Mountain Scholar :: Browsing by Author "Chen, Chaoping, committee member"

Browsing by Author "Chen, Chaoping, committee member"

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Open Access
Biophysical, structural, and functional studies of histone binding proteins
(Colorado State University. Libraries, 2010) Sudhoff, Keely B., author; Luger, Karolin, advisor; Chen, Chaoping, committee member; Henry, Charles, committee member; Woody, Robert, committee member; Hansen, Jeffrey C., committee member
Eukaryotic genomes are extensively compacted with an equal amount of histone proteins to form chromatin. A high level of control over chromatin structure is required to regulate critical cellular processes such as DNA replication, repair, and transcription. To achieve this feat, cells have developed a variety of means to locally or globally modulate chromatin structure. This can involve covalent modification of histones, the incorporation of histone variants, remodeling by ATP-dependent remodeling enzymes, histone chaperone-mediated assembly/disassembly, or any combination of the above activities. To understand how chromatin structure is affected by histones, it is essential to characterize the interactions between histones and their associated proteins. In Saccharomyces cerevisiae, the multi-subunit SWR1 complex mediates histone variant H2A.Z incorporation. Swc2 (Swr1 complex 2) is a key member of the SWR1 complex and is essential for binding and transfer of H2A.Z. Chz1 (Chaperone for H2A.Z/H2B) can deliver H2A.Z/H2B heterodimers to the SWR1 complex in vitro. Swc2 1-179 (a domain of Swc2 that retains histone binding and the apparent preference for variant dimers) and Chz1 are intrinsically disordered, but become more ordered upon interaction with histones. Quantitative measurements done under physiological in vitro conditions demonstrate that Chz1 and Swc2 1-179 are not histone variant-specific. They bind to histones with an affinity lower than that of previously described histone chaperones, and lack the ability to act on nucleosomes or other histone-DNA complexes. Small-angle X-ray scattering demonstrates that the intrinsic disorder of the proteins allows them to adopt a multitude of structural states, perhaps facilitating many different interactions and functions. We show that Swc2 1-179, despite its overall acidic charge, can bind double stranded DNA, in particular, 3-way and 4-way junction DNA. These junctions are thought to mimic the central intermediates found in DNA damage repair. This characteristic is unique to Swc2 1-179. Consistent with this unexpected activity, yeast phenotypic assays have revealed a role for SWC2 in DNA damage repair, as indicated by sensitivity to DNA damaging agent methane methylsulfonate. Importantly, our data has exposed a novel role for Swc2 in DNA damage repair. In an independent study, we investigated the histone chaperone Vps75, a Nap1 homolog. Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone H3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. We show that deletion of VPS75 results in dramatic and diverse mutant phenotypes, in contrast to the lack of effects observed for the deletion of NAP1. The flexible C-terminal domain of Vps75 is important for the in vivo functions of Vps75 and modulates Rtt109 activity in vitro. Our data highlight the functional specificity of Vps75 in Rtt109 activation.
Open Access
Determination of the functions of Rab32, Rab38, and their effector Myosin Vc in the biogenesis of melanosomes
(Colorado State University. Libraries, 2013) Bultema, Jarred, author; Di Pietro, Santiago, advisor; Ross, Eric, committee member; DeLuca, Jennifer, committee member; Chen, Chaoping, committee member; Reddy, Anireddy, committee member
In mammals, pigment produced within specialized cells is responsible for skin, hair, and eye coloration. Melanocytes are specialized cells that produce pigment within an organelle known as the melanosome. Melanosomes are a member of a specialized class of organelles, known as Lysosome-related organelles (LRO), which are responsible for a number of critical functions in mammals such as pigmentation, blood clotting, lung function, and immune function. LROs are related to the ubiquitous lysosome, and are formed using the same molecular mechanisms as lysosomes that rely upon the Adaptor Protein complexes -1 (AP-1) and -3 (AP-3), and the Biogenesis of Lysosome-related Organelles Complex (BLOC)-2 (BLOC-2). These protein complexes are critical for the trafficking of specialized cargoes to melanosomes required for proper melanin synthesis. But, these complexes are also used for the formation of lysosomes, and no mechanism is known to distinguish between trafficking to lysosomes and melanosomes. The melanosome serves as a model system to study the formation of LROs, and insights from the study of melanosomes help explain the biogenesis of other LROs. In this dissertation, I present the finding that Rab32 and Rab38 function as melanosome-specific trafficking factors that allow for the use of AP-3, AP-1, and BLOC-2 in melanosome biogenesis. Using biochemical approaches, I show that Rab32 and Rab38 bind directly to AP-3, AP-1, and BLOC-2 on membranes. In microscopy experiments, I demonstrate that Rab32 and Rab38 localize to early endosomal subdomains where AP-3, AP-1, and BLOC-2 function. Using a combination of biochemical and microscopic approaches, I show that Rab32 and Rab38 serve partially redundant functions in trafficking of specialized cargoes to melanosomes. I report the discovery that Myosin Vc, a class V myosin motor, interacts with Rab32 and Rab38 and serves novel functions in melanosomes trafficking. I show, using biochemical approaches, that Myosin Vc directly binds to several melanosomal Rab proteins and serves as an effector of these proteins in melanosome biogenesis. Using a combination of approaches, I demonstrate that depletion of Myosin Vc from melanocyte cells causes defects in the trafficking of cargoes to melanosomes, but also causes severe defects in the secretion of mature melanosomes. With biochemical and microscopic approaches, I compare the function and localization of Myosin Vc in melanocytes to related proteins Myosin Va and Myosin Vb, and provide evidence to suggest that all three of these proteins function in distinct steps of melanosome trafficking. My results answer outstanding questions about the use of ubiquitous trafficking machinery (AP-3, AP-1, and BLOC-2) in trafficking to a specialized organelle. I provide evidence to answer outstanding questions about the mechanism of action of Rab32 and Rab38 in melanosome trafficking through my studies with Myosin Vc. I also establish new areas of research in the comparison of Myosin Va, Myosin Vb, and Myosin Vc in melanosome trafficking. My results address numerous unknown areas in melanosome biogenesis, expand the knowledge of melanosome biogenesis, and provide numerous new avenues of research to explore to understand specialized trafficking to LROs.
