes. Recently, it was discovered that the eukaryotic initiation factor 2A (EIF2A) – an unconventional translation factor – was uniquely abundant in β-cells compared to other cell types. Past studies using lentiviral transduction to overexpress EIF2A in MIN6 cells experiencing ER stress resulted in increased β-cell survival and reversed the downregulation of global translation that normally occurs as part of the unfolded protein response (UPR), as it seeks to alleviate stress. In this study, we used MIN6 cells and human islets transduced with adenoviruses to induce EIF2A overexpression. Amino acid radiolabeling was used to measure nascent global translation, in combination with immunoprecipitation to isolate and measure proinsulin synthesis. Western blotting was used to quantify changes in steady state protein levels under stressed and unstressed conditions. In MIN6 cells, adenovirus-induced overexpression of GFP-tagged EIF2A did not result in restoration of translation as previously seen, and proinsulin synthesis did not appear to be affected separately. However, EIF2A overexpression appeared to improve proinsulin-insulin conversion under stress. In human islets, regardless of EIF2A overexpression, thapsigargin treatment did not induce a significant repression of translation, nor were we able to measure proinsulin synthesis in any condition using our methods. Western blotting showed that EIF2B subunits were affected differentially by EIF2A overexpression. EIF2B5, which contains the guanine exchange factor catalytic site, was upregulated in EIF2A overexpressing cells in both stressed and unstressed conditions. EIF2B3, which is responsible for GTP binding, was upregulated in EIF2A-overexpressing cells only under stress. The EIF2B2 subunit, which is part of the core that mediates interactions with EIF2S1, showed no trend towards upregulation by EIF2A overexpression. This suggests that EIF2A’s effects on translation may indeed be mediated through EIF2B, but consequently the exact mechanism by which this occurs, and whether or not there is a specific link to insulin synthesis, remain questions in need of further study.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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The dicistrovirus intergenic region internal ribosome entry site (IGR IRES) adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. In this thesis, I describe two studies that attempt to examine how the IGR IRES may have come about. In the first study, I characterized an IGR IRES from a 700-year-old dicistrovirus, named ancient Northwest territories cripavirus (aNCV). From structural prediction of the aNCV IGR sequence and filter binding assays, we showed that the aNCV IGR secondary structure is similar to contemporary IGR IRES structures and could tightly bind to purified human ribosomes. However, there are differences including 105 nucleotides upstream of the IRES of unknown function. We also demonstrated that the aNCV IGR IRES can direct internal ribosome entry in vitro. Lastly, we generated a chimeric virus clone by swapping the aNCV IRES into the cricket paralysis virus (CrPV) infectious clone. The chimeric infectious clone with an aNCV IGR IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus provides a historical framework of the importance of this viral translational mechanism. In the second study, I have examined candidate RNAs that may have IGR IRES-like properties from the Drosophila genome. Previously, we adapted a selective evolution approach to identify RNA elements in the Drosophila genome which have IGR IRES-like properties. From the potential candidate RNAs, RNA3, RNA5 and RNA7 showed tight binding to purified human ribosomes. However, in a competition assay only RNA5 could compete with excess wild-type but not mutant CrPV IGR IRESs for ribosomes. However, we demonstrated that RNA5 did not bind to ribosomal core, as it was accessible by RNase I. Structural predictions were used to identify stemloop (SL) structures of RNA5. Mutations at SL2 altered RNA5 binding affinity, suggesting a potential interaction region. Finally, incubation of RNA5 in rabbit reticulocyte lysate (RRL) did not affect translation in vitro.

