Browsing by Author "Quackenbush, Sandra, committee member"
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Item Open Access A circuitous journey of virus characterization and surveillance in North and Central America(Colorado State University. Libraries, 2023) McMinn, Rebekah J., author; Ebel, Gregory D., advisor; Quackenbush, Sandra, committee member; Brault, Aaron, committee member; Neuwald, Jennifer, committee memberThe burden of ticks and the pathogens they carry is increasing worldwide. Powassan virus (POWV, Flaviviridae: Flavivirus), the only known North American tick-borne flavivirus, is of particular concern due to rising cases and the severe morbidity of human disease. In this dissertation we evaluated the recent emergence of POWV from a culmination of field (chapter 2), in vitro (chapter 3), and in-vivo (chapter 4) studies. In addition, we determined the applicability of a vector-enabled surveillance method (xenosurveillance) in Central America (chapter 5). We first used a genetic approach to evaluate the emergence of lineage II POWV, known as deer tick virus (DTV), in parts of North America where human cases occur. We detected DTV-positive ticks from eight of twenty locations in the northeastern United States with an average infection rate of 1.4%. High-depth whole genome sequencing of eighty-four new and archival POWV and DTV samples allowed us to assess geographic and temporal phylodynamics. We observed both stable infection in the northeastern United States and patterns of geographic dispersal within and between regions. Bayesian skyline analysis demonstrated DTV population expansion over the last fifty years. This is concordant with the documented expansion of Ixodes scapularis tick populations and suggests increasing risk of human exposure as the vector spreads. Finally, we isolated sixteen novel viruses in cell culture and demonstrated limited genetic change after passage, a valuable resource for future studies investigating this emerging virus. We then assessed in vitro phenotypes of POWV on human neuronal cells using 16 genetically diverse isolates obtained from a broad geographic and temporal range. We determined over a 10,000-fold range in peak viral titer and significantly decreased cell mortality for two Midwest DTV isolates, though no clear correlation between in vitro phenotype and geo-temporal characteristics could be made. We then performed whole genome sequencing of virus post neuronal cell passage to identify potential residues of interest. Again, no residues could be linked to phenotype, though several interesting residues with increased frequency post-neuronal cell culture were identified. Based on the significant in vitro diversity observed, we sought to assess pathogenesis and tick transmission phenotypes between isolates. We noted neurological disease in mice in both lineages of POWV, with potential low-virulence strains derived from coastal New York. Additionally, we observed an early neuroinvasion phenotype for a Midwest DTV isolate. The ability to infect I. scapularis ticks was determined by feeding on infected host mice (viremic) and through an artificial infection method. Surprisingly, infection rates in ticks via viremic or artificial infection remained consistent between all five isolates tested, resulting in 12-20% infection rate. Taken together, these data demonstrate potential genotype-independent ability to infect ticks and conversely, strain-dependent differences in pathogenesis. In chapter 5, we evaluated a vector-enabled surveillance method ('xenosurveillance') in rural Guatemala. Surveillance methods that permit rapid detection of circulating pathogens are desperately needed. Xenosurveillance is a novel surveillance approach that takes advantage of mosquito feeding behavior to identify blood-borne pathogens that may be circulating in human and animal hosts. This approach circumvents invasive blood sampling of individuals and results in an abundant sample source derived from both humans and animals. In this study, twenty households from two villages (Los Encuentros and Chiquirines) in rural, southwest Guatemala were enrolled and underwent weekly prospective surveillance for 16 weeks. When febrile illness was reported in a household, recently blood-fed mosquitoes were collected from within dwellings and blood samples taken from each member of the household. Mosquitoes were identified to species and blood sources identified by sequencing. Shotgun metagenomic sequencing was used to identify circulating viruses. Culex pipiens (60.9%) and Aedes aegypti (18.6%) were the most abundant mosquitoes collected. Bloodmeal sources were most commonly human (32.6%) and chicken (31.6%), with various other mammal and avian hosts detected. Several mosquito-specific viruses were detected, including Culex orthophasma virus. Human pathogens were not detected. While more intensive sampling may be needed to detect human pathogens, sampling mosquitoes that feed on humans and domestic animals may prove valuable for monitoring pathogens with