Cell Biology & Molecular Genetics Theses and Dissertations
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Item TRANSLATION, REPLICATION AND TRANSCRIPTOMICS OF THE SIMPLEST PLUS-STRAND RNA PLANT VIRUSES(2024) Johnson, Philip Zhao; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plus (+)-strand RNA viruses are among the most common pathogens of plants and animals. Furthermore, they present model systems for the study of basic biological processes, including protein translation and RNA replication, and shed light on the versatile roles that RNA structures play in these processes. After cell entry, the next step in the (+)-strand RNA viral life cycle is translation of the viral genome to produce the viral RNA-dependent RNA polymerase (RdRp) and associated replication proteins necessary for viral replication to occur. For many (+)-strand RNA viruses lacking a 5´cap and 3´ poly(A) tail, translation depends upon RNA structural elements within their genomes capable of hijacking the host translation machinery, which for plant viruses are commonly located in their 3´ proximal regions and are termed 3´ cap-independent translation enhancer (CITE) elements. In Chapter 2, I report upon my work characterizing a new subclass of panicum mosaic virus-like translation enhancer (PTE) elements, which bind and co-opt for viral use the host translation initiation factor 4E (eIF4E) – the translation initiation factor normally responsible for binding and recognition of mRNA 5´caps during canonical eukaryotic translation initiation. Thus, PTE 3´CITEs present a novel mechanism for co-opting the critical host factor eIF4E. My work characterizing a new subclass of PTE 3´CITEs further revealed characteristics common among all PTE 3´CITEs pertaining to their mechanism of binding eIF4E.After translation of the necessary viral replication proteins, replication of the viral RNA occurs, which again is in large part mediated by RNA structural elements within the viral genome that can bind to the viral RdRp and/or host factors involved in viral replication. Indeed, RNA structural elements often serve dual roles in viral translation and replication and/or are located proximal to RNA structural elements involved in the alternate function. In Chapter 3, I discuss my work characterizing novel replication elements in the 3´ terminal regions of umbraviruses (family Tombusviridae). The uncovered replication elements appear to be specific to umbraviruses and are located immediately upstream of replication/translation elements that are common throughout the Tombusviridae, lending greater complexity to the already complex 3´ proximal structures of umbraviruses. While the study of (+)-strand RNA viruses has historically focused on their protein-coding transcripts, (+)-strand RNA viruses also commonly produce additional non-coding transcripts, including recombinant defective RNAs, typically containing 5´ and 3´ co-terminal viral genome segments, and (+/-)-foldback RNAs, composed of complementary (+)- and (-)-strand viral sequences joined together. Long non-coding RNAs that accumulate to high levels have also been reported for plant and animal (+)-strand RNA viruses in recent years, and truncations of viral transcripts also commonly arise due to host nuclease activity and/or premature termination of replication elongation by the viral RdRp. The rise of long-read high-throughput sequencing technologies such as nanopore sequencing presents an opportunity to fully map the complexity of (+)-strand RNA viral transcriptomes. In Chapter 4, I present my work performing this analysis, employing direct RNA nanopore sequencing, in which the transcripts present in an RNA sample of interest are directly sequenced. This analysis revealed for the umbra-like virus citrus yellow vein-associated virus (CY1): (i) three novel 5´ co-terminal long non-coding RNAs; (ii) D-RNA population dynamics; (iii) a common 3´ terminal truncation of 61 nt among (+)-strand viral transcripts; (iv) missing 3´ terminal CCC-OH motif in virtually all (-)-strand reads; (v) major timepoint- and tissue-specific differences; and (vi) an abundance of (+/-)-foldback RNAs at later infection timepoints in leaf tissues. This work also sheds light on the current shortcomings of direct RNA nanopore sequencing as a technique. Finally, the importance of RNA structural biology in the study of (+)-strand RNA viruses presents the need for specialized RNA structure drawing software with functionality to easily control the layout of nucleobases, edit base-pairs, and annotate/color the nucleobases and bonds in a drawing. It is through the visual exploration of RNA structures that RNA biologists routinely improve upon the outputs of RNA structure prediction programs and perform crucial phylogenetic analyses among related RNA structures. Large RNA structures, such as whole viral genomes thousands of nucleotides long, can only be studied in their entirety with the aid of RNA structure visualization tools. To this end, I have developed over the course of my doctoral education the 2D RNA structure drawing application RNAcanvas, which is available as a web app and has grown popular among the RNA biology community. RNAcanvas emphasizes graphical mouse-based interaction with RNA structure drawings and has special functionality well suited for the drawing and exploration of large RNA structures, such as automatic layout adjustment and maintenance, complementary sequence highlighting, motif finding, and performance optimizations. Large viral structures such as that of the 2.7 kb CY1 genomic RNA could not have been characterized without the aid of