Research Classification
Research Interests
Relevant Thesis-Based Degree Programs
Affiliations to Research Centres, Institutes & Clusters
Research Options
Research Methodology
Recruitment
Projects include
- elucidating virus host interactions such as antiviral innate immunity and viral proteins that modulate cellular processes
- identifying host protein substrates by viral proteinases
- non-canonical protein synthesis mechanisms used by viruses and cellular mRNAs
- mRNA therapeutics
Approaches include biochemical, molecular and cell biology methods, proteomics and genome-wide deep sequencing.
Viruses that we work on:
- dicistrovirus
- poliovirus
- coxsackievirus, CVB3
- Zika
- coronavirus
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Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Viruses must co-opt host cellular functions to facilitate virus infection in cells. To accomplish this, they have evolved diverse strategies to hijack the host cell for its own benefit. One such strategy is through the activity of virus-encoded proteases, which are not only essential for viral polyprotein processing, but also for the cleavage of host cell proteins to modulate cellular pathways such as translation and innate immunity. While enterovirus proteases such as 2A and 3C have been well-studied and target many host proteins, the flaviviral NS2B-NS3 protease is poorly characterized and only a few host protein targets have been identified to date. In this thesis, I hypothesized that the cleavage of currently unidentified host proteins promotes the modulation and hijacking of host cellular processes. The N-terminomics based approach terminal amine isotopic labeling of substrates (TAILS) was employed to identify protease-generated N-termini and host substrates cleaved during virus infection. Three primary aims were addressed: first, characterizing the cleavage of the adapter protein 14-3-3ε by the enteroviral 3Cᴾʳº; next, identifying substrates of the Zika virus (ZIKV) NS2B-NS3ᴾʳº in human A549 cells during infection; finally, to conduct a global analysis of protease-generated N-termini, including both viral and cellular protease substrates, in human and mosquito cell lines. Characterizing the cleavage of 14-3-3ε by 3Cᴾʳº resulted in two key observations: (i) cleavage of 14-3-3ε promotes virus infection and (ii) the newly formed N-terminal fragment represses RIG-I signalling. TAILS analysis of ZIKV-infected A549 cells also uncovered host proteins cleaved during virus infection, including the RNA binding protein HNRNPH3 which was found to be cleaved by NS2B-NS3ᴾʳº. Moreover, TAILS provided insights into the proteolytic activity of viral and cellular proteases during ZIKV infection in human (A549) and mosquito (C6/36) cell lines, shedding light on protease specificity and subcellular localization of these targets. In summary, TAILS identified novel targets of viral and cellular proteases during infection, enhancing our understanding of the biological pathways disrupted during picornavirus and flavivirus infection and offering potential therapeutic or antiviral targets against viral protease activity.
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Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of positive-sense single-stranded RNA viruses. VPg is covalently linked to the 5’ end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses encode multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use the dicistrovirus, Cricket paralysis virus (CrPV), which encodes four non-identical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses encode two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1-4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, from which mutations in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant-negative effect. I hypothesized that the multiple VPgs in dicistroviruses compensate for the relatively weaker translation of the non-structural proteins compared to the structural proteins was tested using minigenome reporter constructs. Viral minigenome reporters that monitor dicistrovirus 5’ untranslated region (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of IGR IRES:5’ UTR IRES translation. We uncover a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote virus infection. We also performed a bioinformatic analysis of VPgs from the family Dicistroviridae and find many novel dicistroviruses containing repeated VPgs, up to eight copies and find that the VPg type but not the number correlates with the RdRp evolution of Dicistroviruses.