zoonotic potential.Item Open Access CD4+ T-cell derived IL-10 mitigates malarial anemia(Colorado State University. Libraries, 2013) Kiser, Patti, author; Avery, Anne, advisor; Callan, Robert, committee member; Olver, Christine, committee member; Quackenbush, Sandra, committee memberIndividuals living in malaria endemic areas develop effective anti-parasite immunity over several years of repeated exposure, but become resistant to severe disease after just one or two infections. This observation suggests that the acquired immune system plays a role in both processes, but may involve different mechanisms. Using the mouse model of malaria caused by non-lethal Plasmodium yoelii, we test the hypothesis that CD4+ T-cell derived IL-10 contributes to disease resistance by mitigating severe anemia. Here we show that IL-10 deficient mice develop significant anemia despite a very low parasite burden. Anemia in this model is mediated primarily by increased erythrocyte destruction and not from suppressed erythropoiesis. Wild type mice that have recovered from P. yoelii infection have an expanded population of IL-10 producing CD4 T cells, with the majority of these cells co-expressing IFN-γ and display a Th1 phenotype. In the absence of IL-10, there is an increase in IFN-γ+ T cells. We demonstrate that IL-10 competent CD4+ T cells protect athymic nude mice from anemia when compared to CD4 T cells taken from recovered IL-10 deficient mice. Utilizing an ex vivo system that tests the function of APCs in activating CD4+ T-cells, we also determined that APCs exposed to P. yoelii in vivo induced a greater population of CD4+ T-cells that express IL-10 compared to naïve APCs. We also demonstrate that IFN-γ is required, with the possible involvement of IL-10 and IL-12, for efficient IL-10 expression in CD4+ T-cells. Our findings suggest that one mechanism by which the acquired immune system contributes to resistance to severe anemia may be the development of CD4 T cells that co-express IL-10 and IFN-γ, thereby self-regulating IFN-γ levels, which then inhibits pro-inflammatory mediated destruction of naïve red blood cells.Item Open Access Dynamics of H3 and H2B octamer variants(Colorado State University. Libraries, 2020) McVay, Abigail Lea, author; Hansen, Jeffrey C., advisor; Quackenbush, Sandra, committee member; DeLuca, Jennifer, committee memberIntroductions of alterations within a nucleosome can lead to drastic changes to the accessibility and stability of genetic material during various phases of the cell cycle. To understand these changes, three octamer mutants were recombined, reconstituted with a high affinity tandem repeat sequence, and tested using a combination of in vitro experimental methods. All Tailless, H2BTL and an H2BTL & H3TL nucleosomal array mutants were sedimented, digested, re-associated, and visualized in order to determine the role of histone tails and their influence on chromatin condensate structure. The All Tailless octamer mutant expressed an inability to form complex molecular structures, suggesting that histone tails are necessary to further the process of chromatin condensate association and subsequent folding. The H2BTL mutant expressed high levels of concentration and an increased level of association compared to the other mutants. This lead to the assumption that the exclusion of only one histone tail lead to a greater ability to associate compared to mutants lacking two or more tails. The H2BTL & H3TL mutant had a possibility of two distinct populations within solution, suggesting that the exclusion of at least two tails led to a loosely compacted and easily accessible chromatin condensate structure. In summary, this data suggests that as histone tails are excluded from octamer mutants, the chromatin condensates expresses a decreased ability to form higher degrees of compaction. Exclusion of histone tails from octamer mutants also resulted in a more accessible 601-12 tandem repeat sequence susceptible to various changes.Item Open Access Effects of major inorganic constituents of asphalt on the rapid determination of plutonium(Colorado State University. Libraries, 2024) Chunko, Raissa, author; Sudowe, Ralf, advisor; Brandl, Alexander, committee member; Quackenbush, Sandra, committee memberIn case of a nuclear incident, standard radioanalytical techniques must be available to analyze radionuclides in unusual matrices. Radiochemical analysis of samples in standard matrices of soil, water, and air are very well established; however, much less research has been conducted on the effect of unusual matrices such as steel, concrete, glass, and asphalt. In the event of a detonation of an improvised nuclear device (IND) in an urban environment, the standard separation techniques used for plutonium separations from asphalt samples originating from roadways and roofing shingles must rigorously be tested to provide useful insight on the characteristics of the special nuclear material. Batch studies were used to determine the changes in uptake of plutonium on extraction