Eric Jan

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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