Browsing by Author "Perera, Rushika, committee member"
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Item Open Access Adenosine triphosphate is an allosteric inhibitor of coxsackievirus B3 3Dpol(Colorado State University. Libraries, 2016) Karr, Jonathan Paul, author; Peersen, Olve, advisor; Cohen, Robert, committee member; Perera, Rushika, committee memberPicornaviruses pose a significant threat to human and animal health, but at present there are no drugs to prevent or treat picornaviral infections. However, intensive study of the picornaviral lifecycle has revealed several promising pharmacological targets, including the RNA-dependent RNA polymerase, 3Dpol, that is responsible for replicating the viral genome. 3Dpol is central in the virus lifecycle, determines the distribution of mutants in viral progeny, and is a very highly conserved protein across picornavirus species. As such, it is an attractive target for antiviral research. The only 3Dpol inhibitors that have been found to date are nucleoside analogues that act directly on the active site, even though global dynamics of the protein that are sensitive to allosteric effects of various mutations have been shown to be important determinants of fidelity. The research presented in this thesis provides the first direct evidence of allosteric regulation of 3Dpol by a small molecule. Inhibition assays investigating the relative affinities of a stalled coxsackievirus elongation complex for non-cognate nucleotides uncovered a mixed inhibition profile of ATP. Among the six picornavirus species tested, this mode of inhibition seems specific to coxsackievirus B3 (CVB3). Engineered mutations in CVB3 3Dpol, including two that were previously found to lower polymerase fidelity, diminish the uncompetitive component of ATP inhibition. ATP inhibition was found to be dependent on the β- and γ-phosphates. The potential role of ATP’s allosteric effect in the virus lifecycle as well as the importance of a biochemically confirmed allosteric site on the polymerase are discussed.Item Open Access An exploration of viral RNA-mediated strategies to stall and repress the cellular exoribonuclease XRN1(Colorado State University. Libraries, 2018) Charley, Phillida A., author; Wilusz, Jeffrey, advisor; Zabel, Mark, committee member; Perera, Rushika, committee member; Reddy, Anireddy, committee memberThe regulation of mRNA decay plays a vital role in determining both the level and quality control of cellular gene expression in eukaryotes. Since they are likely recognized as foreign/unwanted transcripts, viral RNAs must also successfully navigate around the cellular host RNA decay machinery to establish a productive infection. This bypass of the cellular RNA decay machinery can be accomplished in many ways, including the sequestering of regulatory proteins or inactivating enzymatic components. One attractive way for RNA viruses to undermine the cellular RNA decay machinery is to target the cellular exoribonuclease XRN1 since this enzyme plays a major role in mRNA decay, appears to coordinate transcription rates with RNA decay rates, and is localized to the cytoplasm and thus readily accessible to cytoplasmic RNA viruses. We have previously shown that many members of Flaviviridae (e.g. Dengue, West Nile, Hepatitis C and Bovine Viral Diarrhea viruses) use RNA structures in their 5' or 3' untranslated regions (UTRs) to stall and repress XRN1. This results in the stabilization of viral RNAs while also causing significant dysregulation of cellular RNA stability (and thus dysregulation of overall cellular gene expression). In this dissertation we first extend this observation to another member of the Flaviviridae, Zika virus, by demonstrating that structures in the 3' UTR of the viral genomic RNA can stall and repress XRN1. Significantly, we also demonstrate that the 3' UTR of the N mRNA of the ambisense segment of Rift Valley Fever virus, as well as two other phleboviruses of the Phenuiviridae, also can effectively stall and repress XRN1. This observation establishes XRN1 stalling in an additional family of RNA viruses, in this case in the order Bunyavirales. We have mapped the region responsible for XRN1 stalling to a G-rich core of ~50 nucleotides and provide evidence that the formation of a G-quadruplex is contributing to stalling of XRN1. In addition to phleboviruses, we also detected RNA regions that stall XRN1 in the non-coding regions of two other virus families. The 3' UTRs of all four ambisense transcripts of Junin virus, an arenavirus, stall and repress XRN1. This observation was extended to two additional arenaviruses, suggesting that XRN1 stalling may be a conserved property of the 3' UTRs in the Arenaviridae. Finally, we demonstrate that the non-coding RNA from beet necrotic yellow vein virus RNA segment 3 is produced by XRN1 stalling and requires a conserved sequence called the coremin motif. Collectively, these observations establish XRN1 stalling and repression as a major strategy used by many virus families to effectively interface with the cellular RNA decay machinery during infection. We performed two proof of principle studies to extend the significance of the observation of XRN1 stalling during RNA virus infections. First, since XRN1 stalling may be associated with successful viral gene expression as well as cytopathology, we explored whether we could identify a small molecule compound that could interfere with the knot-like three helix RNA junction structure that stalls XRN1 in the 3' UTR of flaviviruses. We tested several triptycene-based molecules, compounds that have been previously shown to intercalate into