chromatography resins in the presence of trace metal components found in asphalt including aluminum, iron, and manganese at possible ranges found in asphalt samples. In these studies, selected cations with a +3-oxidation state had some interesting effects on the uptake of plutonium on the extraction chromatography resins. Aluminum increased the sorption of plutonium only on DGA and TRU resins especially at rising concentrations. Iron very unexpectedly increased the sorption of plutonium on all resins particularly at high concentrations. Additionally, metals found in the +3 oxidation state were shown to interfere with the recovery of plutonium from column studies conducted due to the synergistic effects. From this data, the contaminates found in asphalt with a +3-oxidation state may either compete with plutonium or give rise to a "salting out" effect that increases the sorption on the extraction chromatography resins and will need to be considered during the development of a rapid separation technique for plutonium from asphalt samples.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 Open Access From Retroviridae to Flaviviridae: adventures in molecular virology(Colorado State University. Libraries, 2021) Butler, Molly, author; Rovnak, Joel, advisor; Quackenbush, Sandra, committee member; Stenglein, Mark, committee member; Moreno, Julie, committee memberThe work presented here encompasses two avenues of investigation: the first, regarding the identification of a novel retrovirus in Gunnison's prairie dogs, and the second regarding the role of cyclin-dependent kinases 8 and 19 (CDK8 and CDK19) as transcriptional regulators during infection with dengue virus serotype 2 (DENV2) and during the innate immune response. Part I: During the course of research and wildlife disease surveillance efforts, we identified three cases of thymic lymphoma in free-ranging Gunnison's prairie dogs (Cynomys gunnisoni). As Gunnison's prairie dogs are keystone species, that is, critical for the maintenance of their ecosystems, we investigated the potential for an association between the observed thymic lymphomas and retroviral infection. We identified a novel retroviral sequence which exhibits genetic organization consistent with a type D betaretrovirus and which was highly associated with thymic lymphoma in Gunnison's prairie dogs. The proposed name of this virus is Gunnison's prairie dog retrovirus (GPDRV). Part II: CDK8 and CDK19 are transcriptional regulators which are critical for modulating gene expression changes during induced states such as hypoxia and starvation. We investigated the role of CDK8 and CDK19 in two distinct but related induced states: infection with DENV2 and the type I interferon response. We found that in the context of DENV2 infection, CDK8/19 regulate metabolic gene expression changes, the result of which is a reshaping of the host cell metabolic environment which is ultimately beneficial to viral replication. Therefore, chemical inhibition or reduced expression of CDK8 or CDK19 significantly restricted viral replication. Both within the context of DENV2 infection and with non-viral stimulation of innate immunity, we identified a role for CDK8 and CDK19 as regulators of the type I interferon response. CDK8 and CDK19 have distinct and overlapping functions as regulators of IFN-β expression dependent on the nature of the stimulus. This work not only furthers our understanding of host transcriptional regulation during DENV2 infection and within innate immunity, but also the diverse and complex functions of CDK8 and CDK19 as key modulators of cellular stress responses.Item Open Access Integrating p300 functions in HTLV-1 transcription initiation(Colorado State University. Libraries, 2014) Luebben, Whitney R., author; Nyborg, Jennifer K., advisor; Stargell, Laurie, committee member; Laybourn, Paul, committee member; Prenni, Jessica, committee member; Quackenbush, Sandra, committee memberThe HTLV-1 provirus overcomes a repressive chromatin environment for efficient transcription of its genome. This is accomplished by the robust recruitment of the coactivator protein, p300, to the viral enhancer sites through interactions with DNA bound pCREB and the viral transactivating protein, Tax. Recruitment of p300 to the HTLV-1 promoter results in histone acetylation and nucleosome depletion from the promoter region in the presence of the histone chaperone, Nap1. To study the histone acetylation requirements for Nap1-dependent nucleosome disassembly, we utilized immobilized in vitro assembled chromatin templates containing site specific K→R mutations within the N-terminal tails of the histones. Through these studies, we identified histone H3, lysine 14 as the functionally relevant acetylation site for Nap1-dependent nucleosome disassembly. Additionally, we found a significant correlation between nucleosome disassembly from the HTLV-1 promoter and acetylation-dependent transcription activation. These studies suggest that nucleosome disassembly is a prerequisite for transcription