Browsing by Author "Wilusz, Jeffrey, committee member"

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Open Access
Assessment of mosquito and animal model factors in Aedes-borne arbovirus transmission and disease
(Colorado State University. Libraries, 2021) Miller, Megan Rae, author; Foy, Brian, advisor; Wilusz, Jeffrey, committee member; Kading, Rebekah, committee member; Montgomery, Tai, committee member; Magunda, Forgivemore, committee member
To view the abstract, please see the full text of the document.
Open Access
Dodging wrenches in the time of COVID: exploring flavivirus replication mechanisms and SARS-CoV-2 antibody development
(Colorado State University. Libraries, 2024) Terry, James Steven, author; Geiss, Brian J., advisor; Wilusz, Jeffrey, committee member; Ebel, Gregory, committee member; Snow, Christopher, committee member
Flaviviruses pose a significant threat to global health, threatening hundreds of millions of people who live in endemic areas. Infection with flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV) can trigger symptoms ranging from a mild cold-like illness to microcephaly, encephalitis, hemorrhagic fever, and death. As climate change alters global temperature ranges, habitable environments for the flavivirus arthropod vectors are expanding into previously unexposed regions. Due to a lack of flavivirus vaccines and antivirals, most efforts to combat infection fall within vector population control and palliative care for infected individuals. To develop antivirals and vaccines against flaviviruses, we need to better understand the fundamental mechanisms through with the viruses replicate. By investigating incompletely understood processes in the replication cycle, new antiviral targets can be identified and pursued. This dissertation investigates two components of the flavivirus replication cycle to better understand the key processes necessary for successful flavivirus infection. Additionally, this dissertation reports on efforts during the SARS-CoV-2 COVID-19 pandemic to develop novel reagents to assist in research and diagnostic development. An important determinant of successful flavivirus infection is the generation of subgenomic flavivirus RNA (sfRNA). This RNA is composed of exoribonuclease resistant RNA (xrRNA) structures in the flavivirus 3' untranslated region (3'UTR). These structures allow the flavivirus 3'UTR to withstand degradation by stalling the host Xrn1 exoribonuclease, halting viral RNA degradation and creating sfRNA. The production of sfRNA is critical for flavivirus replication success as the new RNA entity actively suppresses the host cell immune response to viral infection. There are blind spots in our understanding of the key stages of sfRNA generation, namely how the Xrn1 substrate is produced from the flavivirus genome. It has previously been postulated that host decapping enzymes remove the flavivirus Type 1 cap structure, allowing Xrn1 to bind to the 5' monophosphate and degrade the viral RNA. The enzyme responsible for decapping has not yet been identified. Following preliminary evidence from the Geiss Lab, we investigated the host decapping enzyme Dcp2 as the protein responsible for priming flavivirus RNA for Xrn1 degradation and sfRNA production. We developed a pipeline using splint-ligation to specifically label monophosphorylated WNV RNA with an RNA adapter at the 5' end. Following this the ratio proportion of viral RNA that is monophosphorylated is revealed using a qRT-PCR reporting system. With this pipeline, it was determined that suppressing Dcp2 expression increased the proportion of monophosphorylated WNV RNA in infected cells while having no significant effect on monophosphorylated RNA in newly produced virions. Additionally, northern blot analysis revealed that sfRNA generation was not reduced by Dcp2 knockdown. From this study we determined that Dcp2 is not necessary for sfRNA generation, and thus other processes are responsible for the generation of monophosphorylated viral genomes for Xrn1 degradation. One hole in our understanding of flavivirus replication concerns the viral replication compartment. The compartment is an invagination in the endoplasmic reticulum membrane that is formed through viral protein manipulation. This environment then hosts the viral replication machinery, protecting the vulnerable viral RNA from host cell immune detection as new viral genomes are produced. Proper viral protein-protein interactions are critical for the successful formation of this viral RNA factory. While studies have been conducted to determine the replication compartment location and some interactions between nonstructural proteins, our understanding of how these proteins interact with each other in situ is limited. To address this, we employed crosslinking mass spectrometry. First, a flavivirus replication compartment purification and crosslinking pipeline underwent a series of evolutions and significant optimizations followed mass spectrometry data acquisition. Then, a crosslinked protein analysis pipeline using the Bonvin Lab programs DisVis/HADDOCK was validated with crosslinked bovine serum to ensure its utility with crosslinked viral compartment samples. MaxQuant analysis revealed some viral protein crosslinks while highlighting areas for improvement in our methodology. Nevertheless, the identified intramolecular crosslinks within NS1, NS3, and NS5 hint at potential dimer interfaces. An intermolecular crosslink between NS3 and NS4b was identified that supports the observations of previous studies while establishing in situ evidence for interactions along an NS3 N-terminus and NS4b residue K172 interface. The results provided intriguing preliminary evidence for future investigations into the replication compartment protein-protein interactions and established a protocol for analyzing viral proteins with crosslinking mass spectrometry. In addition to chronicling on flavivirus