three helix junctions and identified four triptycene derivatives that interfere with XRN1 stalling. Lastly, we explored whether there might be a cellular exoribonuclease that could navigate through the well-characterized flavivirus structure that effectively stalls XRN1. Our efforts focused on the mammalian Dom3z/DXO enzyme which contains both 5' decapping and 5'-3' exoribonuclease activity. Interestingly, recombinant Dom3z/DXO enzyme did not stall on RNAs containing the 3' UTR of either Dengue virus or the Rift Valley Fever Virus N mRNA. This may suggest that there is a molecular arms race of sorts between the cell and the virus for supremacy of regulating the 5'-3' decay of RNA during infection.Item Open Access Assessing and understanding the generation and function of RNA decay intermediates in non-insect borne flaviviruses(Colorado State University. Libraries, 2019) Mundell, Cary T., author; Wilusz, Jeffrey, advisor; Geiss, Brian, committee member; Perera, Rushika, committee member; Reddy, Anireddy, committee memberCellular gene expression is an intricate process regulated on many levels that allows the cell to react correctly to stimuli or to maintain homeostasis. RNA viruses must act to preferentially drive production of their own messenger RNAs (mRNAs) and proteins in order to successfully replicate and ensure continued infection. Due to the necessity for RNA viruses to remain in the cytoplasm, regulatory factors that affect host mRNAs likely also affect the transcripts of RNA viruses. RNA decay represents a major pathway of regulation for mRNAs. A multitude of RNA viruses possess unique mechanisms that act to prevent the decay of viral transcripts and allow for successful translation. Members of the viral family Flaviviridae are positive sense, single-stranded RNA viruses that do not possess a poly(A) tail. Therefore, it is highly likely that these transcripts would be marked as deadenylated and shuttled down one of the RNA decay pathways that exist in the cell. Interestingly, members of the genera Flavivirus of the family Flaviviridae possess a conserved structured 3' untranslated region (UTR) that acts to interfere with the decay processes of the major cytoplasmic cellular 5'-3' decay enzyme XRN1. In addition, members of the generas Hepacivirus, Hepatitis C Virus (HCV) and Pestivirus, Bovine Viral Diarrhea Virus (BVDV), possess XRN1 stalling elements within their 5' UTRs. These stalling sites block the action of the exonuclease and generate decay intermediates. The generation of these decay intermediates represses XRN1 activity in the infected cell. Herein we demonstrate a new method for studying RNA decay through the use of XRN1-resistant RNAs (xrRNAs). In this method we utilize the well characterized xrRNA of Dengue Virus Type 2 (DENV2) as a readout to study the decay rates of relatively large RNA constructs. We show that not only is utilizing an xrRNA an effective method for confirming XRN1-mediated decay, but that the accumulation of the readout xrRNA can be utilized to understand changes in the decay kinetics of RNA substrates. We further utilize this method to demonstrate a lack of XRN1 stalling elements within the poliovirus internal ribosomal entry site (IRES) element. We provide evidence that the stalling of XRN1 in the 5' UTR of BVDV is dependent on both the presence of the entire IRES structure and the presence of a stem loop 5' to the IRES element through the analysis of a series of truncations. Finally, we demonstrate one possible role for the HCV and BVDV decay intermediates as the truncated IRES element maintains translatability in an in vitro system. Collectively, these data better define the structural requirements for the novel XRN1 stalling elements located in the 5' UTR of non-insect borne members of the Flaviviridae as well as the potential function of the decay intermediates.Item Open Access Characterization of grcC1 and grcC2 prenyl diphosphate synthases potentially involved in menaquinone synthesis in Mycobacterium tuberculosis, and a homologous enzyme (ms1133) in Mycobacterium smegmatis(Colorado State University. Libraries, 2021) Gatlawi, Hana Bashir, author; Crick, Dean C., advisor; Quackenbush, Sandra L., committee member; Stargel, Laurie A., committee member; Perera, Rushika, committee memberCharacterization of GrcC1 and GrcC2 prenyl diphosphate synthases potentially involved in menaquinone synthesis in Mycobacterium tuberculosis, and a homologous enzyme (MS1133) in Mycobacterium smegmatis Biosynthesis pathways provide attractive drug targets in Mycobacterium tuberculosis. Understanding the biochemistry of the enzymes that are involved in those pathways is very important to make an achievement in this field. Menaquinones are the major lipoquinones found in M. tuberculosis and their synthesis has been suggested to be a valid drug target in this bacterium. Menaquinone is a key component of the M. tuberculosis respiratory chain and a major electron carrier during aerobic growth with many electron acceptors. It has also been reported that the isoprenoid side chain is an important antioxidant. The genes grcC1 and grcC2 in M. tuberculosis encode proteins, probably polyprenyl diphosphate synthases, but little information is known about these enzymes although previous studies suggested that polyprenyl diphosphate synthases could be involved in the menaquinone biosynthesis. In this study, we cloned, expressed, purified and biochemically characterized the enzymatic activity of enzymes encoded by grcC1, grcC2 in M. tuberculosis strain H37Rv, and the homologue polyprenyl diphosphate synthase ms1133 in Mycobacterium smegmatis. The