activation, as nucleosome disassembly creates a nucleosome free region within the HTLV-1 promoter, allowing for the subsequent recruitment of Pol II and general transcription machinery for activation of transcription. The identification of a single and specific acetyl-lysine residue led us to the hypothesis that the p300 acetyl-lysine binding domain (bromodomain) was involved in HTLV-1 transcription activation through recognition of H3K14ac. To test this hypothesis, we utilized a p300 bromodomain deletion mutant and a CBP/p300 specific bromodomain inhibitor, (SGC-CBP30), to investigate the involvement of the p300 bromodomain in HTLV-1 transcription activation. Importantly, we found that the p300 bromodomain is not involved in the initial recruitment of the coactivator to the chromatin template as previously proposed, rather the bromodomain functions after recruitment to the promoter and following acetylation of the histone tails. These findings are consistent with a role for the p300 bromodomain in nucleosome disassembly and uncover a novel function for the bromodomain in gene activation.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 Pseudotyping of lentiviral vector with novel vesiculovirus envelope glycoproteins derived from Chandipura and Piry viruses(Colorado State University. Libraries, 2016) Hu, Shuang, author; Akkina, Ramesh, advisor; Quackenbush, Sandra, committee member; Aboellail, Tawfik, committee member; Ryan, Elizabeth, committee memberLentiviral vector system is widely used in gene therapy. Although the envelope glycoprotein of vesicular stomatitis virus (VSV-G) has been mostly used to pseudotype lentiviral vectors, its disadvantages such as low transduction levels in certain cell types and sensitivity to inactivation by human complement hinders the usage of VSV-G pseudotyped lentiviral vectors in some cells or its direct in vivo clinical application. Aiming at overcoming some of these drawbacks of VSV-G, we evaluated two novel vesiculovirus envelope glycoproteins from Chandipura virus and Piry virus (CNV-G and PRV-G), as alternatives to VSV-G. Our results showed that pseudotyped lentiviral vectors could be generated with both these envelopes with high titers and stabilities similar to VSV-G. While displaying a more selective tropism than VSV-G, both CNV-G and PRV-G pseudotypes were found to be efficient in transducing a variety of cell types that include neuronal, fibroblastic and epithelial cells from across different species in addition to a number of human T-lymphocyte cell lines in vitro. Additionally, both the novel pseudotypes were found to be more resistant to human sera inactivation than the VSV-G pseudotype, thus providing better candidates for systemic administration. These data, taken together, establish that both Chandipura and Piry viral glycoproteins are suitable alternative candidates for lentiviral vector pseudotyping with an additional advantage for potential in vivo use in various gene therapy-based applications.Item Open Access Sindbis virus usurps the cellular HUR protein to stabilize its transcripts and promote infections of mammalian and mosquito cells(Colorado State University. Libraries, 2010) Sokoloski, Kevin J., author; Wilusz, Jeffrey, advisor; Wilusz, Carol, advisor; Blair, Carol, committee member; Peersen, Olve, committee member; Quackenbush, Sandra, committee memberMembers of the genus Alphavirus are recognized as significant human pathogens. Infection of vertebrate hosts often results in febrile illness and occasionally severe encephalitis. The archetypical alphavirus is Sindbis virus, which we have utilized in these studies. The genomic and subgenomic RNAs of Sindbis virus strongly resemble cellular mRNAs as they are capped at their 5’ ends and polyadenylated at their 3’ termini. These features allow the viral RNAs to act like cellular mRNAs and make them prime substrates for the cellular mRNA decay machinery. Sindbis virus RNAs are indeed subject to degradation by the cellular mRNA decay machinery in cell culture models of infection. Nevertheless, they decay by a mechanism that is different from the majority of cellular mRNAs as the decay of Sindbis virus transcripts is predominantly deadenylation-independent. As cellular mRNAs are often regulated by elements present in their 3’ untranslated regions (UTR), we hypothesized that these viral 3’UTR elements were functioning similarly to cellular mRNA stability elements resulting in the enhancement of viral infection. The primary goal of the research described in this dissertation was to characterize in mechanistic detail how the Sindbis virus 3’UTR represses deadenylation. To this end we used both cell free extracts and tissue culture systems to assay the effects of the viral 3’UTR on transcript stability. Interestingly, multiple elements were found to be independently repressing deadenylation in mosquito cytoplasmic extracts. Further examination revealed that a major stability determinant was the U-rich element (URE) observed in the 3’UTR of many alphaviruses. The ability