replication cycle studies this dissertation includes a chapter chronicling work during the COVID-19 pandemic on SARS-CoV-2. Monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid protein were generated, characterized, and sequenced during the height of the pandemic. These antibodies were the first of their kind to be published and were made available for use during the global SARS-CoV-2 research effort. This chapter also reports on collaborative efforts surrounding the use of antibodies for diagnostics and predictive computational pipelines. Work was done to assist the Henry Lab in developing inexpensive electrochemical and colorimetric ELISA devices targeting SARS-CoV-2 NP for bedside diagnostic use. Lastly, wet lab verification was performed to validate Jacob Deroo's epitope-predicting PAbFold AlphaFold2 pipeline. The work covered in this dissertation spans five years, two viruses, and three separate target areas. These projects, while varied, are all bound together by the common goal of contributing to the advancement of knowledge and techniques for stopping viral threats to global health. Knowing how a virus creates a safe environment for genome replication or identifying which host proteins help create an immune-modulating viral RNA molecule is important for identifying new paths towards intelligently designed antivirals. Similarly, developing and characterizing antibodies to supply a global research effort and validate cutting-edge computational tools is necessary for actively combatting a global pandemic and preparing for the next one. With this work, scientific inquiry ranging from foundational knowledge to translational science is explored.
Open Access
Exploration of the arthropod virome, its biological impacts on host health, and its potential implementation in biocontrol
(Colorado State University. Libraries, 2020) Cross, Shaun T., author; Stenglein, Mark D., advisor; Wilusz, Jeffrey, committee member; Foy, Brian D., committee member; Metcalf, Jessica L., committee member
With the advent of next generation sequencing, viruses are being discovered at an unprecedented rate. The collection of these viruses, known as the virome, and their impact on the host is relatively understudied compared to the bacterial microbiome. The underlying goal of this thesis work was to better understand how the virome interacts with the host, and this has been accomplished in two ways. First, we biologically characterized predominant virus constituents in arthropod viromes, namely arthropod-infecting partitiviruses. In a subsequent study we measured how one of these partitiviruses, galbut virus, impacted the fitness of Drosophila melanogaster. Second, we searched for evidence that these viruses are active in their interactions with fellow microbial constituents within the host. Specifically, we addressed how the virome may change disease vectors' competence in harboring and transmitting pathogens with a focus on Ixodes scapularis ticks. Partitiviruses are segmented, multipartite dsRNA viruses that until recently were only known to infect fungi, plants, and protozoans. Metagenomic surveys have revealed that partitivirus-like sequences are also commonly associated with arthropods. One arthropod-associated partitivirus, galbut virus, is common in wild populations of D. melanogaster. To begin to understand the processes that underlie this virus's high global prevalence, we established colonies of wild-caught infected flies. Infection remained at stably high levels over three years, with between 63-100% of individual flies infected. Galbut virus infects fly cells and replicates in tissues throughout infected adults, including reproductive tissues and the gut epithelium. We detected no evidence of horizontal transmission via ingestion but vertical transmission from either infected females or infected males was ~100% efficient. Vertical transmission of a related partitivirus, verdadero virus, that we discovered in a laboratory colony of Aedes aegypti mosquitoes was similarly efficient. This suggests that efficient biparental vertical transmission may be a feature of at least a subset of insect-infecting partitiviruses. To study the impact of galbut virus infection free from the confounding effect of other viruses, we generated an inbred line of flies with galbut virus as the only detectable virus infection. We were able to transmit the infection experimentally via microinjection of homogenate from these galbut-only flies. This sets the stage for experiments to understand the biological impact and possible utility of partitiviruses infecting model organisms and disease vectors. Using the galbut virus and D. melanogaster system, we set forth to answer: what are the biological effects, if any, of galbut virus infection on D. melanogaster fitness? Using multiple lines of flies from the Drosophila Genetic Reference Panel (DGRP) that differed only in their galbut virus infection status, a variety of fitness measurements were performed across both sexes. Galbut virus minimally impacted lifespan and had no effects on fecundity, but infection did significantly impact developmental speeds of flies. When challenged with various viral, bacterial, and fungal pathogens, some galbut virus infected flies had altered sensitivity to these pathogens. These susceptibility changes varied by both genetic background and sex. Galbut virus overall has minimal influences on host transcriptional responses, consistent with minimal phenotypic impacts of galbut virus infection. Major constituents of the microbiome were not perturbed by galbut virus infection. All fitness measurements alterations attributable to galbut virus were small, but they were dependent by strain and sex, highlighting the importance of these variables in phenotype outcomes. However, these altered measurements in galbut virus infected flies were dwarfed in comparison to those measurements attributable solely by fly strain