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All viruses must usurp host ribosomes for viral protein synthesis. Dicistroviruses utilize an InterGenic Region Internal Ribosome Entry Site (IGR IRES) to directly recruit ribosomes and mediate translation initiation from a non-AUG start codon. The IGR IRES adopts a three-pseudoknot structure that is comprised of a ribosome binding domain (PKII and PKIII) and a tRNA-like anticodon domain (PKI) connected via a short, 1-3 nucleotide hinge. Recent cryo-EM structural analysis of the dicistrovirus Taura syndrome virus (TSV) IGR IRES bound to the ribosome suggests that the hinge region may facilitate translocation of the IRES from the ribosomal A to P site. In chapter 2, we provide mechanistic and functional insights into the role of the hinge region in IGR IRES translation. Using the honeybee dicistrovirus, Israeli acute paralysis virus (IAPV), as a model, we demonstrate that mutations of the hinge region resulted in decreased IRES-dependent translation in vitro. Toeprinting primer extension analysis of mutant IRESs bound to purified ribosomes and in rabbit reticulocyte lysates showed defects in the initial ribosome positioning on the IRES. Finally, using a hybrid dicistrovirus clone, mutations in the hinge region of the IAPV IRES resulted in decreased viral yield. Our work reveals an unexpected role of the hinge region of the dicistrovirus IGR IRES coordinating the two independently folded domains of the IRES to properly position the ribosome to start translation. The dicistrovirus, Cricket paralysis virus (CrPV), has evolved a novel recoding mechanism whereby ribosomes recruited to the viral internal ribosome entry site (IRES) undergo a ribosome bypass event to either start translation or continue translation 37 nucleotides downstream to direct +1 frame translation of an ORF called ORFx. Although ribosome bypassing has been described in bacteriophage T4 gene 60 and in mitochondria of yeast, to the best of our knowledge, the ribosome bypassing mediated by the CrPV IRES is the first to be reported in higher order eukaryotes. In chapter 3, we employ a mutational analysis to identify genomic elements contributing to IRES mediated ribosome bypassing.

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A subset of RNA viruses can cause significant harm to human health and agriculture. As such, it is important to understand how these RNA viruses replicate and interact with the host machinery in order to identify potential antiviral targets. Some of these RNA viruses contain a Stop-Go sequence, consisting of a conserved C-terminal DxExNPG^P sequence, that mediates co-translational processing in the viral polyproteins. In Stop-Go translation, host ribosomes skip synthesis of a glycine-proline peptide bond, causing release of the translation product. Ribosomes do not dissociate, rather, they continue translating a downstream product. Stop-Go processing occurs without stop codons, and re-initiation occurs without initiation factors. The precise Stop-Go translation mechanism remains unclear. This study aims to better understand the Stop-Go mechanism, its sequence requirements in a dicistrovirus model of infection, and identify inhibitors.I performed a mutational analysis of the Dicistrovirus Cricket Paralysis Virus (CrPV) Stop-Go sequence to investigate effects of mutations on viral infection, yield, and processing. I used various biochemical methods including CrPV RNA transfections, infections, and viral titers to examine effects of these mutants on CrPV infection. To evaluate effects of Stop-Go processing in viral polyprotein processing, I used [₃₅S]-Cysteine-Methionine to conduct in-vitro-translation reactions of mutants and pulse-chase labelling in infected S2 cells. Results of the mutational analysis confirm that wildtype Stop-Go sequences are required for viable CrPV protein production. Certain mutations diminish infectivity, while others ablate viral infection. Viral titers reveal requirements for Stop-Go sequence amino acids under CrPV infection. In-ivvitro-translation and immunoblotting data reveal that Stop-Go processing releases the multifunctional 1A protein to promote infection, suggesting that timing of 1A release is critical in infection.I next aimed to identify the first inhibitors of Stop-Go translation using high-throughput screening methods. I developed a yeast one-hybrid-based high-throughput chemical screen for Stop-Go inhibitors measuring yeast growth as a readout. Using this system, I screened 3346 compounds and identified but did not validate multiple potential inhibitors of Stop-Go translation. This chapter also describes new tools developed to perform secondary validation of hits in yeast, drosophila, mammalian cells, and in sf-21 insect translation extract.

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Many positive-sense single-stranded RNA (+ssRNA) viruses encode an open reading frame that is translated as a polyprotein. This viral polyprotein is subsequently cleaved by its virally encoded protease or in some instances with the aid of host proteases. It has been well established that +ssRNA viruses, such as poliovirus encode protease(s) that can cleave and target host protein substrates in order to facilitate viral infection. The Dicistrovirade family, are +ssRNA viruses that primarily infect arthropods such as honey bees, shrimp, and crickets and can have an impact on agriculture and the economy. Dicistroviruses encode a cysteine protease, 3C, that is responsible for the cleavage of its own polyprotein. To date little is known about dicistrovirus protease structure, catalytic efficiency, cleavage site specificity and substrate specificity. Cricket paralysis virus (CrPV), a dicistrovirus, has been well characterized within its family. CrPV has been characterized for its translation mechanism as well as a few of its encoded proteins such as 1A, thus making it a good model to study. Given that other +ssRNA viral 3C proteases, such as poliovirus, cleave host substrates during infection, it could be thought that the CrPV 3C protease cleaves target host proteins during infection. In order to better understand the fundamental processes that are regulated during infection, CrPV was chosen as a model. In this thesis CrPV 3C protease was purified to address two aims. 1) Purify and verify activity of CrPV 3C protease and 2) Determine cleavage site specificity of CrPV 3C protease. This will help give a better understanding of the catalytic efficiency and target substrate specificity of the purified protease.