Open Access
Development and efficacy testing of broad alphavirus vaccines and antivirals and characterization of alphavirus neuroinvasion
(Colorado State University. Libraries, 2016) Rico, Amber, author; Olson, Ken, advisor; Ebel, Greg, committee member; Powers, Ann, committee member; Bowen, Richard, committee member; Chen, Chaoping, committee member
Alphaviruses are mosquito-borne pathogens that cause worldwide disease and death in humans and animals. Several alphaviruses are select agents and are a legitimate biosafety and bioweapon concern. Additionally, several alphaviruses are emerging infectious diseases. Climate change and urbanization have expanded mosquito populations and increased human-mosquito interactions within this decade and will continue into future decades. As mosquito populations expand, naïve human populations are exposed to arthropod-borne viruses, including alphaviruses, and vector-borne diseases have surged. The increasing prevalence of arthropod-borne disease has highlighted the global need to develop measures that prevent or treat arthropod-borne disease infection. Currently, vaccines to prevent alphavirus infection are limited to investigational new drug status and no therapeutics are available to treat alphavirus disease. This dissertation will describe projects aimed at preventing or treating alphavirus infection and characterizing the process of alphavirus neuroinvasion. To address the concern of potential outbreaks of an intentional or natural nature, alphavirus vaccines based on the ectodomain of alphavirus E1 were designed and tested. Cationic liposomes complexed with nucleic acid adjuvants and alphavirus E1 protein (lipid-antigen-nucleic acid complexes; LANACs) provided the best platform for alphavirus E1 vaccination. Interestingly, western equine encephalitis virus (WEEV) E1 (LANAC WEEV E1) protected against both mouse WEEV and eastern equine encephalitis virus (EEEV) challenge but not Venezuelan equine encephalitis virus (VEEV); whereas, VEEV E1 (LANAC VEEV E1) protected against both VEEV and EEEV challenge but not WEEV. LANAC VEEV E1 + WEEV E1 vaccination protected mice against EEEV, VEEV, and WEEV challenge. Mice immunized with LANACs (LANAC WEEV E1, LANAC VEEV E1 or LANAC VEEV E1 + WEEV E1) mounted strong humoral immune responses, but were lacking neutralizing antibody. Hamsters immunized with LANAC WEEV E1 failed to mount humoral immune responses and were not protected from challenge. Antibody derived from E1 vaccination binds infected cells and purified E1, but not intact virions. E1 antibody is non-neutralizing yet protective against CHIIKV, EEEV, SINV, VEEV, and WEEV in vitro. In vivo we have demonstrated that antibody is protective against all three new world alphaviruses (NWAs0. Antibody affects late stages of the viral life cycle and likely inhibits virus release or cell death. Following a screen, conducted by our collaborators, of FDA approved and ex-US approved compounds for effectiveness against viral encephalitic and hemorrhagic fever viruses, we tested compounds for anti-alphavirus activity to develop therapeutics to treat alphavirus infection. Yield reduction assays identified four compounds that inhibited virus replication by two to four logs. These compounds were further tested in vitro for mechanism of action. Compounds P-75802, P-75803, P-75805, and P-75811 affected early stages of replication. Compound P-75802 was tested in vivo and was found to possess limited antiviral activity. The process of alphavirus neuroinvasion, particularly following peripheral inoculation, is poorly defined. In the studies described here, encephalitic alphavirus neuroinvasion following EEEV, VEEV, and WEEV intranasal and subcutaneous inoculation were described relying on bioluminescent in vivo and ex vivo imaging, CLARITY imaging, and histopathology. We found that neuroinvasion occurs through two routes dependent on inoculation method. The cranial nerves, particularly the olfactory tract nerve, were involved in neuroinvasion following intranasal inoculation. Neuroinvasion from the periphery, footpad inoculation, occurred via a route independent of the olfactory tract. Neuroinvasion occurred in areas where the blood brain barrier is naturally absent including the hypothalamus, anterioventral third ventricle region (AV3V), area postrema, and the pineal body.
Open Access
Discovery and characterization of the SLAC complex and its role in actin polymerization during clathrin-mediated endocytosis
(Colorado State University. Libraries, 2013) Feliciano, Daniel, author; Di Pietro, Santiago, advisor; Bamburg, James, committee member; Curthoys, Norman, committee member; Chen, Chaoping, committee member; Reist, Noreen, committee member
Endocytosis is the process by which cells control the lipid and transmembrane protein compositions in order to comply with certain requirements essential for cellular function. The different forms of endocytosis provide the cell with a discriminatory system where specific cargoes are selected, packed and internalized when there is a particular physiological demand. Given the importance of endocytic internalization routes for a variety of cellular processes, it is not surprising that defects in the protein machinery involved in these pathways leads to pathologies. Examples of some metabolic disorders associated to defects in adaptor or receptor function include autosomal recessive and familial hypercholesterolemia. In other cases, mutations in actin regulatory proteins, such as WASp, can cause many blood disorders that include primary immunodeficiency and thrombocytopenia. Clathrin-mediated endocytosis (CME) is a fundamental pathway conserved from yeast to humans that proceeds by forming a clathrin coat at the plasma membrane followed by the recruitment of proteins that promote membrane curvature, actin polymerization, and scission. CME is the mayor route for nutrient uptake, distribution of membrane components, and receptor internalization. During CME, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. Las17 (WASp) is the strongest activator of the Arp2/3 complex in yeast cells, it is not autoinhibited, and arrives to endocytic sites 20 seconds before actin polymerization begins. One of the most outstanding questions in the field has been how Las17 is inhibited during the initial 20 seconds after its arrival to sites of endocytosis. In this dissertation, the discovery and characterization of a stable complex between Las17 and the clathrin adaptor Sla1 is described, in which Las17 is inhibited. This interaction is direct, multivalent, and strong, and was mapped to novel Las17 polyproline motifs that are simultaneously class I (RxxPxxP) and class II (PxxPxR). In vitro pyrene-actin polymerization assays established that Sla1 inhibition of Las17 activity depends on a new class I/II Las17 polyproline motifs. The inhibition is based on competition between Sla1 and monomeric actin for binding to sequences comprising a novel G-actin binding site in Las17 that is also characterized. The Las17 novel G-actin binding module 1 (LGM1) requires two sets of arginine-rich sites for normal Las17 function in vitro and in vivo. Furthermore, live cell imaging showed the interaction with Sla1 is important for normal Las17 recruitment to endocytic sites, its inhibition during the initial 20 seconds, and for efficient endocytosis. Within this complex, Las17 requires full length Bzz1, a membrane tubulation protein, for its activation in vitro through a mechanism that does not depend on complex dissociation. Since Sla1 and Las17 regulate actin polymerization during clathrin-mediated endocytosis, this complex has been named SLAC. The discovery and characterization of the SLAC complex help to define the negative and positive mechanisms regulating Las17 activity and answer one of the most outstanding questions in the field. This work also sets the stage to decipher the roles of other WASp homologues in mammalian cells. Overall, findings reported here advance our understanding of the regulation of actin polymerization by Las17 during clathrin-mediated endocytosis.