enzymes were active and catalyzed the condensation reaction that added [14C]IPP to allylic substrates of varying chain-lengths, including dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP). The purified expressed proteins generally preferred the longer chains allylic substrates of 10 or more carbon atoms. GPP is likely the preferred substrate of GrcC1, whereas FPP is likely the preferred substrate of GrcC2 and MS1133. The values of Vmax, Km and Kcat have been calculated and the final products of these enzymes were determined. The final products of all of these enzymes are long chain polyprenyl diphosphates. GrcC1 catalyzed the formation of solanesyl diphosphate with nine isoprene units (C45) which is needed for the menaquinone biosynthesis in M. tuberculosis. Since the role of the enzyme in the menaquinone biosynthesis pathway has been suggested elucidation of the properties of this enzyme and obtaining more information on the biological role helps to demonstrate the involvement of the enzyme in menaquinone biosynthesis in M. tuberculosis. Our results support the hypothesis that the enzyme is integral part of menaquinone synthesis in mycobacteria. This dissertation also illustrates the essentiality of the studied genes. Our constructed knock-out mutations tested the essentiality of the genes grcC1, grcC2 and ms1133 for the survival and growth of the pathogen M. tuberculosis and its lab surrogate M. smegmatis. The results demonstrated that grcC1 is an essential gene for M. tuberculosis survival. grcC2 and ms1133 both are not essential for survival, but the absence of these genes delays bacterial growth in M. tuberculosis and M. smegmatis.Item Embargo Characterizing the effects of bluetongue virus coinfection in Culicoides sonornesis(Colorado State University. Libraries, 2023) Carpenter, Molly Jean, author; Mayo, Christie, advisor; Mathiason, Candace, committee member; Perera, Rushika, committee member; Simpson, Katie, committee memberBluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides biting midges. Infection of domestic and wild ruminants with BTV can result in devastating disease and significant economic losses. In concert with climate change, BTV outbreaks have been characterized by an expanding geographical range and incursions of novel serotypes into endemic regions. As a virus with a segmented genome, reassortment between BTV strains may increase genetic diversity which can alter BTV transmission dynamics and generate epizootic events. While factors driving BTV's expansion are poorly understood, reassortment between virus strains may enhance BTV's ability to spread to new regions. The following studies aimed to investigate different facets of BTV coinfection and reassortment in the Culicoides vector including temperature effects, BTV serotype infection titers, and virus coinfection dissemination. While warmer temperatures have been demonstrated to increase virogenesis, temperature effects on reassortment is not known. The first aim was to evaluate how temperature affects Culicoides survivorship, virogenesis, and progeny virus genotype outcomes in BTV coinfected Culicoides sonorensis. C. sonorensis were provided bloodmeals containing BTV serotype 10 (BTV-10), BTV serotype 17 (BTV-17), or both BTV serotypes and maintained at different temperatures (20°C, 25°C, or 30°C). Every other day, C. sonorensis were collected and processed for BTV qRT-PCR to track virogenesis over time. Co-infected C. sonorensis collected were processed for BTV plaque-isolation (a technique to visualize replicating virus units). The complete genotypes of isolated plaque progeny were determined using shotgun next-generation sequencing. Results indicate that C. sonorensis maintained at warmer temperatures had productive virogenesis earlier in infection than C. sonorenesis held at cooler temperatures. However, C. sonorensis maintained at cooler temperatures had longer mean survival times. Most of the plaque progeny virus genotypes aligned with parental serotype BTV-17, while a few plaques had both parental serotypes represented. While warmer temperatures may accelerate virogensis for earlier potential transmission, there is a trade-off with C. sonorensis mean survival times. Most of the plaque progeny genotypes aligned with BTV-17 indicating that it may be the more fit serotype in the C. sonorensis system. To further explore why the majority of progeny virus aligned with BTV-17, the second aim was to evaluate if different coinfection ratios of BTV-10 and BTV-17 affect progeny virus genotype outcomes. In prior in vitro BTV coinfection and modeling studies, progeny genotypes were dominated by the parental strain with the higher initial multiplicity of infection. To recapitulate this in an in vivo model, C. sonorensis were fed a blood meal containing BTV-10, BTV-17, or both BTV strains with contributing titers of BTV-10 ATCC: BTV-17 at either 90:10, 75:25, 50:50, 25:75, or 10:90 ratios. Pools of five midges were collected in triplicate every other day and processed for pan BTV qRT-PCR to track virogenesis over time. Day ten post-infection midges were collected in pools of ten and processed for plaque isolation and propagation. The complete genotypes of isolated plaques were identified using shotgun next-generation sequencing. Plaque progeny virus genotyping demonstrated an overall trend of progeny virus aligning with the parental serotype with the higher titer. However, a few plaques at a subset of co-infection ratios demonstrated reassortant genotypes with patterns that were suggestive of preferred segment combination. While the parental serotype with the higher contributing