and sex. These findings further support a trend of predominately cryptic phenotypes of partitivirus infections. To understand how the virome interacts with other microbial constituents, we specifically searched for polymicrobial interactions within the field of vector-borne diseases. I. scapularis ticks harbor a variety of microorganisms, including eukaryotes, bacteria and viruses. Some of these can be transmitted to and cause disease in humans and other vertebrates. Others are not pathogenic but may impact the ability of the tick to harbor and transmit pathogens. A growing number of studies have examined the influence of bacteria on tick vector competence but the influence of the tick virome remains less clear, despite a surge in the discovery of tick-associated viruses. In this study, we performed shotgun RNA sequencing on 112 individual adult I. scapularis collected in Wisconsin, USA. We characterized the abundance, prevalence and co-infection rates of viruses, bacteria and eukaryotic microorganisms. We identified pairs of tick-infecting microorganisms whose observed co-infection rates were higher or lower than would be expected, or whose RNA levels were positively correlated in co-infected ticks. Many of these co-occurrence and correlation relationships involved two bunyaviruses, South Bay virus and blacklegged tick phlebovirus-1. These viruses were also the most prevalent microorganisms in the ticks we sampled and had the highest average RNA levels. Evidence of associations between microbes included a positive correlation between RNA levels of South Bay virus and Borrelia burgdorferi, the Lyme disease agent. These findings contribute to the rationale for experimental studies on the impact of viruses on tick biology and vector competence. Follow-up analyses on a second population of I. scapularis ticks derived from New York, USA revealed that these potential functional relationships may be population-specific. When evaluating South Bay virus, blacklegged tick phlebovirus-1, and B. burgdorferi in these individual ticks, no correlative or cooccurrence associations were observed. The lack of concordance between populations suggests that interactions between microbial constituents may be fluid, and change based upon location and populations. To characterize the biology of tick-associated viruses, an attempt to isolate South Bay virus was performed. Despite using mammalian and tick cell lines, we were unsuccessful in isolating South Bay virus through in vitro cell culture. The lack of success accents the challenge for understanding the biology of these arthropod-specific viruses. Further additional attempts to acquire infectious South Bay virus, such as creating a reverse genetics system, are warranted for its biological characterization.
Open Access
Identification of direct targets of serine/arginine-rich 45 protein isoforms by TRIBE (Targets of RNA-binding proteins Identified By Editing) in Arabidopsis thaliana
(Colorado State University. Libraries, 2021) Huynh, Nikki, author; Reddy, A. S. N., advisor; Garrity, Deborah, committee member; Wilusz, Jeffrey, committee member
To view the abstract, please see the full text of the document.
Open Access
In vitro and in vivo studies on pre-mRNA splicing in plants
(Colorado State University. Libraries, 2017) Albaqami, Mohammed M., author; Reddy, A. S. N., advisor; Wilusz, Jeffrey, committee member; Ben-Hur, Asa, committee member; Montgomery, Tai, committee member
To view the abstract, please see the full text of the document.
Open Access
Long range interaction networks within 3Dpol and the roles they play in picornavirus genome replication and recombination
(Colorado State University. Libraries, 2020) Watkins, Colleen L., author; Peersen, Olve B., advisor; Cohen, Robert, committee member; Ho, P. Shing, committee member; Wilusz, Jeffrey, committee member
Picornaviruses contain a single-stranded positive sense RNA genome approximately 7.5kb in length. The genome encodes for a single polyprotein that can future be divided into three functional regions; the P1 region containing the viral capsid proteins, the P2 region whose proteins function primarily in membrane rearrangement during viral replication, and the P3 region which contains four protein responsible for RNA replication. The final protein in the P3 region is 3Dpol, an RNA-dependent RNA polymerase (RdRP) whose structure is analogous to a "right hand" with fingers, palm and thumb domains, and around which this dissertation will be centered. Section one of this work investigates the roles three regions within the fingers domain play in the catalytic cycle of 3Dpol: "The kink" located within the index finger, "the gateway" found on the pinky, and "the sensor", which bridges the two beta-strands of the middle finger. This study demonstrates that the kink residues are involved in RNA binding as mutations to these residues result in decreased initiation time and elongation complex lifetime. The gateway residues are found to act as a molecular stop against which the template-RNA strand positions itself post-translocation, eventually resetting the active site for the next round of nucleotide incorporation. Lastly the sensor residues serve two key functions: 1) A final checkpoint to determine the correct nucleotide has entered the active site, and 2) As a possible source for proton donation to the pyrophosphate leaving group formed during catalysis. The inter-connected nature of the residues investigated in this section give rise to the idea that it is not individual residues alone that control major steps during the catalytic cycle, but instead that long ranging interaction networks within the different polymerase domains are ultimately responsible for controlling different actions carried out by the polymerase. Section two of this work looks at the long-range interaction networks within 3Dpol by dissecting the roles each polymerase domain plays in catalytic