Open Access
DNA repair proteins Metnase and PARP1 regulate DNA integration
(Colorado State University. Libraries, 2015) Nie, Jingyi, author; Nickoloff, Jac A., advisor; Bailey, Susan M., committee member; Liber, Howard L., committee member; Chen, Chaoping, committee member
DNA integration occurs naturally in various formats and plays important roles in evolution. DNA integration also affects human and animal health. Various genome-editing tools have been developed based on site-specific DNA integration. In mammalian cells, DNA integration is largely random. The mechanism of random DNA integration is not fully understood but has close association with repair of double-strand DNA damage. There are two major pathways to repair double-strand breaks (DSBs), homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, NHEJ occurs more frequently than HR, possibly explains why random integration is more efficient than homology-directed integration or gene targeting. Proteins function in DSB repair pathways often engage in DNA integration. Metnase is a fusion protein that only expresses in higher primates, including humans. Metnase contains a SET methyltransferase domain and a transposase domain. Metnase promotes efficiency and accuracy of NHEJ and promotes DNA integration. The SET domain dimethylates histone H3K36 at DSB sites, and the transposase domain binds to the human Mariner transposon Hsmar1 terminal inverted repeat (TIR) sequence specifically. Both domains have been shown to be important for the role of Metnase in NHEJ. In this study, we tested the role of Metnase in promoting plasmid integration. We hypothesized that Metnase promotes plasmid integration through its functions in the NHEJ pathway. Metnase enhances the efficiency and accuracy of NHEJ, we predict that overexpression of Metnase can prevent integrating plasmid and genomic DNA at integration sites from large deletions. Besides, if the specific TIR binding of Metnase can direct more DNA integration into the TIR sequence in the human genome, overexpression of Metnase would increase the ratio of DNA integration found at or nearby TIR region. To test this hypothesis, we mapped plasmid integration in the human cell line HEK293T at low and high levels of Metnase expression. Our results demonstrated that Metnase promotes plasmid DNA integration independently of TIR sequence in the human genome. Overexpression of Metnase suppressed microhomology-mediated DNA integration, supporting our hypothesis that Metnase promotes DNA integration through classical NHEJ (cNHEJ). In contrast to cNHEJ, alternative NHEJ (aNHEJ) utilizes a different set of core proteins to rejoin broken ends. Compared to cNHEJ, aNHEJ is more error-prone and considered as the major generator of chromosomal translocations. Initiation of aNHEJ requires end resection. PARP1 plays an important role in initiating aNHEJ by recruiting end resection factors to DSBs. PARP1 has also been shown to promote DSB-induced chromosomal translocations. Based on the structural similarity between chromosomal translocations and DNA integration, we hypothesized that PARP1 may promote a sub-set of DNA integration, possibly through aNHEJ. We tested the effects of two PARP1 inhibitors PJ34 and Olaparib on DNA integration. Surprisingly, the two inhibitors showed opposite effects on DNA integration. PJ34 suppressed DNA integration, while Olaparib promoted DNA integration. We then confirmed PARP1 promoted DNA integration in a stable PARP1 knockdown cell line. Future studies are needed to understand the engagement of PARP1 in DNA integration and interpret the result where Olaparib promotes DNA integration.
Open Access
Evaluation of novel therapeutics for HIV prevention and treatment in a humanized mouse model
(Colorado State University. Libraries, 2011) Neff, Charles Preston Tagg, author; Akkina, Ramesh, advisor; Aboellail, Tawfik, committee member; Callahan, Gerald, committee member; Chen, Chaoping, committee member; Garrity, Deborah, committee member
In the absence of an effective HIV-1 vaccine finding alternative therapeutics and preventative methods has become essential. In this regard preventative approaches such as pre-exposure chemo-prophylaxis that employ either topical applied microbicides or systemically administered anti-retroviral drugs show great promise. In these studies, we evaluated two new classes of clinically approved drugs with different modes of action namely, an integrase inhibitor raltegravir and a CCR5 inhibitor maraviroc as potential systemically and topically applied pre-exposure chemo-prophylaxis. Additionally, therapeutic strategies designed to combat HIV/AIDS using siRNAs show considerable promise. However, targeted delivery of these synthetic molecules into infected cells in vivo has been a formidable challenge. In addressing this need we sought to evaluate the efficacy of a chimeric construct consisting of an HIV-1 gp120 specific aptamer with viral neutralization capacity fused to a siRNA with proven efficacy against tat/rev viral transcripts. We also sought to evaluate the efficacy of structurally flexible, cationic PAMAM dendrimers as a siRNA delivery system. For these novel therapeutic strategies to succeed it is important to evaluate them in both in vitro and in vivo. The rhesus macaque has been a valuable research tool for comparative HIV-1 studies. However, aspects of this model render its usefulness limited considering its expensive nature and not utilizing HIV-1 itself. In this regard the recently developed humanized mouse model that permits multi-lineage human hematopoiesis is an excellent alternative to the non human primate model. To generate humanized mice, neonatal Rag2-/-yc-/- or Rag1-/-yc-/- mice were xenografted with human CD34+ hematopoietic stem cells, resulting in a model that can permit HIV-1 infection. Upon infection by HIV-1 chronic viremia develops with a subsequent loss of CD4 T cells. These mice also successfully mimic the predominant mode of HIV-1 transmission via the sexual vaginal route which also results in chronic viremia and helper T cell loss. Thus this small animal model permits the rapid preclinical evaluation of potential candidates for pre-exposure prophylactic (PrEP) efficacy as well as novel RNA-based therapeutics. Here we utilize these humanized mouse models to evaluate the PrEP efficacies of the drugs named above as well as the in vivo efficacy of siRNAs delivered by utilizing a chimeric aptamer construct or a PAMAM dendrimer. Our results showed that both of these approaches using either a chimeric aptamer or a PAMAM dendrimer resulted in suppression of viral loads in vivo and most importantly also resulted in protection from T-cell depletion, making these compounds attractive therapeutic candidates for the treatment of HIV-1 infection. Lastly, using the same humanized mouse model we also successfully tested a gene therapy strategy employing lentiviral vectors having RNA-based anti-HIV-1 constructs convey intracellular immunization against HIV-1 in vivo.
Open Access
Genomic characterization, detection and molecular evolution of arthropod-borne viruses of the family Bunyaviridae
(Colorado State University. Libraries, 2010) Lambert, Amy Julia, author; Blair, Carol D., advisor; Chen, Chaoping, committee member; Lanciotti, Robert S., committee member; Miller, Barry R., committee member; Black, William C., committee member
The genomic characterization, detection and evolution of arthropod-borne human pathogens and related viruses of the family Bunyaviridae are presented. This study began with the determination of primary nucleotide sequence data for a diversity of bunyaviruses of interest that were not previously characterized at the genetic level. Following molecular characterization, an RT-PCR strategy was designed, according to previously determined and newly derived nucleotide sequence data, to target S genomic segments of 47 viruses, including 29 arthropod-borne human pathogens, of the family Bunyaviridae. Following development, this strategy was used, in some instances with an expanded capacity for the detection of multiple segments of the bunyavirus genome, for the identification of arthropod-borne bunyaviruses of medical importance and novel circumstance at a global level. Phylogenetic analyses performed on nucleotide sequence data generated by these efforts facilitate an ancestral understanding of the discovered agents. Finally, advanced molecular and phylogenetic analyses of a subset of newly derived sequences are described here to facilitate an enhanced understanding of the evolution of Bunyaviridae.
