Relevant Degree Programs
Affiliations to Research Centres, Institutes & Clusters
- 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
Approaches include biochemical, molecular and cell biology methods, proteomics and genome-wide deep sequencing.
Viruses that we work on:
- coxsackievirus, CVB3
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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2021)
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.
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.
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.
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.
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.
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.
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.
Master's Student Supervision (2010 - 2020)
No abstract available.
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.
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.
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.