titer may have more representation in progeny virus, reassortment events can provide genetic diversity. As reassortment between BTV-10 and BTV-17 was infrequent, it was conjectured that the two parental BTV serotypes did not routinely coinfect the same cells. Thus, the third aim was to determine extent of dissemination and characterize tropism of BTV coinfection in C. sonorensis. In situ hybridization approach was employ using the RNAscope® platform to detect patterns of BTV infection in histologic cross sections of coinfected C. sonorensis. Upon assessment by microscopy, mosaic patterns in which serotypes did not often overlap, suggest that coinfection at the cellular level may not be abundant with these two serotypes. This could be a consequence of superinfection exclusion. Understanding BTV coinfection and its biological consequences will add an important dimension to the modeling of viral evolution and emergence.Item Open Access Extrinsic and intrinsic drivers of feline immunodeficiency virus evolution in the mountain lion(Colorado State University. Libraries, 2018) Malmberg, Jennifer L., author; VandeWoude, Sue, advisor; Crooks, Kevin, committee member; Quackenbush, Sandra, committee member; Perera, Rushika, committee memberViruses are among the most rapidly evolving entities in biology and are so intricately associated with their obligate hosts that the boundary between host and pathogen, and thus the study of one versus the other, is blurred by intimate interactions at scales ranging from proteins to populations. Viral genetic variation is both ecologically and molecularly determined, and thus viruses serve as measurably evolving populations that provide a window into adaptations and behaviors of their vertebrate hosts. Of all viral families, the biology of retroviruses is coupled especially tightly to that of the host due to permanent integration of viral DNA into eukaryotic chromosomes, producing an inherently dynamic infection that persists for life. Feline immunodeficiency virus (FIV) is among the oldest of viruses in the Lentivirus genus and puma (Puma concolor) are the most extensively ranging New World terrestrial mammal. We used molecular analyses to investigate the host-pathogen interactions between pumas and FIV across geographic and temporal space, within and across populations, and among FIV subtypes. In Chapter One, we investigate cross-species transmission of FIV from bobcats to pumas and compare the outcome of spillover infections in two populations separated by vast geographic space. Our findings reveal that the puma is typically a dead-end host of bobcat FIV infection, although altered population dynamics can promote stuttering chains of infection following spillover events. In Chapter Two, we employed a novel next generation sequencing technique to investigate the impact of management interactions such as population supplementation on FIV dynamics in the endangered Florida panther. Results from this chapter show evidence for cointroduction of one subtype of FIV with translocated pumas from Texas, followed by local extinction of the previously circulating, 'less fit' subtype in the puma host. Chapter Three describes an important intrinsic driver of viral evolution through characterization of the APOBEC3 protein A3Z3 in the puma, a primary cellular restriction factor against FIV. We show evidence that at least one geographically associated genotype of puma FIV is able to evade lethal hypermutation typical of A3Z3 activity despite a deficiency in the viral counter protein Vif. The collective findings of this work explore the ancient relationship between a vastly ranging apex predator and a chronic lentiviral infection by applying both novel and conventional methodologies to a unique, naturally occurring host-pathogen system. Although our questions were specific to FIV in pumas, the methodologies described here can be applied to other systems and models to address inherent limitations of opportunistic field studies including DNA degradation and sequencing of low copy number templates from archival biological samples. Ancient viral infections have the potential to elucidate the life history of mammalian hosts, which is particularly useful in the study of elusive and broadly ranging carnivores threatened by urbanization and habitat fragmentation. Future objectives of this work will expand analyses to incorporate additional populations, such as the modern Texas puma, and more thoroughly investigate genotype variation in Vif-A3Z3 interactions. Collectively, our results will inform additional studies that seek to elucidate determinants of host-pathogen interactions in naturally-occurring systems across diverse ecosystems and broad spatiotemporal scales.Item Embargo Gene-targeted mouse models provide novel insights into strain diversity and interspecies transmission of chronic wasting disease(Colorado State University. Libraries, 2022) Sun, Julianna, author; Telling, Glenn, advisor; Ross, Eric, committee member; Chanda, Soham, committee member; Perera, Rushika, committee memberPrion diseases are fatal, transmissible neurodegenerative diseases that affect humans and other animals and are caused by the aberrant misfolding of the prion protein (PrP) to a disease-causing form. The term 'prion' was coined in 1982 by Stanley Prusiner to denote a small proteinaceous infectious particle which is now known to be the cause of scrapie in sheep, transmissible mink encephalopathy (TME), bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in cervids such as deer and elk. Additionally, humans can develop prion diseases via multiple routes – spontaneously in