Open Access
HIV prophylaxis: an essential role for T cells and adjuvants in recombinant mucosal Lactobacillus acidophilus vaccines
(Colorado State University. Libraries, 2016) LeCureux, Jonathan Spicer, author; Dean, Gregg, advisor; Aboellail, Tawfik, committee member; Chen, Chaoping, committee member; Zabel, Mark, committee member
Current HIV vaccines have poor efficacy, with inconsistent levels of protection following mucosal HIV exposure. Lactic acid bacteria offer an alternative vaccine vector targeting the primary site of HIV infection, the mucosa. In these studies we evaluated the immunogenicity of several strains of Lactobacillus acidophilus expressing HIV membrane proximal external region (MPER), a portion of HIV envelope that contains broadly neutralizing antibody binding sites. We evaluated MPER-only expressing strains along with strains expressing adjuvants (interleukin-1β or flagellin) to improve immunogenicity against the HIV MPER. We compared the adjuvant strains to the MPER-only strain by oral administration in BALB/c mice to observe these improvements, and in CD40L-/- mice to observe if T cell help was necessary. Some BALB/c animals were also placed on a modified diet supplemented with prebiotic rice bran to observe any influence on vaccine immunogenicity. Resulting antibody responses and interleukin-17 levels were measured by ELISA, and T and B cell levels were measured by flow cytometry. Here we show that the addition of adjuvants, including dietary rice bran, to L. acidophilus vaccine strains improves their immunogenicity against HIV MPER. Our results indicate that anti-MPER IgG and IgA levels, as well as the number of anti-MPER antibody secreting cells, are improved with adjuvants, and that T cell help is required for an effective immune response. These results, combined with the many advantages offered by this lactic acid bacteria vaccine system make L. acidophilus an attractive vaccine vector for primate and human trials.
Open Access
HIV-1 Gag trafficking and assembly: mathematical models and numerical simulations
(Colorado State University. Libraries, 2013) Munoz-Alicea, Roberto, author; Liu, Jiangguo, advisor; Tavener, Simon, advisor; Chen, Chaoping, committee member; Mueller, Jennifer, committee member; Shipman, Patrick, committee member
AIDS (acquired immune deficiency syndrome) is an infectious disease that takes away many people's lives each year. Group-specific antigen (Gag) polyprotein precursor is the major structural component of HIV, the causing agent of AIDS. Gag is essential and sufficient for the formation of new HIV virus-like particles. The late stages of the HIV-1 life cycle include the transport of Gag proteins towards the cell membrane, the oligomerization of Gag near the cell membrane during the budding process, and core assembly during virion maturation. The mechanisms for Gag protein trafficking and assembly are not yet fully understood. In order to gain further insight into the mechanisms of HIV-1 replication, we develop and analyze mathematical models and numerical algorithms for intracellular Gag protein trafficking, Gag trimerization near the cell membrane, and HIV-1 core assembly. Our preliminary results indicate that active transport plays an important role for Gag trafficking in the cytoplasm. This process can be mathematically modeled by convection-diffusion equations, which can be solved efficiently using characteristic finite element methods. We employ differential dynamical systems to model Gag trimerization and HIV-1 core assembly. For the Gag trimerization model, we estimate relationships between the association and dissociation parameters as well as the Gag arrival and multimerization parameters. We also find expressions for the equilibrium concentrations of the monomer and trimer species, and show that the equilibrium is asymptotically stable. For HIV-1 core assembly, we first consider a model developed by Zlonick and others, which regards assembly as a polymerization reaction. We utilize theoretical and numerical tools to confirm the stability of the equilibrium of CA intermediates. In addition, we propose a cascaded dynamical system model for HIV-1 core assembly. The model consists of two subsystems: one subsystem for nucleation and one for elongation. We perform simulations on the nucleation model, which suggests the existence of an equilibrium of the CA species.
Open Access
Identification of Culex tarsalis D7 salivary protein and role of salivary protein vaccine on subsequent West Nile virus infection
(Colorado State University. Libraries, 2010) Reagan, Krystle Lynn, author; Blair, Carol D., advisor; Olson, Kenneth Edward, committee member; Foy, Brian D., committee member; Chen, Chaoping, committee member; Wang, Tian, committee member
Mosquito salivary proteins (MSPs) modulate the host immune response, leading to enhancement of arboviral infections. Identification of protein factors in saliva responsible for immunomodulation should lead to new strategies to prevent and protect against arboviral infection. D7 salivary proteins are among the most abundant in mosquito saliva, and they function as both vasodilators and suppressors of local inflammation. Here we identify D7 salivary proteins in Culex tarsalis, an important disease vector in the western United States. Recombinant D7 proteins were used to analyze the systemic and local immunomodulatory properties of the host. In this project, we immunized mice with recombinant D7 and tested for protection against subsequent challenge with West Nile virus (WNV) (NY99) delivered by mosquito bite. The vaccine was able to elicit a specific immune response. However, it enhanced WNV infection in the mouse model. We suggest that WNV enhancement is due to three factors. First, vaccinated mice had significant cellular infiltrates at the mosquito bite site, which included WNV permissive monocytes and dendritic cells. Increases in these cell populations at the mosquito bite site leads to an increase in initial viral infection and dissemination. Others have shown that higher peripheral viral levels lead to a worse disease outcome from infection. Secondly, mortality curves in infected mice receiving passive transfer of serum containing antibodies from vaccinated mice mimicked those from vaccinated animals. Antibody neutralization of mosquito salivary proteins that are critical in completing a successful blood meal may lead to increased probing time by the mosquito. Increased probing by the mosquito results in an increase in amount of saliva being deposited, therefore an increase in initial viral dose. Lastly, the cytokine profile observed in vaccinated mice showed an increase in the Th2 cytokine IL-4 and regulatory cytokine IL-10 and a decrease in Th1 cytokines such as IL-12p70 and IFNγ. A protective immune response to WNV includes high levels of Th1 cytokines. Production of IL-4 by mice that received the rD7 vaccine directly inhibits the Th1 cytokines necessary for protection. This work has increased our understanding of the complex nature of immunity to MSPs. Vector saliva vaccines have been successful in protecting against other blood feeding arthropods transmitted diseases. Nevertheless, differences in vector and pathogen physiology may preclude this approach from being successful for mosquito virus systems.