RNAcanvas. In Chapter 5, I present my work developing RNAcanvas.Item Characterization of chromatin assembly dynamics mediated by the histone H3.3 chaperone HIRA and implications of innate immunity during Human Papillomavirus infection(2024) Della Fera, Ashley Nichole; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The circular double-stranded DNA genome of Human papillomavirus (HPV) is chromatinized throughout its viral lifecycle and relies on numerous host chromatin assembly processes, epigenetic modifications, and immune evasion to ensure genomic stability and productive infection. Despite its chromatinization, the HPV genome remains susceptible to innate immune pathways that sense and respond to foreign DNA. In this work next generation sequencing (RNAseq) was utilized to profile changes in the host transcriptome following cellular differentiation and HPV infection in keratinocyte cell lines. Global alterations in keratinocyte differentiation were observed upon HPV infection, and unexpectedly, upregulation of innate immune signaling upon differentiation. Recent findings indicate that packaged HPV genomes are enriched in histone H3.3. Notably, the replication-independent histone H3.3 chaperone HIRA has been implicated in several pro- and anti-viral responses, but its function during HPV infection has yet to be elucidated. Using in-situ approaches, the role of HIRA during the late phase of the HPV lifecycle was evaluated, which showed that HIRA and other chromatin assembly factors localize to sites of HPV replication. Here the requirements for this localization were further characterized, and the impacts of HIRA on HPV genome amplification and viral transcription during the late stage of the HPV life cycle were assessed. Moreover, histone H3.3 phosphorylated at serine 31 was shown to be highly associated with HPV replication factories. HIRA, in part through association with the PML nuclear body associated protein Sp100, has also been reported to promote innate immune responses following infection with other DNA viruses. Here, HIRA localization to PML-NBs was identified to increase following stimulation with IFN in an Sp100-dependent manner. However, while Sp100 is required for localization of HIRA at PML-NBs, it was not required for HIRA localization at sites of HPV replication. In summary, this work highlights the broad changes in the host transcriptome following cellular differentiation and HPV infection, elucidates a previously undescribed role for histone H3.3 chaperone HIRA during the late phase of the HPV life cycle, and further characterizes the relationship between HIRA and Sp100 at PML-NBs.Item Engineering physiologically-relevant model systems to understand the requirements of rhinovirus C infection(2023) Goldstein, Monty Eli; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Rhinovirus (RV) is the most prevalent etiologic agent of the common cold, and infections by RV species C (RV-C) are often associated with more severe illness, and have been strongly correlated with childhood development of asthma. Due to lack of in vitro and in vivo model systems capable of supporting the RV-C life cycle, few details of RV-C biology are understood about this recently discovered, clinically-relevant respiratory pathogen. To reveal the nature of virus-host interactions and study viral pathogenesis, the application of physiologically-relevant model systems that capture relevant cell types, differentiation states, and microenvironmental cues is essential. Applying these principles to our investigations of RV-C, I engineered in vitro and in vivo model systems to better understand the requirement of specific host factors for RV-C replication in human and mouse cells. Specifically, I utilized a pseudostratified in vitro model of human airway epithelium (HAE) to study RV-C replication, and applied CRISPR/Cas9 technology in these cultures to assess the specific role for stimulator of interferon genes (STING) in promoting viral replication. Since RV-C species tropism is highly restricted, I then applied our knowledge of RV-C replication in HAE cultures towards building an improved RV-C mouse model. Here, I first characterized RV-C replication in mouse lung cells in vitro, and demonstrated that human STING expression enhanced viral replication; second, I applied these findings in vivo, where I generated a transgenic mouse expressing the human ortholog of the RV-C receptor, cadherin-related family member 3 (CDHR3), along with human STING. While these mice lack overt symptoms typically associated with viral infection, they exhibited significantly increased viral replication 24 hours-post infection. Finally, to support ongoing efforts to further develop these mice as a robust small animal model of RV-C, I developed several novel cell lines which represent important tools to interrogate the impacts of other host factors on RV-C replication in mouse cells, which upon validation, can be re-engineered into these transgenic mice.Item STRATEGIES AND RESOURCES FOR RATIONAL VACCINE DESIGN AND ANTIBODY-ANTIGEN DOCKING AND AFFINITY PREDICTION(2022) Guest, Johnathan Daniel; Pierce, Brian G.