to repress deadenylation in our cell free extract system was similarly observed with the UREs of Venezuelan equine, eastern equine, western equine and Semliki Forest viruses. Taken together, these data strongly assert that the repression of deadenylation via the URE is evolutionarily conserved. Prior to this study, the URE had no ascribed function. The repression of deadenylation imparted by the URE correlated with the binding of a cellular 38kDa factor. This 38kDa factor was determined to be the cellular HuR protein. Both the human and mosquito HuR proteins were found to bind with high affinity to the Sindbis virus 3’UTR. Reduction of cellular HuR protein levels using RNAi resulted in an increase in the rate of viral RNA decay. Furthermore, a significant decrease in the titer of progeny virus was observed. A similar effect on viral titer was observed when the predominant HuR binding site, the URE, was deleted from the viral 3’UTR. Taken together these observations identify a novel Alphavirus/ host interface that significantly impacts viral biology. Furthermore these studies have confirmed our hypothesis that the members of genus Alphavirus have indeed evolved RNA stability elements that resemble cellular mRNA stability elements for the purpose of enhancing viral infection. Furthermore these studies identify a potential therapeutic anti-viral target - the cellular HuR protein.Item Open Access Understanding regulation of HIV-1 protease precursor autoprocessing(Colorado State University. Libraries, 2019) Tien, Chih-Feng, author; Chen, Chaoping, advisor; Bamburg, James, committee member; Peersen, Olve, committee member; Quackenbush, Sandra, committee memberThe HIV-1 protease (PR) is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. Protease autoprocessing is generally referred to proteolytic reactions catalyzed by the precursor itself leading to liberation of free, mature PR in a highly regulated manner. We study the precursor autoprocessing mechanism using engineered fusion precursors carrying the p6*-PR miniprecursor sandwiched between various proteins and/or epitope peptides expressed in transfected mammalian cells. The studies reported here examined and identified factors involved in regulation of precursor autoprocessing. Modulation of precursor autoprocessing activity and outcomes by the 26 amino acid maltose binding protein signal peptide (SigP) mimicking the proviral constructs. A H69D mutation in PR abolished autoprocessing of SigP-containing fusion precursors or Gag processing in viral particles whereas it only partially suppressed autoprocessing of fusion precursors lacking SigP. The mature PRs released from SigP-carrying precursors or associated with the viral particles are both resistant to self-degradation whereas those released from SigP-lacking fusion precursors are prone to self-degradation. Furthermore, the PR-containing autoprocessing intermediate fragments released from a SigP fusion precursor or a proviral constructs showed protease inhibitor response profiles distinct to those released from the corresponding fusion precursor lacking SigP. These findings of context-dependent modulation reveals the complexity of precursor autoprocessing regulation that most likely accompanies sequence variation imposed by the evolution of the upstream Gag moiety. We also examined trans proteolysis for its functional correlation with precursor dimerization. Fusion enzymes carrying GST, a well-known dimer forming protein, processed the GST-fused substrate in trans as expected. Interestingly, positive trans processing was also detected between enzyme and substrate precursors carrying maltose binding protein (MBP), a known monomeric tag, or lacking any dimer-inducing tag, suggesting that a dimer-inducing flanking tag is not required for trans proteolysis in the transfected cells. Sucrose gradient sedimentation analysis detected dimeric substrates, with or without dimer-inducing GST, as the major complexes in transfected cell lysates. In the presence of a protease inhibitor (PI) at high enough concentrations, dimeric enzymes were predominantly detected. Without PI treatment, fusion enzymes with different tags and varied p6* sequences showed monomers or dimers or mixtures, suggesting modulation of enzyme dimerization by p6* peptides and flanking tags. Precursors carrying two PRs in tandem tethered by a GGS linker demonstrated higher propensities of forming inter-molecular dimers than intra-molecular dimers, indicating a role of p6* peptide in regulating precursor dimerization. Collectively, our results decoupled the requirement of a dimer-inducing tag upstream of the p6*-PR miniprecursor for precursor trans proteolysis and demonstrated elements within and beyond p6*-PR miniprecursor that collectively influence precursor dimerization, which revealed additional complexity involved in precursor autoprocessing regulation. In summary, this dissertation highlights complicated regulations and more than one productive pathway involved in HIV-1 protease precursor autoprocessing.