cycle. Through generation of chimeric polymerases it was determined that the pinky finger, with some influence by the fingers domain, controls RNA binding, the palm domain dictates nucleotide discrimination, and nucleotide capture and active site closure rates. It was also established that the thumb domain controls translocation, and an interaction between the palm and thumb domains was needed to generate a viable virus, supporting the idea of interface I, a protein-protein interface that was discovered in the first 3Dpol crystal structure. What is most striking about these findings is that unlike other single subunit polymerases that perform translocation by using a large swinging motion within the fingers domain, viral RdRPs use an entirely different domain altogether. The last section of this work deals with viral recombination, an event that is carried out at a low frequency during virus replication. Recombination is proposed to be a mechanism by which mutations can be purged from the genome independent of polymerase fidelity. This study carries out a mechanistic investigation into how mutation of residue 420 from a leucine to an alanine affects polymerase replication kinetics. It also takes a look at the mutation of residue 64 from a glycine to a serine, a previously identified mutation that results in a high-fidelity polymerase, in the presence and absence of L420A. This work revealed that mutations L420A and G64S operate independently of each other by affecting different steps in the catalytic cycle with G64S increases in fidelity predominately from monitoring nucleosugar positioning while L420A affects nucleobase positioning and polymerase grip on the product RNA strand.
Open Access
RNA interference as an alternative preventive measure for avian influenza in poultry
(Colorado State University. Libraries, 2014) Linke, Lyndsey M., author; Salman, Mo, advisor; Landolt, Gabriele, committee member; Olea-Popelka, Francisco, committee member; Wilusz, Jeffrey, committee member
Avian influenza virus (AIV) is a viral pathogen that causes a wide range of disease in poultry, from subclinical to severe clinical illness and can often result in death. In 1878, AIV was first described as a disease affecting poultry. Nearly 80 years later this disease-causing agent was identified as influenza A virus and a member of the family Orthomyxoviridae. AIV was not considered a significant human pathogen until 1997, when high pathogenic AIV H5N1 emerged from the wildfowl reservoir and was directly transmitted from domestic poultry to humans. Despite a long history of outbreaks in animals, this incident propelled AIV into a globally recognized disease associated with socioeconomic and animal health consequences. Each AIV outbreak highlights ways to improve upon current control strategies and stimulates new ideas for developing novel approaches and technologies to better mitigate AIV outbreaks worldwide. AIV is a dynamic pathogen to study. Host range and adaptation, pathogenicity, pathology, molecular composition, and the epidemiology of AIV all make this virus particularly challenging to control in poultry. Vaccines against AIV are available but the protection they provide for poultry is limited, especially when administered at the onset or in the midst of an outbreak. The most efficacious vaccines must be administered subcutaneously or intramuscularly, an impediment to successfully immunizing large numbers of poultry in a short window of time. Frequently, improper storage and handling leads to vaccine failure. To elicit efficient protection the vaccine must be HA-subtype specific to the outbreak virus. Often stockpiles of vaccines become obsolete and new vaccines must be generated. This is a time consuming process and can take months to secure and additional time to disseminate and administer. In the naive animal, protective antibody production takes two to three weeks to acquire following vaccination. Even if the decision to vaccinate during an outbreak is rapid and an appropriate vaccine is available for immediate use in poultry, vaccination alone would do little to protect against the threat of infection and break the chain of transmission, especially in areas lacking appropriate biosecurity measures. These limitations convey a genuine need to develop a prophylactic that would offer universal protection against any subtype or strain of AIV and would provide rapid protection in the face of an outbreak. Using RNA interference (RNAi) methodologies, this dissertation focuses on developing an innovative antiviral prophylactic that works rapidly to protect poultry against AIV shedding and transmission. The innovation behind this prophylactic technology lies in combining RNAi with the transkingdom RNAi (tkRNAi) delivery platform. This anti-AIV technology specifically targets conserved viral gene segments using small interfering RNA (siRNA) generated and delivered to chicken mucosal respiratory tissues using the tkRNAi system. The work presented in this dissertation details the steps taken to show proof of concept for using this technology to prevent AIV replication and shedding in vitro using an avian cell model and in vivo using commercial chickens. The overarching vision for this anti-AIV technology is to provide a cost effective means to protect commercial and backyard flocks against AIV outbreaks. The long-term goal is to promote this prophylactic as a complement to vaccination with the intention of developing a more effective and robust control plan against AIV in poultry. If this technology is successful, it could be applied in the face of an outbreak to reduce the shedding and transmission of virus within poultry, between farms, and across borders, thereby improving animal health and reducing the economic impact of outbreaks worldwide. Additionally, this work could provide the framework and valuable evidence for developing a similar anti-influenza prophylactic for humans.