Open Access
Increasing dengue virus vaccine safety and immunogenicity by manipulating antigenic determinants of the flavivirus envelope protein
(Colorado State University. Libraries, 2010) Hughes, Holly Ruth, author; Blair, Carol D., advisor; Chang, Gwong-Jen J., advisor; Chen, Chaoping, committee member; Bowen, Richard Arnold, committee member
Dengue virus (DENV), which exists as four closely related serotypes, is a mosquito-borne pathogen causing significant global disease burden, either as classic dengue fever (DF) or in its most severe manifestation, dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). Severe dengue disease is often associated with secondary DENV infection and hypothesized to frequently be induced by cross-reactive, weakly neutralizing antibodies, a process referred to as antibody-dependent enhancement of infection (ADE). Due to the complex humoral immune response to DENV infection and the complexity of severe DENV disease, currently no licensed DENV vaccine is available. The goal of this dissertation is to increase the safety and immunogenicity of DENV vaccination through a better understanding of the antigenic properties of the envelope protein. To these ends the objectives of this research were to: 1) use site-directed mutagenesis and monoclonal antibody mapping to identify possible cross-reactive epitopes of the DENV-2 E protein which could contribute to ADE (Chapter 2), 2) use B cell epitope modification to construct a serotype-specific DENV-2 vaccine with reduced potential of vaccine-induced ADE (Chapter 3), and 3) identify potential dominant T cell epitopes in West Nile virus E which could act as immunological adjuvants for DENV-2 vaccines (Chapter 4). Humoral immune responses to DENV infection are complex and can exacerbate pathogenicity, yet are essential for immune protection. DENV-2 E protein epitope-specific antigens were created and used to measure immunoglobulin responses to three distinct epitopes in serum samples from DENV-2 infected humans. Immunoglobulin responses to DENV-2 infection exhibited significant levels of individual variation. Antibody populations targeting broadly cross-reactive epitopes centered on the fusion peptide in structural domain II were large, highly variable, and greater in primary than in secondary sera from DENV-2 infected patients, confirming previous studies and identifying the fusion peptide as an immunodominant epitope. E protein domain III cross-reactive immunoglobulin populations were similarly variable and much larger in IgM than in IgG. DENV-2 specific domain III IgG formed a very small proportion of the antibody response, yet was significantly correlated with DENV-2 neutralization, suggesting that the highly protective IgG recognizing this epitope in murine studies plays a role in humans as well. These results begin to tease apart complex humoral immune responses to DENV infection and thus are important for improving our understanding of dengue disease and immunological correlates of protection relevant to DENV vaccine development and testing. DENV vaccines must induce a balanced protective immunity to all four serotypes to reduce the possibility of cross-reactive antibody induced severe disease upon subsequent infection. By modification of immunodominant B cell epitopes of E, cross-reactivity reduced (CRR) DENV-2 DNA vaccine candidates were developed and tested in mice for immunogenicity and potential reductions in developing vaccine induced ADE in mice. Unlike wild-type vaccine, CRR vaccine immunized mouse sera neutralized virus and did not enhance viral infection in vitro. Thus, reducing cross-reactivity in the envelope glycoprotein of DENV may provide a solution to increase vaccine safety and resolve the long-standing obstacle of immune enhancement in dengue vaccine development. DNA vaccination is a highly pursued vaccine platform for its safety, stability, and ease of development, use and manufacturing. However, DNA vaccination is hindered by lower immunogenicity. Methods investigated to increase the immunogenicity of DNA vaccines have included the use of immunological adjuvants; however, few are approved for human use. A dominant, naturally occurring CD4 T cell epitope located in the transmembrane domain of West Nile virus was identified and its ability to increase the immunogenicity of heterologous flavivirus vaccines was investigated. The incorporation of the West Nile virus CD4 epitope into a DENV-2 DNA or protein vaccine significantly increased neutralizing antibody titers compared to parental vaccines without the CD4 epitope. Identifying differential antigenic properties of vaccines may alleviate concerns of imbalanced immunity associated with multivalent vaccinations.
Open Access
Lipid raft signaling in cofilin-actin rod formation induced by amyloid-β and TNFα
(Colorado State University. Libraries, 2012) Mi, Jianjie, author; Bamburg, James R., advisor; Chen, Chaoping, committee member; Partin, Kathryn M., committee member
Rod-like inclusions (rods), composed of actin saturated with cofilin, are induced in neurons by energetic and oxidative stresses, excitotoxic levels of glutamate, and amyloid beta treatment. Cofilin is an F-actin assembly regulatory protein critical to various actin-dependent processes, such as cytokinesis, cell migration, and neurite formation. Overexpression or hyperactivation (excessive dephosphorylation) of cofilin coupled with its oxidation can lead to formation of rods. Rods represent a likely mechanism to explain the synaptic loss associated with early stages of Alzheimer's disease (AD) and thus represent a novel target for therapeutic intervention. In live neurons, the study of cofilin-actin rod formation induced by specific mediators of stress has been limited because overexpression of fluorescent protein-tagged wild type (WT) cofilin results in formation of considerable numbers of spontaneous rods. A fluorescent cofilin mutant that could incorporate into induced rods but form no spontaneous rods even when overexpressed would offer a useful alternative for live-cell imaging. The R21Q mutant cofilin-RFP has been reported to not induce rods when overexpressed but incorporates into rods containing endogenous cofilin, thus serving as a rod marker in live cells. Here we show that expression of WT cofilin driven by promoters that result in a high or moderate steady-state level of exogenous protein produces a significant number of spontaneous rods, three to four fold over controls. However, R21Q cofilin-RFP expressed behind these same promoters will only incorporate into rods formed from endogenous protein, but not enhance spontaneous rod, even when accompanied by the photo stress induced by microscopic observation. Using the R21Q cofilin- RFP to measure rod formation, we then showed that the proinflammatory cytokine (TNFα) induced about a 3 fold increase in rod formation over untreated controls quantified either as the percent of neurons with rods (percent rod index) or as the number of rods per field (number rod index). Amyloid beta dimer/trimer (Aβd/t) induced about a 2.5 fold increase over controls in the percent of neurons with rods, and close to a 2 fold increase in the number of rods per field. To determine the fidelity of the R21Q cofilin-RFP in labeling all of the rods, we induced rods in control infected or R21Q cofilin-RFP expressing neurons with ATP depletion for 30 min, or with either Aβd/t (250 pM) or TNFα (50 ng/ml; 2.9 nM) for 24 h. Neurons were fixed and immunostained with a primary antibody for cofilin and an Alexa 647 nm-labeled secondary antibody. The percent of rods in RFP expressing cells that co-labeled with mRFP and Alexa 647 were then quantified. Although 100% of rods induced by ATP depletion co-labeled, surprisingly only 48% of the rods induced by TNFα co-labeled, similar to Aβd/t treatment. The reasons for this are not clear but taken together, our results demonstrate that R21Q cofilin-RFP can be used for a live cell marker for following induced rod formation but not as a quantitative measure of the total rod response. Induction of cofilin-actin rods by amyloid beta and TNFα is mediated by the cellular prion protein, a component of lipid raft domains which can signal to activate NADPH oxidase. Lipid rafts are cholesterol/sphingolipid enriched detergent resistant membrane domains in which many membrane receptors associate. Rafts can be visualized with an Alexa labeled cholera toxin B subunit which binds to GM1 ganglioside. Here we used neurons expressing R21Q cofilin-RFP to determine if rod formation is associated with coalesced lipid raft domains and if the coalesced lipid rafts form before or after rods are visible. In the three rods we visualized forming during the period in which lipid rafts were labeled we saw no lipid raft coalescence at sites of the newly formed rods. If we looked at the total R21Q cofilin-RFP labeled rods, about 45% of them co-localize with enlarged lipid raft domains. Thus results suggest that rods may bring about the reorganization of the membrane raft domains, although more data are required to make a definitive conclusion.