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Precise control of the levels and spatiotemporal domains of protein synthesis is fundamental to cellular processes. Regulation of protein synthesis largely occurs at the rate-limiting step of translation initiation in which the translation start site is selected by the scanning ribosomal pre-initiation complex (PIC) and its associated initiation factors. Upstream open reading frames (uORFs) are prevalent regulatory elements located in the 5’ untranslated regions (5’UTR) of approximately 50% of mammalian transcripts. Generally, uORFs are viewed as constitutive repressors of translation of the downstream coding sequence (CDS) by sequestering ribosomes. Recent genome-wide studies have revealed that uORFs have widespread regulatory functions in different biological contexts, however our understanding of the roles played by uORFs is still in its infancy. In Drosophila melanogaster, the spatial and temporal expression of the transcription factor grainy head (grh) must be tightly controlled to ensure proper epithelial and central nervous system development. Intriguingly, grh’s eight mRNA isoforms display uORF-containing 5’UTRs ranging from 1 to 24 uORFs. To test for a role of these uORFs in Grh function, this thesis attempts to characterize the role of grh-RJ’s eleven uORFs in modulating the downstream CDS translation in order to fine-tune Grh’s spatiotemporal expression throughout Drosophila development. In this study, both in vitro translation assays and in vivo genetic analyses were used to analyze the regulatory role of grh-RJ’s uORFs on the downstream CDS translation. Our in vitro results showed that grh-RJ’s eleven uORFs severely repressed translation of the downstream CDS in translation extracts. Meanwhile, our transgenic in vivo results showed that that grh-RJ’s uORFs spatially restricted and repressed reporter expression in the Drosophila embryo. In general, we found that the role of grh-RJ’s uORFs is to repress translation of the downstream CDS, including restricting the spatial expression of Grh during Drosophila development. Together with the widespread prevalence of uORFs among species, this research suggests an extensive role of uORFs in regulating the level and spatiotemporal expression of proteins, which will likely contribute greatly to a fundamentally novel understanding of biological systems.

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Endoplasmic reticulum (ER) stress activates an integrated stress response which causes inhibition of overall protein synthesis via phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha). However, ER stress also results in selective translation of mRNAs, one of which is a transcription factor ATF4. ATF4 activates transcription of downstream stress-induced genes such as growth arrest and DNA-damage inducible gene 34 (GADD34) under ER stress. The function of GADD34 is to dephosphorylate eIF2alpha by interacting with protein phosphatase 1, thus leading to recovery of overall protein synthesis and translation of stress-induced transcripts through a negative feedback mechanism. In this thesis, we showed that GADD34 is not only transcriptionally induced, but also translationally regulated for maximal expression under ER stress. Translational regulation of GADD34 was mediated by its 5’ untranslated region (5’ UTR), which was found to contain two upstream open reading frames (uORFs) in human and mouse. It was revealed that the downstream uORF2 is required for basal repression and translational upregulation under ER stress, while the upstream uORF1 is dispensable in this regulation. In addition, the uORF2 is readily recognized and translated, but the uORF1 is bypassed by the scanning ribosomes. Further mutational analysis on the GADD34 5’ UTR demonstrated that the uORF2 and the intercistronic region between the uORF2 and the main ORF are sufficient to direct translation when eIF2alpha is phosphorylated. In this process, the amino acid/nucleotide identity of the uORF2 was not required, but its conserved size was important. The sequence conservation within the intercistronic region also was identified, but changing the length and pyrimidine:purine ratio in this region did not significantly affect translational regulation. Finally, we set up in vitro translation systems where cap-dependent translation is compromised by inhibiting ternary complex and eIF4F formation in order to test GADD34 translational regulation. The results from the current thesis suggest that GADD34 translation is mediated through its 5’ UTR via a unique mechanism, which may serve as a model to understand translational regulation of other uORFs-containing mRNAs under cellular stress.

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