Open Access
Small RNAs in Aedes aegypti: one giant step for virus control in mosquitoes
(Colorado State University. Libraries, 2022) Williams, Adeline E., author; Olson, Ken, advisor; Antolin, Mike, committee member; Calvo, Eric, committee member; Franz, Alexander, committee member; Wilusz, Jeffrey, committee member
Aedes aegypti mosquitoes are key vectors of medically relevant arthropod-borne (arbo) viruses such as Zika (ZIKV), dengue (DENV1-4), and yellow fever (YFV). When Ae. aegypti become infected with arboviruses, RNA interference (RNAi) is a critical antiviral immune mechanism that is a key determinant for successful virus transmission. The major antiviral pathway is the RNAi small-interfering RNA (siRNA) pathway, although evidence shows that the Piwi-interacting RNA (piRNA) pathway also acts as an important RNAi mechanism for controlling persistently infective viruses. The overarching goals of this work were twofold: (1) to determine the potency of the Ae. aegypti siRNA pathway against Zika virus and (2) to understand molecular mechanisms underlying piRNA-mediated antiviral immunity and its implications on mosquito vector competence. To achieve these goals, we (1) engineered transgenic Ae. aegypti mosquitoes that synthetically triggered the endogenous siRNA pathway against ZIKV and then quantified virus resistance in these mosquitoes, (2) sequenced small RNAs (sRNAs) of the mosquito virome that may impact vector competence and virus persistence, and (3) characterized structural features of Piwi4, an antiviral protein, involved in sRNA binding and subcellular localization to gain insights on its role in the piRNA and siRNA pathways. A major challenge in the fight against arboviruses is the lack of effective vaccines and limited therapeutic options. Vector control remains the primary method of preventing disease, and integrated vector management (IVM), including the genetic control of mosquitoes, is imperative to prevent emerging arboviral diseases. To this end, we designed an antiviral effector gene – a ZIKV-specific double stranded (ds) RNA –that synthetically triggered the mosquito's siRNA pathway after a bloodmeal in transgenic Ae. aegypti. Small RNA analyses in transgenic midguts revealed ZIKV-specific 21 nucleotide (nt) siRNAs 24 hours after a non-infectious bloodmeal. Nearly complete (90%) inhibition of ZIKV replication was found 7-to-14 days post-infection (dpi); furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that the siRNA pathway can be synthetically exploited to generate ZIKV-resistant Ae. aegypti mosquitoes. In the context of gene drive, antiviral effectors expressed in transgenic Ae. aegypti will be an invaluable tool for a population replacement vector control approach. piRNA-mediated antiviral immunity involves an endogenous viral element (EVE) – viral derived cDNA (vDNA) integrated into host genomes – as well as infection with a cognate virus, which together trigger piRNA amplification and lead to virus silencing. EVEs are from viruses that infected a population in previous generations, and most are derived from insect-specific viruses (ISVs) that persistently infect Ae. aegypti. We hypothesized that ISVs and ISV-derived piRNA populations, like EVEs, have geographic structure and impact vector competence to arboviruses. To test this hypothesis, we sequenced sRNAs from geographically distinct Ae. aegypti and characterized virus-derived sRNAs (vsRNAs). Overall, the distribution of total sRNAs was highly variable. Small RNAs derived from ISVs were diverse and dependent on geographic origin. We next infected Ae. aegypti from Poza Rica, Mexico with DENV2 and analyzed changes in the sRNA virome. DENV2 intrathoracic inoculation resulted in DENV2-specific siRNAs and piRNAs. We also found increased loads of sRNAs against the ISVs verdadero (Partitiviridae: unclassified), Aedes anphevirus (Xinmoviridae: Anphevirus), and chaq-like virus (Partitiviridae: unclassified) after DENV2 infection compared to ISV-derived sRNAs in controls. Overall, our study highlights the diversity of infective ISVs and the complexity of the sRNA virome across Ae. aegypti populations, which likely has consequences on sRNA crosstalk, virus replication, and vector competence. To gain insights on how Piwis, piRNA-binding proteins, are involved in virus control, we characterized structural features of an antiviral Piwi, Piwi4, involved in RNA binding and subcellular localization. We found that Piwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3'-terminal ends of piRNAs, bound to mature (3'-terminal 2'-O-methylated) and 3'-terminal unmethylated RNAs with similar micromolar affinities (KD = 1.7 ± 0.8 μM and KD of 5.0 ± 2.2 μM, respectively) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins had significant impacts on binding behaviors, likely by impacting protein secondary structure. We also found that Piwi4 was both cytoplasmic and nuclear in mosquito tissues, and we identified a Piwi4 nuclear localization signal in the N-terminal region of the protein. These studies provide insights on the dynamic role of Piwi4 in RNAi and pave the way for future studies aimed at understanding Piwi4 interactions with diverse RNA populations.