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 member
Dengue 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.
Open Access
Mathematical models for HIV-1 viral capsid structure and assembly
(Colorado State University. Libraries, 2015) Sadre-Marandi, Farrah, author; Liu, Jiangguo, advisor; Tavener, Simon, advisor; Chen, Chaoping, committee member; Hulpke, Alexander, committee member; Zhou, Yongcheng, committee member
HIV-1 (human immunodeﬁciency virus type 1) is a retrovirus that causes the acquired immunodeﬁciency syndrome (AIDS). This infectious disease has high mortality rates, encouraging HIV-1 to receive extensive research interest from scientists of multiple disciplines. Group-specific antigen (Gag) polyprotein precursor is the major structural component of HIV. This protein has 4 major domains, one of which is called the capsid (CA). These proteins join together to create the peculiar structure of HIV-1 virions. It is known that retrovirus capsid arrangements represent a fullerene-like structure. These caged polyhedral arrangements are built entirely from hexamers (6 joined proteins) and exactly 12 pentamers (5 proteins) by the Euler theorem. Different distributions of these 12 pentamers result in icosahedral, tubular, or the unique HIV-1 conical shaped capsids. In order to gain insight into the distinctive structure of the HIV capsid, we develop and analyze mathematical models to help understand the underlying biological mechanisms in the formation of viral capsids. The pentamer clusters introduce declination and hence curvature on the capsids. The HIV-1 capsid structure follows a (5,7)-cone pattern, with 5 pentamers in the narrow end and 7 in the broad end. We show that the curvature concentration at the narrow end is about five times higher than that at the broad end. This leads to a conclusion that the narrow end is the weakest part on the HIV-1 capsid and a conjecture that “the narrow end closes last during maturation but opens first during entry into a host cell.” Models for icosahedral capsids are established and well-received, but models for tubular and conical capsids need further investigation. We propose new models for the tubular and conical capsid based on an extension of the Caspar-Klug quasi-equivalence theory. In particular, two and three generating vectors are used to characterize respectively the lattice structures of tubular and conical capsids. Comparison with published HIV-1 data demonstrates a good agreement of our modeling results with experimental data. It is known that there are two stages in the viral capsid assembly: nucleation (formation of a nuclei: hexamers) and elongation (building the closed shell). We develop a kinetic model for modeling HIV-1 viral capsid nucleation using a 6-species dynamical system. Numerical simulations of capsid protein (CA) multimer concentrations closely match experimental data. Sensitivity and elasticity analysis of CA multimer concentrations with respect to the association and disassociation rates further reveals the importance of CA dimers in the nucleation stage of viral capsid self-assembly.
Open Access
Molecular determinants of dengue virus type-2 critical for early events in antibody-dependent enhancement of infection
(Colorado State University. Libraries, 2013) Chotiwan, Nunya, author; Blair, Carol, advisor; Huang, Claire, advisor; Chen, Chaoping, committee member
Antibody-dependent enhancement (ADE) of infection might be one of the major factors in the development of more severe forms of dengue disease in patients undergoing a secondary infection of dengue virus (DENV). The ADE is caused by cross-reactivity of subneutralizing or non-neutralizing antibodies (Abs), which form virus-Ab complexes and enhance virus infection by binding to the Fcγ receptors (FCγR) on FcγR-bearing cells. The early events in non-ADE infection have been previously studied, but the virus entry pathway and the DENV molecular determinants involved in ADE are still largely unclear. There are two hypotheses for the early entry pathway of ADE infection: (1) Ab opsonized DENV binds to FcγR and directly enters cells through phagocytic pathway; (2) FcγR plays an auxiliary role in concentrating the opsonized virus to the cell surface, but other cellular receptors are still required for virus entry into the endocytosis pathway. Herein, we investigated contributions of the DENV2 E proteins to ADE infection, as well as the role of FcγRIIA in the enhancement of infection and possible entry route of the Ab-opsonized virion. ADE of wild type (WT) DENV2 infection can be promoted in FcγRIIA-bearing K562 cells by using subneutralizing cross-reactive flaviviral monoclonal antibody (MAb), subcomplex DENV MAb or serotype-specific DENV2 MAb against the E protein, as well as non-neutralizing anti-DENV-prM MAb. The enhancement of infection was analyzed by comparing DENV2 infection under ADE conditions with DENV-Ab complex and non-ADE conditions with DENV alone. Numbers of DENV infected cells were determined by flow cytometry of infected cells stained with labeled MAb 2H2-AlexaFluor-488, while increases in viral output were quantified by qRT-PCR of viral genomes. We investigated multiple DENV mutants generated by reverse genetic technology to identify molecular determinants in the envelope protein (E) of DENV-2 that are critical for DENV attachment and viral-endosomal membrane fusion in both non-ADE and ADE infection of the virus in K562 cells. We determined that binding of virus-Ab complex with FcγRIIA alone is not sufficient for virus entry during ADE infection. Furthermore, the molecular determinants of E protein critical for virus entry and virus-mediated endosomal membrane fusion involved in non-ADE infection were also required for ADE infection. ADE was also tested in FcγRIIA-transfected CV-1 cells (CV-1- FcγRIIA). Surprisingly, no enhancement occurred with any of the tested MAb in this cell type. Numbers of cells expressing FcγRIIA and density of FcγRIIA molecules expressed on CV-1- FcγRIIA cell surface were similar to those of K562 cells. These results supported our findings that FcγRIIA alone was not sufficient to enhance viral infection. The results also suggested that cellular components that are present in K562 cells but absent in CV-1- FcγRIIA cells play a major role in ADE of infection.