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Stress granules (SG) are ribonucleoprotein aggregates that accumulate during cellular stress when translation is limited. Inhibition of SG assembly has been observed under virus infection across species, suggesting a conserved fundamental viral strategy. However, the significance of SG modulation during virus infection is not fully understood. The 1A protein encoded by the model dicistrovirus, Cricket Paralysis Virus (CrPV), is a multifunctional viral protein that can inhibit SG formation and bind to and degrade Argonaute-2 (Ago-2) in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway. Moreover, the R146 residue at the C-terminus of 1A is necessary for virus infection in Drosophila S2 cells and flies. Here, I uncouple CrPV-1A's functions and provide insights into its underlying mechanism for SG inhibition. CrPV-1A’s ability to inhibit SG formation does not require the Ago-2 binding domain but does require the E3 ubiquitin ligase binding domain. Overexpression and infection studies in Drosophila and human cells showed that wild-type CrPV-1A but not mutant R146A CrPV-1A localizes to the nuclear membrane, which correlates with nuclear enrichment of poly(A)+ RNA. Transcriptome analysis demonstrated that a single R146A mutation dramatically dampens host transcriptome changes in CrPV-infected cells. Finally, inhibition of SG formation by CrPV-1A requires Ranbp2/Nup358 in an R146-dependent manner. I propose that CrPV utilizes a multifaceted strategy for productive virus infection whereby the CrPV-1A protein interferes with a nuclear event that contributes to the suppression of SG assembly.
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Dicistroviruses possess a positive-sense, monopartite single-strand RNA genome that encodes two open reading frames containing the nonstructural and structural polyproteins (ORF1 and ORF2) separated by the intergenic region (IGR) internal ribosome entry site (IRES). Translation of each ORF is directed by distinct IRESs, a 5’ untranslated region (UTR) and an IGR IRES. Previous bioinformatic studies have shown that a subset of dicistroviruses contain an overlapping gene in the +1 translational reading frame within the structural polyprotein gene near the IGR IRES region. We hypothesize that the IGR IRES directs translation of two overlapping ORFs, a novel +1 frame ORFx and the 0 frame ORF which encodes the viral structural polyprotein. In this thesis, using Israeli acute paralysis virus (IAPV) as a model, the existence and start site of ORFx were identified using mutagenesis and Mass Spectrometry analyses. In addition, the structural elements within the IAPV IGR IRES that determine alternative reading frame translation initiation were explored. Lastly, the localization of overexpressed tagged-ORFx in Drosophila S2 cells was examined to gain insights of its function. Summarizing, we have discovered a novel mechanism that increases the coding capacity of a virus through an IGR IRES. These studies of IAPV IGR-IRES will further our understanding of IRESs mediated translation initiation and reading frame decoding.
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Viruses exist as obligate intracellular parasites, with one of the largest classifications of viruses being the positive single stranded RNA viruses ((+)ssRNA). Viral families in this group are incredibly diverse in their replication schemes and host tropisms. Despite this, there exist fundamental principles between them. Unravelling these common mechanisms can give rise to a greater understanding of virus biology and lead to the development of novel antiviral therapies and biotechnology. Members of the Dicistroviridae contain monopartite, (+)ssRNA genomes, between 8 to 10 kilobases in size. Infectious to agriculturally and economically important arthropods, these viruses have served as model systems to study fundamental cellular processes such as translation and innate immunity. Dicistroviruses contain two open reading frames (ORFs), which are translated by two distinct internal ribosome entry sites (IRESs). The 5’ untranslated region IRES drives translation of the viral non-structural proteins encoded in ORF1, whereas the intergenic region (IGR) IRES directs translation of the viral structural proteins of ORF2. The scheme by which these viruses replicate is poorly described. Here, we develop the first infectious clones of the dicistrovirus type species, Cricket paralysis virus (CrPV), termed CrPV-2 and -3. We demonstrate that this clone is fully infectious in Drosophila S2 cells and causes mortality when injected into adult flies. Utilizing this clone, we examined how specific mutations in the IGR IRES affect viral gene expression in vivo. Moreover, we demonstrate that the CrPV IGR IRES uses an unusual mechanism for +1-frame translation of a hidden overlapping ORF, which is important for viral pathogenesis. Finally, using a combination of biochemical and mass spectrometry based approaches we show that CrPV may usurp cellular pathways to obtain an envelope. This thesis offers insights into the complex replication scheme of dicistroviruses and provides a foundation for future studies into the life cycle of these viruses.