the case of sporadic Creutzfeldt-Jakob disease (CJD), inherited in the cases of fatal familial insomnia (FFI) and Gerstmann-Straussler-Scheinker (GSS) syndrome, or acquired in the cases of variant CJD and Kuru. In addition to classical prion diseases, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Frontotemporal dementia have recently been classified as prion-like diseases due to similar protein misfolding mechanisms critical to these disease pathogeneses. Thus, the exploration of prion disease mechanisms has implications for a variety of neurodegenerative diseases. The main focus of this thesis will be the characterization of CWD strains and pathogenesis using mouse models of CWD. The disease was first described in Colorado in the 1960s in mule deer and rocky mountain elk and since then has expanded in has expanded in both geographical range and host species range including white-tailed deer, moose, red deer and reindeer. In North America, CWD has now been documented in 30 American states and three provinces in Canada. In addition to cases in North America, CWD has been identified in South Korea as a result of accidental transmission of subclinically infected cervids from Canada. In 2015, Norway reported a case of CWD in a herd of reindeer, and shortly after also reported cases of CWD in three free-ranging moose, marking the first cases of CWD in Europe. As a result, surrounding countries increased CWD surveillance, and Finland reported two cases of CWD in moose, and Sweden reported four cases of CWD in moose. At the time of writing, 20 reindeer, 11 moose, and two red deer in Norway, four moose in Sweden, and two moose in Finland have been diagnosed as CWD positive in Europe. The persistent spread of CWD raises both ecological and economical concerns thus the characterization of CWD pathogenesis is of utmost importance. Prions are unlike viral and bacterial pathogens in that their infectious component is entirely proteinaceous. The templated conversion of PrPC to PrPSc is driven by PrPSc imposing its infectious conformation onto PrPC. In other words, there are no primary structural differences between PrPC and PrPSc and thus higher order structural differences between PrPC and PrPSc must account for infectivity of PrPSc. This is confirmed by recently solved cryogenic-electron microscopy structures of PrPSc which show an insoluble, β-sheet rich protein structure, as opposed to the soluble, α-helical rich PrPC conformation. Though all heritable information is encoded in protein conformation, prions can exhibit strain characteristics similar to other pathogens. Strains are operationally defined by characteristics such as time to disease onset, clinical signs, and neuropathology. While these characteristics can be defined in the natural disease host, the use of the mouse bioassay has facilitated the ease of strain typing. Since the primary structure of mouse PrP is slightly different than cervid PrP, transmission of CWD to mice is generally inefficient. Our and other labs combat this by the design of transgenic mice expressing cervid-PrP. Specifically, the Telling lab designed prototype transgenic overexpressing cervid-PrP mice, expressing either glutamate (E) or glutamine (Q) at residue 226 of PrP. This is the only primary structural difference between CWD susceptible cervid species: North American elk express E226, while deer, moose, and reindeer express Q226. Our lab then designed gene-targeted mice which express endogenous levels of cervid-PrP, either E226 or Q226 expressing. These mice serve as a proxy to characterize CWD strain characteristics and lend insight into the pathogenesis of CWD. The work included in this thesis largely utilizes these mice to answer fundamental questions pertaining to CWD. These questions include: 1. What effect does the polymorphism at residue 226 of cervid PrP have on CWD pathogenesis? 2. How do the strain profiles of emergent cases of Nordic CWD compare to well-characterized cases of North American CWD? 3. Did CWD originate from a cross species transmission, and what is the potential for further cross species transmission of CWD?Item Open Access Host directed therapy targeting M. tuberculosis infected macrophages(Colorado State University. Libraries, 2016) Lakey, Natalie, author; Basaraba, Randall, advisor; Podell, Brendan, committee member; Perera, Rushika, committee member; Chicco, Adam, committee memberWith the rise of drug resistant strains of Mycobacterium tuberculosis (Mtb) and lags in antimicrobial drug development, it is imperative to explore alternative methods of treatment through host-directed adjunct therapies. Hallmarks of Mtb infection are altered host cell glucose metabolism and non-diabetic hyperglycemia, which increase disease severity and bacterial burden. This can be targeted using a combination of metformin and 2-deoxyglucose (2DG) to lower systemic blood glucose and increase metabolic stress in infected macrophages to induce apoptotic cell death, enabling Mtb clearance and antigen presentation to activate cell-mediated immune responses. We hypothesized that bacterial survival is aided by glycolysis-dependent macrophages, which can be targeted using a combination of metformin and 2DG to strengthen host immune responses. Using an in vitro model of guinea pig bone marrow derived macrophages under normal and high glucose conditions, we determined that both basal respiration and glycolytic activity increased after infection. When singly treated, metformin inhibited basal respiration while increasing glycolysis while 2DG inhibited both processes. In combination metformin and 2DG treatment inhibited basal