Open Access
Molecular determinants of vector specificity in Highlands J virus
(Colorado State University. Libraries, 2014) Borland, Erin M., author; Quackenbush, Sandra, advisor; Powers, Ann, advisor; Chen, Chaoping, committee member
Highlands J Virus (HJV) is an understudied alphavirus that is closely related to western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV). HJV is not known to cause disease in humans or equids, but it is a known avian pathogen with potential to significantly disrupt commercial production flocks. These studies compared the sequences of multiple strains of HJV in order to better characterize and compare the range of available strains. Strain B230, the prototype strain, was used to compare all other strains tested. Strain 64A-1519 was most similar to B230 with 22 nucleotide substitutions, only 5 of which resulted in a change in amino acid residues. Strain WX3-2AP was the most divergent with 167 nucleotide changes resulting in 8 amino acid residue changes. Four distinct lineages were identified through phylogenetic analysis. Lineage 1 consisted of strains B230 and 64A-15191. Lineage 2 consisted of strains AB-80-9, RU-M-80-259, and 73V-2540. Lineage 3 consisted of strains W17791 and two GenBank strains (744-01 and 585-01). Strain WX3-2AP was the sole member of Lineage 4. Vector capacity studies for HJV in live Culex tarsalis mosquitoes have not been published prior to these experiments. Cx. tarsalis mosquitoes were orally infected with one of four strains of HJV: B230, 64A-1519, WX3-2AP, or AB-80-9. The heads and bodies of mosquitoes were separated and processed independently to assess viral presence by cytopathic effects (CPE). The experiments were run in duplicate and at different times to ensure accuracy of results. Two infection patterns emerged: Lineage 1 strains had low infection and dissemination rates at all three time points, while Lineage 2 and 4 strains had high infection and dissemination rates which were more similar to those previously published for WEEV Imperial strain in Cx. tarsalis. Virus titrations were performed on mosquito heads and bodies, and Lineage 4 strain WX3-2AP was found to have the highest average titers. Mosquito bodies had the highest average titer on day 8 post infection and average titers for bodies ranged from 6.60 to 7.26 log10 pfu equivalents/body. Heads had no discernable pattern among titers or strains, but titers ranged between 6.01 and 6.80 log10 pfu equivalents/head. Saliva was collected from mosquitoes infected with Lineage 2 strain AB-80-9 to assess the potential presence of a salivary gland barrier resulting in lack of transmission. Twenty-two of 49 mosquitoes tested transmitted detectible levels of virus, meaning 44.9% of orally infected mosquitoes were able to actively transmit the virus. While the titer of the saliva on a per mL basis was impossible to compute since the volume of saliva could not be quantified, the titers of the samples collected ranged between 1.68 and 5.81 log10 pfu equivalents/saliva sample. By combining the data obtained by sequencing the strains with the data from the mosquito infections, a single amino acid difference between the attenuated Lineage 1 strains and the more virulent Lineage 2 and 4 strains was identified as being potentially responsible for the differences in infectivity. The mutation was located at genome nucleotide position 8605, E2 glycoprotein amino acid 69. This change caused the non-polar glycine in the attenuated Lineage 1 strains to be replaced with an acidic glutamic acid in the more virulent Lineage 2 and 4 strains. Additional studies are needed to assess the in vivo effects of this amino acid change in Cx. tarsalis mosquitoes.
Open Access
Mutagenesis of the dengue virus envelope glycoprotein gene can significantly alter virus infectivity phenotypes in cultured cells and live mosquitoes
(Colorado State University. Libraries, 2011) Erb, Steven Michael, author; Blair, Carol D., advisor; Roehrig, John T., committee member; Olson, Kenneth E., committee member; Chen, Chaoping, committee member
The dengue virus (DENV) envelope (E) glycoprotein is the primary determinant for initiation of host cell infection. To date, studies investigating the contribution of DENV genetics to mosquito infection are limited. A infectious clone cDNA of DENV type 2 strain 16681 (30P-NBX) provided the ability to introduce site-specific amino acid (AA) mutations into the E protein. The results of the studies herein analyze the effects that AA mutations in the E protein have on infectivity of cultured cells and live mosquitoes. The ability of 30P-NBX to infect Aedes aegypti RexD strain mosquitoes after oral infectious blood-meal was investigated and showed that both 30P-NBX and the parent virus 16681 have low, but equivalent midgut infection rates (MIRs). Mosquito midgut infection with 30P-NBX is not affected by the virus titer in the blood-meal as long as titers are above 6 log 10 pfu/ml or 7 log 10 TCID 50 /ml. Additionally, multiple experimental repetitions with at least 20 mosquitoes per infectious blood-feed were required to obtain an accurate average MIR for 30P-NBX. Serial passage of 30P-NBX in RexD mosquito midguts identified a single AA mutation at position 122 in domain II of the E protein from lysine to glutamic acid that correlated with increased MIRs. Introduction of this AA mutation into the infectious clone (mutant virus K122E) reproduced the results from the serial passage experiment. Compared to 30P-NBX, K122E was not only shown to infect a higher proportion of mosquitoes as early as day 2 post blood-feed, but also to produce a disseminated infection in a higher proportion of mosquitoes by day 6 post blood-feed. Also, K122E consistently produced a midgut infection that spread throughout the entire tissue while 30P-NBX stayed restricted by comparison. Virus attachment to midgut cells was compared and showed that 30P-NBX and K122E could attach with equal efficiencies via our midgut-virus attachment assay. Additionally, incorporation of a single AA mutation into the infectious clone at E protein AA 120 from arginine to threonine significantly enhanced mosquito midgut infection compared to 30P-NBX. This is the first time that mosquito infection determinants have been identified in the DENV E protein. Amino acid mutations were engineered into the E protein on the lateral ridge of domain III, the fusion peptide at the distal end of domain II, and the molecular hinge region between domains I and II. Mutant virus phenotypes were analyzed in cell culture and live mosquitoes. In contrast to previous suggestions, domain III mutant virus phenotypes showed that the FG loop structure (previously suggested as a mosquito-specific infection determinant) and not the specific AA sequence is important for infection of mammalian cells and live mosquitoes, while the structure and sequence of the FG loop is dispensable for infection of cultured C6/36 cells. Additionally, mutations that remove positively charged residues from the A strand in DIII significantly attenuate infection of mosquitoes after oral infectious blood-meal and completely abrogate infection in mammalian cells. The results of this study suggest that there may be multiple structures in the E protein that are contributing to virus-receptor interactions. Viruses with mutations in the fusion peptide and hinge region of the E protein were intrathoracically (IT) inoculated into mosquitoes and showed variable infectivity phenotypes. All of the mutants except for one virus from both the fusion peptide and hinge region viruses attenuated infection of mosquito tissues outside the midgut. Importantly, considering that almost all of these viruses were able to replicate as efficiently as wild type in C6/36 cells, the IT inoculation results provide evidence that C6/36 cells are not a complete surrogate for DENV replication in mosquitoes.