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The Dicistroviridae intergenic region internal ribosome entry site (IGR IRES) exhibits the remarkable ability to bind the conserved core of the ribosome with high affinity. By mimicking the conformation of a tRNA, the IGR IRES can bypass the requirement for canonical initiation factors and Met-tRNAi, and initiate translation from a non-AUG start codon in the ribosomal A site. The pseudoknot (PKI) domain of the IRES engages the decoding center upon initial ribosome binding, and subsequently translocates into the P site to allow delivery of the incoming aminoacyl-tRNA. Within the P site, the IRES adopts a conformation that is reminiscent of a P/E hybrid state tRNA to effectively co-opt the canonical elongation cycle. How the IGR IRES establishes the translational reading frame in the absence of initiation factors remains an outstanding question. Here, we elucidate the mechanism of reading frame selection by performing mutagenesis and biochemical assays to explore the function of specific IRES structural elements. We demonstrate that constituents of the Cricket paralysis virus PKI domain, including the helical stem, anticodon:codon-like base-pairing, and the variable loop region are optimized for IRES-mediated translation. Additionally, we reveal through extensive structural and biochemical studies that stem-loop III of the Israeli acute paralysis virus (IAPV) IRES mimics the acceptor stem of tRNA and functions in supporting efficient 0 frame translation. Finally, we established an infectious chimeric clone to investigate how translational regulation by the IAPV IRES affects the viral life cycle. Studies using this chimera demonstrate that formation of stem-loop VI upstream of the IAPV IRES contributes to optimal IRES activity and viral yield. Our findings suggest that extensive and complete tRNA-mimicry by the IAPV IGR IRES facilitates IRES-mediated translation and reading frame selection.
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Viruses have exploited strategies of proteolysis for the purposes of processing viral proteins and manipulating cellular processes to direct synthesis of new virions and subvert host antiviral responses. Many viruses encode proteases within their genome, of which many have been well studied among the family of positive-sense single-stranded RNA picornaviruses. A subset of host proteins have already been identified as targets of picornaviral proteinases; however, the full repertoire of targets is not known. In this thesis, a novel proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) was used to conduct a global analysis of protease-generated N-terminal peptides by mass spectrometry and identify novel substrates of the 3C (3Cpro) and 2A (2Apro) proteinases from poliovirus and coxsackievirus type B3 (CVB3). TAILS was performed on HeLa cell extracts subjected to purified poliovirus 3Cpro or CVB3 2Apro, and on mouse HL-1 cardiomyocyte extracts subjected to purified CVB3 3Cpro. A list of high confidence candidate substrates for all three proteinases was generated, which included a peptide corresponding to the known poliovirus 3Cpro substrate polypyrimide tract binding protein at a known cleavage site, thus validating this approach. Furthermore, three identical peptides in both the poliovirus and CVB3 3Cpro list of high confidence substrates were identified, suggesting that cleavage of these substrates may contribute to general strategy of picornaviral infection. A total of seven high confidence substrates were validated as novel targets of 3Cpro in vitro and during virus infection. Moreover, mutations in the TAILS-identified cleavage sites for these candidates blocked cleavage in vitro and during infection. Depletion of these proteins by siRNAs modulated virus infection, suggesting that cleavage of these substrates either promotes or inhibits virus infection. In summary, an in vitro TAILS assay can be utilized to identify novel substrates of viral proteinases that are cleave during infection. Moreover, TAILS can identify common substrates of viral proteinases between different viral species, revealing general strategies of infection utilized by related viruses. Finally, the identification of novel host substrates provides new insights the viral-host interactions mediated by viral proteinases that are required for successful infection.