respiration more effectively than metformin treatment alone and inhibited glycolysis as effectively as 2DG by itself. Efficacy of metformin-2DG treatment is dependent on high cellular glycolytic activity, a characteristic of granulomatous cells. Metformin-2DG treatment decreased cell survival 48 hours post-treatment by increasing apoptotic cell death and decreased Mtb survival more effectively than metformin or 2DG alone. We conclude that apoptotic induction of macrophages by metformin-2DG treatment may be a viable adjunct treatment to antimicrobial drugs to reduce bacterial burden and increase an effective host adaptive immune response.Item Open Access Macrophage immunometabolism during flavivirus infection(Colorado State University. Libraries, 2022) Donkoh, Jasmine, author; Rovnak, Joel, advisor; Quackenbush, Sandra, committee member; Foy, Brian, committee member; Perera, Rushika, committee member; Chen, Chaoping, committee memberDengue virus (DENV) and Zika virus (ZIKV) are mosquito borne flaviviruses that are transmitted by the Aedes spp. mosquito and have caused outbreaks in Africa, Asia, the south Pacific, and the Americas. Infection with DENV can cause severe illness, such as dengue hemorrhagic fever and dengue shock syndrome, while infection with ZIKV can result in congenital abnormalities, such as microcephaly, and spontaneous abortions. Although disease outcome for these viruses is markedly different, both DENV and ZIKV both target monocytes and macrophage for pathogenesis. Macrophage are among the first cells to be infected by DENV and ZIKV and are disseminated throughout the body. While macrophage are an important cell in flavivirus pathogenesis, the mechanisms by which viruses modulate macrophage function are not fully understood. In this dissertation, I present data that attempts to explain the interaction between macrophage and flaviviruses, as well as investigate the mechanisms in which DENV and ZIKV control macrophage gene expression and metabolism. The most widely used macrophage cell line, THP-1 cells, are cultured as immature monocytes. To become naïve macrophage, these cells are treated with phorbol 12-myristate- 13 acetate (PMA). Once THP-1 monocytes are differentiated into naïve macrophage, they can be polarized into different macrophage subsets. Even though THP-1 macrophage are widely used, the protocols in which to differentiate and polarize cells are not consistent. In chapter 2, we optimize methods to differentiate and polarize THP-1 cells. We measure gene expression and cellular metabolism during differentiation and polarization to characterize macrophage phenotype. These data, coupled with published literature, show that this model is a reliable system to study macrophage biology and flavivirus-macrophage interactions. We use the methods developed in this aim throughout the dissertation. Macrophage metabolism and phenotype determine immune function. Inflammatory (M1) macrophage are inflammatory and mount a strong anti-viral response, while anti-inflammatory (M2) macrophage dampen anti-viral responses. Viruses can alter macrophage phenotype for efficient replication and immune evasion. In chapter 3 we elucidated the role of macrophage polarization on DENV replication, showing that M1 macrophage have suppressed DENV replication while M2 macrophage support replication. In addition, we characterized the impact of DENV infection on M1 and M2 gene expression and metabolism. DENV infection resulted in an upregulation of inflammatory and anti-inflammatory genes in both M1 and M2 macrophage. Infection resulted in similar metabolic profiles in M1 and M2 cells, suggesting that DENV infection reprograms cellular metabolism in a way that is favorable for replication, regardless of macrophage phenotype. The key difference between M1 and M2 cells was the upregulation of interferon genes, where M1 mounted a strong interferon response, M2 mounted a subdued response. The difference in the interferon response could explain the difference in DENV replication observed in the two phenotypes. These data add to the ongoing literature on immunometabolism and its impact on viral pathogenesis. Cyclin dependent kinase 8 (CDK8) and CDK19 are transcriptional cofactors that regulate expression of inflammatory and anti-inflammatory genes. In addition, inhibition of CDK8/19 during DENV infection leads to decreased replication, as well as metabolic shifts in Huh7 cells, a liver cell line. In chapter 4, we investigate the role of CDK8/19 on viral replication and inflammatory/ anti- inflammatory gene expression. We found that inhibition of CDK8/19 kinase activity increased DENV replication and anti-inflammatory gene interleukin 10 (IL-10) expression. In contrast, inhibition of kinase activity decreased expression of inflammatory genes C-X-C motif chemokine ligand 10 (CXCL10). Furthermore, I found distinct mechanisms for each kinase through analysis of DENV-infected CDK8 and CDK19 knockdown cells. Knockdown of CDK8 mimics chemical inhibition of CDK8/19, while knockdown of CDK19 did not change expression in CXCL10 or IL-10. These data indicate that CDK8 and CDK19 regulate the transcription of different genes during DENV infection in macrophage. These data contribute the basic understanding of CDK8/19 regulation during viral infection. Macrophage phenotype plays a large role in ZIKV pathogenesis, where macrophage found near the placenta are an anti-inflammatory phenotype and are susceptible to infection. In chapter 5, we investigated the role of cyclin dependent kinase 8 and phenotype in Zika virus pathogenesis. We found CDK8 gene expression increase throughout