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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 Approaches include biochemical, molecular and cell biology methods, proteomics and genome-wide deep sequencing
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2019)
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.
No abstract available.
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 - 2018)
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.