Open Access
Strange translation - investigating ires-mediated and codon non-optimal translation dynamics at the single mRNA level in living cells
(Colorado State University. Libraries, 2021) Koch, Amanda Lynn, author; Stasevich, Timothy, advisor; Munsky, Brian, committee member; Markus, Steven, committee member; Peersen, Olve, committee member; Wilusz, Jeffrey, committee member
With the advent of Nascent Chain Tracking (NCT), a technique used to visualize single-molecule events of translation in living cells, answering detailed questions about how, when, and where translation is occurring in living cells is possible. Since its publishing debut in 2016, NCT has provided a wealth of information about translation initiation and elongation dynamics, subcellular localization, translation site structure, and reaction to stress for both canonical and non-canonical translation in living cells. Here, we slightly modify the NCT assay to quantify translation dynamics when a ribosome is recruited to an mRNA in a non-canonical fashion and when a ribosome encounters codon non-optimal stretches on a transcript. The first step of translation requires a primed ribosome to be recruited to a readied mRNA. Canonically, this recruitment takes place on the 5' cap of an mRNA and is termed cap-dependent initiation. However, some eukaryotic messages and many viral RNAs use an internal ribosome entry site (IRES) to recruit ribosomes and initiate translation in a cap-independent manner. Specifically, viruses use IRES elements to hijack host ribosomes to translate viral proteins and properly propagate in host cells. While well- studied in bulk, the dynamics of IRES-mediated translation remain unexplored at the single-molecule level. Here, we developed a bicistronic biosensor encoding distinct repeat epitopes in two open reading frames (ORFs), one translated from the 5'-cap, the other from the Encephalomyocarditis Virus IRES. When combined with a pair of complementary probes that bind the epitopes co-translationally, the biosensor lights up in different colors depending on which ORF is translated. Using the sensor together with single-molecule tracking and computational modeling, we measured the kinetics of cap- dependent versus IRES-mediated translation in living human cells. We show that bursts of IRES translation are shorter and rarer than bursts of cap translation, although the situation reverses upon stress. Collectively our data support a model for translational regulation primarily driven by transitions between translationally active and inactive RNA states. Once the ribosome has been recruited to the mRNA and a start codon located, the ribosome will begin decoding the mRNA in nucleotide triplets or codons to ultimately create a protein. In some cases, the ribosome encounters a codon that it cannot decode efficiently. The relationship between codons and ribosome efficiency is termed codon optimality. It has been shown that codon non-optimal mRNA are less stable in cells. However, little is known about the effects of codon non-optimality on translation kinetics and overall translation regulation. In an ongoing collaboration with the Rissland group, we use bulk assays and NCT to address unanswered questions about how codon non- optimality leads to translation regulation along with mRNA instability. Thus far, we have evidence to support that translation repression is occurring in codon non-optimal conditions through inhibition of ribosome initiation and slower elongation. Further investigations of exact translation repression mechanisms are ongoing.
Open Access
The DXO decapping exonuclease is a restriction factor for RNA viruses
(Colorado State University. Libraries, 2019) Lynch, Erin R., author; Geiss, Brian, advisor; Wilusz, Jeffrey, committee member; Perera, Rushika, committee member; Stasevich, Tim, committee member
Cellular RNA exonucleases, such as XRN1 and DXO, aid in the destruction of defective cellular mRNAs and help maintain overall cellular health. The RNA decay system, however, also serves another purpose – degrading viral RNAs. The XRN1 exonuclease is known to be a major antagonist of RNA virus genomes, but the role of other cellular RNA decay enzymes in controlling viral infection is less clear. The cellular 5' decapping exonuclease DXO is able to recognize, de-cap, and degrade RNAs lacking 2'-O-methylation on the first nucleotide after the 5' cap, helping the cell to discriminate self from non-self RNAs. Preliminary data we have developed indicate that flaviviruses and alphaviruses replicate to much higher levels in DXO deficient cells than in cells containing DXO, indicating that DXO may also act as a cellular viral restriction factor. Interestingly, flavivirus genomes contain a 5' cap that is generally 2'-O-methylated at the first base of the transcript, providing a potential mechanism to evade DXO degradation. Overall, our results indicate that the DXO decapping exonuclease helps control the replication of positive strand RNA viruses in cells and represents a new viral restriction factor.