infection, while CDK8 kinase inhibition decreased viral replication. Furthermore, inhibiting CDK8/19 kinase activity led to a decrease in CXCL10 and an increase in IL-10, as seen in a DENV model of infection. We also found that M2 macrophage were more susceptible to infection than M0 or M1. These data suggest that CDK8/19 kinase activity could be a pan-flavivirus mechanism to regulate host gene expression during infection.Item Open Access Mexican mosquitoes: overcoming barriers for dengue and Zika virus infection(Colorado State University. Libraries, 2017) Garcia Luna, Selene M., author; Black, William C., IV, advisor; Ebel, Gregory D., committee member; Perera, Rushika, committee member; Hess, Ann M., committee memberThe mosquito transmitted arboviruses cause an important burden of disease worldwide. In Latin America dengue disease is endemic with more than 1 million dengue fever cases reported yearly. In addition to dengue, chikungunya and Zika viruses have been also circulating since their introduction in 2014 and 2015 respectively. For a mosquito-borne infection to occur susceptible humans, the mosquito vector and the virus should coincide. This dissertation was focused in the mosquito vector and its ability to acquire, maintain and then transmit the virus, termed vector competence. The vector competence was a fundamental measure for the research chapters in which we studied different aspects on the interactions between Aedes aegypti and Aedes albopictus mosquitoes and Dengue-2 and Zika viruses. This dissertation includes three research chapters which were based on the following specific aims. Specific aim 1: Determine the patterns of gene flow and vector competence for DENV-2 of Aedes aegypti from around the Mexican Neovolcanic Axis. It was previously reported that the intersection of the Neovolcanic axis (NVA) with the Gulf of Mexico coast in the state of Veracruz acts as a discrete barrier to gene flow among Ae. aegypti populations north and south of the NVA. These collections also differed in their vector competence (VC) for Dengue virus serotype 2 (DENV-2). Therefore, the goal of the present study was to determine if the same patterns remained 8 years later in collections from 2012. For which haplotype variation for the mitochondrial ND4 and the nuclear genes Dicer-2 and Argonaute-2 was analyzed for north and south of the NVA mosquito populations. Also, the VC of those populations for DENV-2 was determined (Chapter 2). Specific aim 2: Profile the microRNA response of Aedes aegypti midguts to DENV-2 exposure and DENV-2 infection. The microRNA pathway has been found to modulate important physiological mechanisms in mosquito vectors. Therefore in the context of DENV infection, miRNA modulation may provide information about key genes that are important for infection. Differential expression patterns of miRNAs from mosquito midguts upon infection have been unexplored. Therefore, we explored on the involvement of the miRNA pathway in persistently DENV-2 infected mosquitoes, for which DENV-2 virus was detected at 14 days post-infection (dpi). Two comparisons were included in the study. In the first group, DENV-2 infected midguts that produced a disseminated infection (did not have a midgut escape barrier) were contrasted with those that were given a non-infectious blood meal. Also, we included a comparison group from a subset of mosquitoes from the same cohort that were exposed to DENV-2 regardless of their midgut infection status contrasted to unexposed mosquitoes. Analysis of miRNA regulation in mosquitoes may help us to understand more about the intricate interactions between the virus and the vector host (Chapter 3). Specific aim 3: Assess the variation in competence for Zika virus transmission by Aedes aegypti and Aedes albopictus from Mexico. Previous studies have reported low Zika virus (ZIKV) transmission rates for the Asian lineage of ZIKV using mosquitoes from a wide geographical range from the Americas. Beside low transmission rates we hypothesized that VC is variable and is highly dependent upon the geographic origin of the mosquito populations. Hence, we analyzed the ZIKV transmission potential of recently colonized Aedes collections. Ten Ae. aegypti and three Ae. albopictus collections from different locations across Mexico were analyzed for ZIKV (strain PRVABC59 Asian genotype) vector competence at 7 and 14 dpi. We calculated the additive contribution of each of the four transmission barriers to ZIKV infection. In addition, we evaluated the contribution of both mosquito species to ZIKV transmission in areas where their distributions overlap (Chapter 4).Item Open Access SARS-CoV-2 viral RNA biology and its impact on the infected cell(Colorado State University. Libraries, 2023) Altina, Noelia H., author; Wilusz, Jeffrey, advisor; Argueso, Lucas, committee member; Geiss, Brian, committee member; Perera, Rushika, committee member; Yao, Tingting, committee memberThe fine-tuning of the replication and transcription of RNA viruses often requires the interaction of viral RNAs with cellular RNA binding proteins. This project addresses fundamental knowledge gaps on the molecular mechanisms that underlie SARS-CoV-2 gene expression, regulation, and viral RNA-host interactions. After infection, SARS-CoV-2 generates a large set of sub-genomic mRNAs, each containing an identical ~70 base 'leader' region in their 5'UTR (from position 14 to position 75 in RefSeq NC_045512) and a 229 base region at the 3'UTR (from position 29,675 to position 29,903 in RefSeq NC_045512) generated by discontinuous transcription. The accumulation of a considerable amount of these