Open Access
Novel therapeutics, associated adverse effects, and changes in immune responses during pulmonary infection with Mycobacterium tuberculosis
(Colorado State University. Libraries, 2024) Ali, Malik Zohaib, author; Gonzalez-Juarrero, Mercedes, advisor; Basaraba, Randall J., committee member; Moreno, Julie A., committee member; Chen, Chaoping, committee member
Patients diagnosed with multidrug resistant (MDR) or extensively drug resistant (XDR) tuberculosis (TB) have limited treatment options. The Nix-TB clinical trial evaluated a new 6-month regimen containing three-oral-drugs; bedaquiline (B), pretomanid (Pa) and linezolid (L) collectively termed as BPaL for the treatment of TB. This regimen achieved remarkable results as almost 90% of the participants suffering from MDR- or XDR-TB had favorable outcomes. Despite the extraordinary outcomes, many patients also developed severe adverse effects (AEs) which were associated with the long-term administration of the oxazolidinone protein synthesis inhibitor linezolid. Spectinamide 1599 (S) is also a potent protein synthesis inhibitor of Mycobacterium tuberculosis (Mtb) with an excellent safety profile, but which lacks oral bioavailability. In chapter 2, we hypothesized that inhaled spectinamide 1599, combined with BPa ––BPaS regimen ––, has similar efficacy to that of BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the BALB/c (permissive resistant) and C3HeB/FeJ (permissive susceptible) murine chronic TB efficacy models. Both regimens promoted similar bactericidal effects in lung and spleen of both models after 4 weeks. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL treatment also decreased myeloid to erythroid ratio and increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. During therapy both regimens improved the lung lesion burden, reduced neutrophil and cytotoxic T cell counts while increased the number of B and helper and regulatory T cells. This combined data suggests that inhaled spectinamide 1599 combined with BPa is an effective TB therapy that avoids L-associated AEs. The granuloma formation is the pathological hallmark of TB, and several studies suggest that there are temporal and spatial changes in their distinct immune responses. These changes differ not only from one granuloma to another in a single individual but also depend on the severity of the disease. In chapter 3, we attempted to understand longitudinal changes in immune cells, their relationships, and their spatial distribution in granulomas of Mtb infected BALB/c and C3HeB/FeJ mouse models using a novel technique of multispectral imaging microscopy. Multiplex fluorescence immunohistochemistry (mfIHC) is unique in its ability to provide both expression and location of several immune cells along with their co-localization in a single tissue section while preserving tissue architecture and spatial context. The results showed that as the infection progresses, there are also dynamic changes in the immune phenotypes forming the granulomas and those located within the parenchymal tissue. Moreover, the histologically similar granulomas manifested complexity in their immune cell composition mainly due to the presence of adaptive immune responses. The advanced cellular granulomas in BALB/c TB model were mainly predominated by CD4 and CD8 T cells, Ly6G stained neutrophils, B220 B cells and all these were surrounded by F4/80 macrophages. With time post infection, there was an increased uniform recruitment of CD4 and Foxp3 T cells, F4/80 macrophages and Ly6G neutrophils within granulomas compared to parenchymal tissue where IFNγ and IL-10 secreting cells were in abundance. Moreover, B220 B cells and CD8 T cells also showed increased but heterogeneous distribution among the advancing granulomas especially B220 B cells formed clusters. The spatial analysis showed an increased median distance for Ly6G neutrophils, whereas this distance was decreased for B220 B cells when measured from CD4 and CD8 cells. In summary, combining the spatial and temporal data in addition to the mere cell counts helps to uncover interactions and relationships between different immune cells within the granuloma.
Open Access
RNA replication by poliovirus RNA-dependent RNA polymerase: effects of residue 5 on elongation complex stability and processivity
(Colorado State University. Libraries, 2011) Hobdey, Sarah Elizabeth, author; Peersen, Olve B., advisor; Stargell, Laurie, committee member; Chen, Chaoping, committee member; Wilusz, Carol, committee member
The poliovirus (PV) RNA-dependent RNA polymerase (RdRP) is a small, single-subunit, enzyme that is responsible for the replication of the viral genome. PV genome replication is much more involved than just repetitively incorporating a single nucleotide into an oligonucleotide primer. The polymerase must first go through multiple steps of initiation before processive replication can happen. During primer-dependent initiation, the polymerase must bind the primer/template RNA substrate and undergo a conformational change to form a stable RNA-polymerase complex. After the stable RNA-polymerase complex is formed the polymerase undergoes a second conformational change associated with the addition of the first nucleotide to the primer. It is only after these few steps of initiation that the polymerase can begin processive elongation. The work presented in this dissertation addresses a structure-function relationship related to initiation and processivity of the PV RdRP. Specifically, my work shows that the PV RdRP residue 5 is involved in forming and maintaining the stable, elongation-competent complex. Also, the complex stability can be modulated by downstream RNA interactions or by the number of nucleotides that are incorporated to form the stable complex. Lastly, my data demonstrate evidence of an RNA rearrangement during elongation complex formation and show that the maintenance of a stable elongation complex is required for processive RNA replication, which is required for virus replication. To end, these data elucidate a more complete understanding of the structure-function relationships of the viral RNA polymerase and could eventually facilitate in the design of specific polymerase inhibitors.
Open Access
Stable kinetochore-microtubule attachment is sufficient to satisfy the spindle assembly checkpoint
(Colorado State University. Libraries, 2016) Tauchman, Eric Cary, author; DeLuca, Jennifer G., advisor; Chen, Chaoping, committee member; Ross, Eric, committee member; Wilusz, Carol, committee member
During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. The kinetochore proteins that bind spindle microtubules are exquisitely regulated to ensure correct segregation of genetic material at mitotic exit. Aurora B Kinase (ABK) phosphorylates Hec1, a protein that directly binds microtubules. This is critical for enabling the release of incorrect kinetochore-microtubule attachments. Hec1 has nine ABK phosphorylation sites on its tail domain allowing for precise control over binding affinity. We find that at least 7 of these sites are required for wild-type kinetochore-microtubule (K-MT) attachment stability as evaluated by inter-kinetochore distance measures and chromosome alignment capability. We further observe that several sites may have more influence on K-MT attachment stability than others. Hec1 mutations preventing phosphorylation increase kinetochore-microtubule attachment stability. In the absence of stable kinetochore–microtubule (K-MT) attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable Hec1mutant, a core component of the attachment machinery. This mutant reduced the ability of ABK to cause release of erroneous K-MT so we could test the hypothesis that stable K-MT attachments satisfy the SAC even if those attachments deviate from the canonical bipolar form. We find that stable attachments are sufficient to satisfy the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal. These results indicate a conformational change(s), within the kinetochore that occurs upon stable kinetochore-microtubule binding causes the eviction of SAC proteins. This advance in our understanding of SAC function offers insight into the mode of action and the variation in cellular response to mitotic arrest therapies often used in treatments of cancers.