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Viruses are obligate parasites that have evolved strategies to recruit the translational machinery and inhibit antiviral defences. A relatively abundant family of positive-sense, monopartitate single stranded RNA viruses, dicistrovirus, remains relatively uncharacterized. Dicistroviruses are infectious to arthopods and have impacted a number of agricultural industries. Dicistroviruses, as indicated by their name, contain two open reading frames (ORFs). The 5'-untranslated internal ribosome entry site (5'-UTR IRES) directs translation of ORF1 which encodes non-structural proteins and the intergenic (IGR) IRES directs translation of ORF2 which encodes structural proteins. How dicistroviruses affect the host is not completely understood. My thesis focuses on several host pathways that are modulated during cricket paralysis virus (CrPV) infection, a model dicistrovirus. During CrPV infection, I discovered stress granule (SG) formation is inhibited but granules containing poly(A)+ mRNAs form. Furthermore, I discovered a viral protein, CrPV 1A, that inhibits the SG pathway. Upon further characterization of CrPV 1A, I discovered the viral protein also stimulates 5'-dependent translation and 5'IRES dependent translation. Finally, I found IGR IRES-dependent translation is delayed compared to 5'-UTR IRES-dependent translation, thus providing a viral strategy of expressing non-structural proteins such as the replicase and protease prior to the synthesis of structural proteins for viral packaging. This thesis provides insights into the key strategies of dicistrovirus infection, its viral life cycle and the innate immune responses in insect cells.
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Eukaryotes differ from other domains of life in many respects, but at the level of gene architecture, it is the presence of interrupting sequences in their genes that serve as the defining feature. These must be removed with absolute precision in order to maintain the reading frame during translation of the encoded protein; thus it is not surprising that errors in this process, known as precursor messenger RNA (pre-mRNA) splicing, have been linked to a wide variety of human diseases. U6 small nuclear ribonucleoprotein (snRNP) is an essential component of the spliceosome, the large RNA-protein complex that is responsible for catalyzing pre-mRNA splicing. Although U6 small nuclear RNA (snRNA) plays a critical role in catalyzing the splicing reactions, very little is known about the mechanism of converting catalytically inert U6 snRNA in free U6 snRNP into a catalytic component of the assembled spliceosome. Here I present a model for free U6 snRNA secondary structure in free U6 snRNP that suggests that U6 becomes active for splicing through a mechanism that is dependent on its interaction with a second splicing factor, U4 snRNA. I propose that the U6 snRNP-associated protein, Prp24, is responsible for retrieving U6 snRNA from the disassembling spliceosome following splicing of a substrate, and then holds U6 snRNA in a conformation that masks catalytic sequences. I provide evidence, both from the literature and from my own genetic analysis, that the first two RNA Recognition Motifs of Prp24 bind a region of U6 snRNA known as the telestem, presenting U6 in a manner that is favorable for interaction with U4 snRNA. As a step toward solving the crystal structure to test this model, I have developed a system for the simultaneous recombinant expression of all components of U6 snRNP from a single expression vector, followed by purification of the pre-formed complex under non-denaturing conditions. I have subjected these particles to low-resolution negative stain electron microscopy and have also obtained a small angle X-ray scattering model of a sub-complex of free U6 snRNP, the LSm complex. This work has laid the foundation for understanding the structure/function relationship for U6 snRNP.
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The IRES found in the intergenic (IGR) region of viruses belonging to the Dicistroviridae family is remarkable for its ability to bind directly to the ribosome with high affinity and initiate translation without the requirement for any initiation factors by mimicking a P/E hybrid tRNA. Here, we have conducted an in-depth biochemical characterization of the CrPV IGR IRES. We have found that the L1.1 region of the IRES is responsible for 80S assembly and reading frame maintenance, and may play an additional role downstream of ribosome binding. Additional studies on the modularity of the IRES showed that the two domains of the IGR IRES work independently, but in concert with one another to manipulate the ribosome. We then addressed the question of how the IGR IRES recruits ribosomes during periods of cellular stress, when inactive 80S couples accumulate in the cell. Here, we found that the IRES is able to bind directly to eEF2-associated 80S couples, providing a rationale as to how the IRES remains translated during these periods. Finally, we developed a new in vitro translation system to assess the functionality of specialized ribosomes, and used this system and the IGR IRES in order to ask questions about the pathology of dyskeratosis congenita.Though divergent from other viral IRESs, the simplicity of this tRNA-like IRES serves as a powerful model for understanding IRES functions in general, the role of tRNA/ribosome interactions that occur normally during translation, and how these processes are linked to the greater context of the cell.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
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 -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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