leader/3'UTR regions during the infection represents a possible sink for cellular RNA binding proteins. We demonstrated that PTBP1, a cellular protein involved in the regulation of alternative splicing, binds to the SARS-CoV-2 leader region. SARS-CoV-2 infection critically impacted the splicing of several cellular pre-mRNAs that are normally regulated by PTBP1. Mechanistically, we suggest that SARS-CoV-2 sequesters and influences the re-localization of shuttling splicing factors like PTBP1 from the nucleus to the cytoplasm resulting in significant effects on the host cell splicing machinery leading to changes in cellular mRNA splicing patterns during SARS-CoV-2 infection. Given the current extensive interest in epigenetic methylations of both cellular and viral RNAs, our study also explored the role of post-transcriptional RNA modifications on viral mRNAs. We demonstrated that SARS-CoV-2 can usurp the cellular enzyme, namely PCIF1, to place the m6Am modification at the cap proximal position in its mRNAs. This double methylation is usually found on all host mRNAs that initiate with an adenosine residue, and thus SARS-CoV-2 likely installs this modification on its mRNAs to avoid host immune recognition. Interestingly, we also discovered that capping and m6Am modification are tightly regulated throughout the infection. The highest levels of these 5' end RNA modifications were observed at 12 hours post infection, correlating with the full establishment of viral gene expression in infected cells. These findings indicate that 5' end modification of SARS-CoV-2 transcripts is not simply a default process but rather undergoes unanticipated regulation throughout the infection. Collectively, the data presented provide not only new insights into the complex interactions that SARS-CoV-2 has with the RNA biology of the cell during infection, but also identify attractive potential targets for developing novel anti-coronavirus drugs to treat future emerging coronavirus diseases.Item 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 memberCellular 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.Item Open Access Using a precision-cut lung slice co-culture paradigm to increase T-cell populations and model infection ex vivo(Colorado State University. Libraries, 2024) Ehrlich, Alexis T., author; Tobet, Stuart, advisor; Perera, Rushika, committee member; Magee, Christianne, committee memberPrecision cut lung slices (PCLS) bridge a gap between in vivo and in vitro studies by maintaining anatomical organization with structural integrity and intercellular signaling pathways. Applications of PCLS have included the modeling of inflammatory lung diseases, metabolism studies, and drug development. In the lungs, immune responses are carried out by a network of T- and B- cells, the latter of which are resident. The limited resident T-cell population of the lung diminishes accurate representations of pathogen response capacity in PCLS. Addressing this, we set out to increase pulmonary T-cell populations ex vivo. We hypothesized that thymus and bone marrow-derived T-cells would work synergistically to populate the lung in co-culture experiments. A murine organotypic lung co-culture model was developed and characterized for tissue health and T-cell recruitment over 3 days ex vivo using adult neurobasal media with 4 mM glucose + 2% B27 supplement. Lung slices were cultured independently, with bone marrow, thymus, or both. Immune colonization of the lung was assessed using immunohistochemistry for CD3+ T-cells, CD19+ B-cells and ACK2+ cells. Cells were counted in alveolar and airway spaces after 3 days of culture. Our results demonstrate that lung co-cultured with thymus and bone significantly increased T-cell populations ex vivo, whereas lung co-cultured with thymus or bone alone did not significantly alter T-cell counts. Additionally, B-cells and C-Kit+ cell populations were not influenced by the culture paradigm. Using this paradigm, we went on to explore this lung co-culture paradigm when stimulated by an immune modulatory agent – LPS- and when an active lung infection is present using Pseudomonas aeruginosa. Lung and lung co-cultures had increases in T-cell counts after immune stimulation and infection. Additionally, the co-cultures further increased T-cell counts after treatment. More strikingly, the co-cultures influenced the degree of bacterial infection in the lung slices without altering the B-cell populations among cultures. Viral infections are also common pathogens that affect the lungs, so we examined then examined the ability of viral pathogens to infect precision cut lung slices. As we did not get an infection with live virus, we explored the effect of a viral mimic – Resiquimod- on lung co-cultures on the immune responses. Resiquimod and the co-cultures are both able to increase T-cell populations in lung slices ex vivo. These results suggest that the increased T-cell population corresponding with thymus and bone marrow co-culture could be a result of cell-cell interaction or the secretion of growth factors. Cell secretions or growth factor release could stimulate thymic secretion of T-cells or could stimulate T-cell proliferation in the lung, suggesting that co-culture with thymus and bone marrow can elicit a T-cell response ex vivo. T-cells are necessary for host-pathogen immune responses, most commonly by CD8+ T-cells but there are other populations of T-cells. Although there are limitations to the use of lung slices in infection studies, the results of the co-culture and increasing T-cells is a promising step to studying pathogen response capacity in the future ex vivo.