Open Access
The impact of insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) in human and canine osteosarcoma
(Colorado State University. Libraries, 2019) Alyami, Nouf Mahdi, author; Duval, Dawn, advisor; Wilusz, Jeffrey, committee member; Argueso, Lucas, committee member; Stargell, Laurie, committee member
Osteosarcoma (OS) is a malignant bone tumor that afflicts over 10,000 dogs. Most dogs and approximately 30-40% of children with OS succumb to metastatic disease. We identified elevated insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) as one of the biomarkers of poor prognosis in canine OS. IGF2BP1 is an oncofetal protein that regulates mRNA subcellular localization, nuclear export, stability, and translation. IGF2BP1 controls the expression of oncogene targets and correlates with poor outcome in a variety of human cancers. Using microarray analysis, we identified elevated insulin-like growth factor II mRNA binding protein 1 (IGF2BP1) expression as a biomarker of poor prognosis in canine osteosarcoma. Also, our preliminary data show that IGF2BP1 knockdown (shRNA) in a human OS cell line increased sensitivity to doxorubicin by ≥ tenfold compared to control. Significant reductions in cellular migration, invasion, proliferation, and tumor growth in nude mice were also observed (p < 0.05). The current research explores mechanisms for increased IGF2BP1 expression in panels of human and canine osteosarcoma cell lines. Gene amplification, hypomethylation, increased transcription, and alterations in microRNA (miRNA) regulation directly or through 3'UTR shortening have all been hypothesized by many studies as mechanisms to increase IGF2BP1 expression in cancer. We evaluated the expression and alternative polyadenylation of IGF2BP1 using RT-qPCR and western blot analysis in human and dog osteosarcoma cell lines. We assessed transcriptional activation of IGF2BP1 using luciferase reporters containing promoter sequences from the human and canine IGF2BP1 genes. To detect genomic amplification and methylation, we used qPCR to assess gene copy numbers and treatment with the DNA methylase inhibitor, 5-Azacytidine, to explore activation of gene expression through hypomethylation. Using qPCR analysis, we observed genomic amplification in 35% of canine tumors and cell lines and correlated amplification with IGF2BP1 transcript expression (p = 0.0006, Pearson r = 0.88). We observed no genomic amplification in human cell lines. Significant loss of 3'UTR regulatory sequences was found in 20% of canine cell lines (p < 0.05). The promoter analysis showed that most regulatory elements were located within ~580bp from the translational start site in both species. Using pathway-focused luciferase reporter assays, we identified activation of the following factors: MYC, NF-Kappa B, AP-1, and TCF4: β-catenin. Thus, our data show that multiple mechanisms can contribute to elevated IGF2BP1 expression, and these results can be used to develop new treatment strategies that target elevated IGF2BP1 or regulatory mechanisms. Using the McKinley canine OS cell line, we generated and validated stable overexpression of IGF2BP1 (IGF2BP1-pLVX-Puro, Clontech). The stable OS cell line pool and individual clones with a corresponding empty vector control were analyzed and tested for migration, invasion, proliferation, and resistance to standard chemotherapeutic agents. We analyzed migration and invasion using a scratch wound assay and measured cellular proliferation as a surface confluence for 90 hours on an IncuCyte Zoom. We also assessed the clones' sensitivity to doxorubicin over 48 hours using a bioreductive resazurin-based fluorometric assay. We assessed changes in transcript expression in response to IGF2BP1 from isolated total RNA analyzed on Affymetrix Canine 1.0ST microarrays (University of Colorado Cancer Center Genomic and Microarray Shared Resource). The overexpressing IGF2BP1 clones had increased resistance to doxorubicin compared to the control, and the IC50 levels correlated with IGF2BP1 mRNA levels (p < 0.05, r2 = 0.89). For cellular proliferation, we found that only the IGF2BP1-expressing pool, that represents random insertion of the plasmid without selecting isolated clones, exhibited a significantly higher rate of proliferation relative to the empty vector control (p < 0.05). However, one of the highest expressing IGF2BP1 isolated clones had significantly greater cellular mobility and invasion than this pool, and both the pool and isolated clone had significantly higher rates of migration and invasion that cells transfected with the empty plasmid (p < 0.05). Microarray analysis of control and over¬expressing cells was used to detect global changes in gene expression and to identify potential targets of IGF2BP1. Differentially expressed genes were cross ¬referenced to the RNA¬ Binding Protein Immunoprecipitation database, published by Conway et al. (2016) using human stem cells, to identify direct mRNA targets bound by IGF2BP1. We identified 162 genes that were differentially expressed between control and overexpressing cells (FC ≥2, FDR< 0.05), and 13 of those genes have been previously reported to bind IGF2BP1 directly. Pathway analysis of these 13 genes identified enrichment for genes involved in the regulation of cell adhesion, migration, and the extracellular matrix. Altered expression and IGF2BP1 binding of a subset of these transcripts were confirmed using RNA immunoprecipitation and RT-qPCR. Our data suggest that IGF2BP1 plays a significant role in human and canine osteosarcoma. This study revealed the functional relevance of IGF2BP1 and identified it as a biomarker for aggressiveness in osteosarcoma. With this knowledge, new treatment strategies can be developed that target IGF2BP1 or it is signaling pathways for osteosarcoma, or any cancer that expresses high levels of IGF2BP1. This treatment may have a high impact on the cell's ability to metastasize.
Open Access
The role of plants as an environmental reservoir of chronic wasting disease prions
(Colorado State University. Libraries, 2016) Ortega, Aimee Elise, author; Zabel, Mark, advisor; Mathiason, Candace, committee member; Leach, Jan, committee member; Wilusz, Jeffrey, committee member
Transmissible Spongiform Encephalopathies (TSEs) are a group of diseases caused by an abnormal version, PrPRES, of the normal cellular host protein prion protein (Prnp) termed PrPC. Disease is fatal resulting in amyloid deposits and spongiform degeneration in the brain in most but not all cases. Clinical signs can include wasting, increases in salivation, and general motor impairment but many other clinical signs exist and can vary between TSEs. PrPRES is incredibly resistant to inactivation and can withstand radiation, formalin treatment, and autoclaving to name a few tried decontamination methods whereas PrPC is degraded normally. This difference in degradation allows for differentiation between the two protein forms as PrPRES is resistant to degradation by Proteinase K. In the early 1980s this abnormal protein was discovered to be the sole causative agent of the various TSEs which at the time was a novel finding and a novel method of disease transmission. It is thought that slightly misfolded forms of PrPC occur which can then misfold further eventually forming PrPRES. PrPRES then has the ability to act as a template for conversion, converting PrPC. Numerous TSEs exist that affect both humans and a variety of animals. One of the animal TSEs is Chronic Wasting Disease (CWD) which affects cervids such as elk, deer, and moose (Cervus candensis, Odocoileus hemionus, Alces alces) and has become endemic in both free-ranging and captive herds. The exact mechanisms behind spread of CWD are unknown but research has shown that environmental reservoirs play a role in transmission dynamics. We chose to explore whether PrPRES can be detected on or inside grasses and plants naturally exposed to prions in CWD endemic areas by use of Protein Misfolding Cyclic Amplification (PMCA). Here we present novel environmental evidence showing that PrPRES can be found on the surface of multiple plants from Rocky Mountain National Park and mice inoculated with these samples are showing clinical signs of disease.
Open Access
The spliceosome recycling factor, SART3, regulates H2B deubiquitination by Usp15
(Colorado State University. Libraries, 2014) Long, Lindsey J., author; Yao, Tingting, advisor; DeLuca, Jennifer, committee member; Luger, Karolin, committee member; Wilusz, Jeffrey, committee member
In eukaryotes plasticity of chromatin architecture is paramount to allow proper regulation of processes such as transcription regulation, DNA repair, and DNA replication. Modulation of chromatin dynamics is primarily achieved via signaling to chromatin modifiers and remodelers though a complex code of histone post-translational modifications (PTMs). These PTMs include methylation, acetylation, phosphorylation, and ubiquitination. In comparison to other histone PTMs, attachment of the 8.5 kDa ubiquitin (Ub) protein stands out due to its considerable size. The majority of histone monoubiquitination occurs on histones H2A and H2B (at lysine residues 119 and 120, respectively), and these modifications have roles in the regulation of many cellular processes including transcription, pre-mRNA processing, and DNA damage repair. To uncover the mechanisms underlying various functions associated with ubiquitinated histones, we generated non-hydrolyzable Ub-histone mimics and assembled them into H2A/H2B dimers or nucleosomes. Quantitative mass spectrometry was employed to identify proteins that bound to unmodified or modified histone dimers and mononucleosomes. We also found that, within the context of a mononucleosome, Ub, when attached to H2B, partially obscures the H2A/H2B acidic patch. Among the proteins that were identified, a deubiquitinating enzyme (DUB), Usp15, exhibited high affinity and specificity towards ubiquitinated histone dimers. Further characterization demonstrated that Usp15 is a bona fide histone DUB and preferentially deubiquitinates Ub-containing histone octamers versus Ub-containing mononucleosomes. Usp15 associates with the U4/U6 spliceosome recycling factor, SART3, which we found also bound to histones. These interactions result in more efficient histone deubiquitination by Usp15. In cells, depletion of SART3 results in elevated ubH2B levels that we show is due to a decreased rate in H2B deubiquitination. These observations indicate SART3 may play a role in regulating ubH2B dynamics as a possible mechanism by which regulate alternative splicing and transcription. Depletion of SART3 also alters transcriptional and alternative splicing patterns. By chromatin immunoprecipitation, we confirmed that SART3 localizes to at least a subset of genes whose transcription decreased upon SART3 depletion. Future studies will be designed to elucidate the mechanism by which Usp15, SART3, and ubH2B work together to regulate transcription and alternative splicing.