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Antibody recognition of antigens is a unique class of protein-protein interactions, and increased knowledge regarding the determinants of these interactions has advanced fields such as computational vaccine design and protein docking. However, the diversity and flexibility of antibodies and antigens can hinder generation of potent vaccine immunogens or prediction of correct antibody-antigen interfaces, slowing progress in the design of vaccines and antibody therapeutics. In this thesis, we present strategies to design vaccine candidates for a difficult viral target and describe expanded resources for benchmarking and training antibody-antigen docking and affinity prediction algorithms.We utilized rational design to develop candidate immunogens for a vaccine against hepatitis C virus (HCV), which represents a global disease burden despite recent advances in antiviral treatments. This design strategy produced a soluble and secreted E1E2 glycoprotein heterodimer with native-like antigenicity and immunogenicity by fusing ectodomains with a leucine zipper scaffold and a furin cleavage site. We developed additional constructs that incorporated synthetic or non-eukaryotic scaffolds or alternative ectodomains that included consensus sequences designed using a large reference database. Finally, we utilized previously published data on HCV antibody neutralization and E1E2 mutagenesis to predict residues that impact antibody neutralization and E1E2 heterodimerization, offering potential insights that can aid vaccine design. To improve our knowledge of and accuracy in modeling antibody-antigen recognition, we assembled a set of antibody-antigen complex structures from the Protein Data Bank (PDB) that expanded Docking Benchmark 5, a widely used benchmark for protein docking. These complexes more than doubled the number of antibody-antigen structures in the benchmark and, based on tests of current algorithms, highlight significant challenges for docking and affinity prediction. Building on this resource, we assembled and curated a dataset of ~400 antibody-antigen affinities and corresponding structures, forming an expanded and updated benchmark to guide ΔG prediction of antibody-antigen interactions. Using this dataset, we retrained combinations of terms from existing scoring functions and potentials, demonstrating that this resource can be used to improve antibody-antigen ΔG prediction. Overall, these findings can advance HCV vaccine design and antibody-antigen docking and affinity prediction, helping to better elucidate the determinants of antibody-antigen interactions and to better display vaccine immunogens for induction of neutralizing antibodies.Item MUCIN-MEDIATED AND INTERFERON-DRIVEN DEFENSE MECHANISMS AGAINST INFLUENZA VIRUS INFECTION IN HUMAN AIRWAY EPITHELIUM(2022) Iverson, Ethan; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The human airway epithelium represents the primary site of infection for many respiratory viruses, including influenza A virus (IAV). To safeguard this tissue and maintain the functionality of the lung, humans possess a two-layer, extracellular, mucus barrier composed predominantly of individual proteins termed mucins. Additionally, underlying epithelial cells produce interferons upon virus detection that promote the establishment of a local antiviral state through autocrine and paracrine signaling. However, despite these protective measures, IAV continues to cause significant annual morbidity and mortality across the globe. Therefore, we sought to further investigate how specific mucin molecules interact with IAV, and how interferon drives intrinsic antiviral defense in the context of a human airway epithelial (HAE) culture system. By utilizing fluorescently-labeled influenza virus particles we further elucidate the adhesive interactions between mucus and influenza virus while also detailing, for the first time, real-time IAV diffusivity within patient-derived mucus samples. These results reveal that the polymeric structure of mucus greatly influences the mobility of IAV within human secreted mucus. Additionally, we investigate the interaction between influenza virus and tethered mucin 1 (MUC1), finding that MUC1 expression is enhanced by virus-driven inflammation and interferon signaling. Moreover, by establishing a genetically-tractable airway epithelial model, we detail the protective role MUC1 plays in preventing the initial establishment and spread of influenza virus in HAE. Specifically, we find that the loss of MUC1 significantly enhances IAV uptake and spread. Finally, we observe that the directionality of IFN exposure at airway epithelial surfaces impacts the magnitude of protection against IAV and SARS-CoV-2. We then detail the cellular composition of our HAE culture system and define a shared IFN response profile across all HAE component cell types as well as cell type-specific interferon stimulated genes. Together our work provides novel insight into the innate and intrinsic anti-viral properties of the human airway epithelium.Item KEEPING IT IN FRAME: MONITORING RIBOSOMAL FRAMESHIFTING DURING TRANSLATION(2022) Kelly, Jamie; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Programmed -1 ribosomal frameshifting (-1 PRF) is a molecular mechanism that redirects translating ribosomes into a new reading frame. It is widely used by RNA viruses to conserve genome space while expanding the viral proteome and it can help regulate gene expression in eukaryotic cells. Strict regulation of both programmed and non-programmed frameshift events are essential to translational fidelity. This dissertation explores the -1 PRF element of SARS coronavirus 2 (SARS-CoV-2) and the -1 PRF inhibitor, Shiftless. We comparatively analyzed the structural and functional conservation of -1 PRF elements in SARS-CoV and SARS-CoV-2. Both -1 PRF structure and frameshift efficiency were highly conserved between the two viruses and a small molecule effective against SARS-CoV -1 PRF significantly decreased frameshift efficiency in SARS-CoV-2. This suggests -1 PRF is an attractive antiviral target and could be a useful tool to combat the SARS-CoV-2 pandemic or future outbreaks of similar coronaviruses. The innate immune system targets viral frameshifting using an interferon-stimulated -1 PRF inhibitor called Shiftless (SFL) that binds, arrests, and terminates translation of -1 frameshifted ribosomes. We found that SFL is not only expressed in response to interferon but that it may have a role in general translational fidelity. SFL is constitutively expressed at low levels in human-derived cell lines and its effects are not limited to -1 PRF signals. Disruption of SFL homeostasis results in reciprocal 2-fold changes to recoding efficiencies in a panel of human and viral-derived translational recoding signals, decreases reporter gene expression, and decreases mRNA steady state abundances. Additionally, SFL over or under expression combined with knockdown of prominent ribosome-associated protein quality control (RQC) proteins reveals that SFL is epistatic to RQC. These results suggest that SFL has a role in general translational fidelity monitoring for spontaneously frameshifted ribosomes in addition to its role as a member of the innate immune response.Item TRANSLATION AND MOVEMENT OF AN INFECTIOUS UMBRAVIRUS-LIKE RNA CITRUS YELLOW VEIN ASSOCIATED VIRUS(2021) Liu, Jingyuan; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)While the citrus yellow vein disease was first reported more than 60 years ago, its causal agent, citrus yellow vein associated virus (CYVaV), was only sequenced in 2013. CYVaV was recently structurally characterized and phylogenetically classified as a Class 2 umbravirus-like associated RNA (ulaRNA), a new category of coat-protein dependent subviral RNA replicons. There is a dearth of structural and biological characterizations of ulaRNAs as well as studies on their translation regulation. CYVaV has a limited genome size (2.7 kb), and contains only two ORFs that encode replicase proteins p21 and p81. Here I show that CYVaV transcripts are infectious in Arabidopsis protoplasts and Nicotiana benthamiana plants, and CYVaV can systemically infect the latter using agro-infiltration, despite the absence of encoded movement proteins or silencing suppressors. Fluorescent in situ hybridization (FISH) revealed that CYVaV is phloem-limited, and restricted to sieve elements, companion cells, and phloem parenchyma cells. In this work, the secondary structures of the CYVaV 5ʹ end and 3ʹ UTR were determined using SHAPE structure probing and phylogenic comparisons, and were used to infer the putative structures of other Class 2 ulaRNAs, revealing a number of distinctive structural features. Here I report the identification of a novel 3ʹCITE in the 3ʹUTR of CYVaV that is strongly conserved in Class 2 ulaRNAs and structurally resembles an I-shaped structure (ISS) 3ʹCITE. However, unlike ISS, the CYVaV structure binds to eIF4G and no long-distance interaction is discernible between the CYVaV ISS-like structure and sequences at or near the 5ʹ end. We also report that the ~30 nt 5ʹ terminal hairpin of CYVaV and related ulaRNAs can enhance translation of reporter constructs when associated with either the CYVaV 3ʹCITE, or the 3ʹCITEs of umbravirus PEMV2, or even independent of a 3ʹCITE. These findings introduce a new type of 3ʹCITE and provide the first information on translation of ulaRNAs.Item Chromatin Control of Papillomavirus Infection(2020) Porter, Samuel Stephen; McBride, Alison A; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The genomes of papillomaviruses are packaged into chromatin throughout the entire viral lifecycle. A peculiar feature of papillomaviruses genome organization is that the viral DNA is associated with host histones even inside the virion particle. However, little is known about the nature of the epigenome within papillomavirions, or its biological impact on early infection. Here, we use three approaches to study the epigenome of papillomavirions. Papillomaviruses can be assembled in packaging cells by expression of the capsid proteins in the presence of the viral genome. We have optimized and manipulated this process to generate viruses with replicated and genetically modified virion DNA and have used these “quasivirions” to evaluate early infection of primary human keratinocytes. We have also profiled the histone modifications on chromatin extracted from native virions isolated from human and bovine warts. We find that, compared to host cells, the viral chromatin is enriched in histone modifications associated with transcriptionally active chromatin (including histone acetylation), and depleted in those associated with transcriptional repression. To examine the biological role of histone acetylation in the early virus lifecycle, we produced HPV quasivirions with highly acetylated chromatin by assembling the virions in cells treated with histone deacetylase inhibitors. We show that acetylation of viral chromatin results in a reduction of early viral transcription in primary keratinocytes indicating that the histone modifications on virion chromatin do influence the early stages of infection. Collectively, these studies demonstrate that histone modifications on virion chromatin are important for the HPV infectious cycle.Item HIGH-RESOLUTION ANALYSIS OF HIV ENVELOPE-SPECIFIC ANTIBODY RESPONSES TO ACCELERATE RATIONAL IMMUNOGEN DESIGN(2020) Lei, Lin; Li, Yuxing; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The recent isolation of HIV broadly neutralizing antibodies (bNAbs) from HIV infected individuals has reinvigorated efforts to develop B cell-based vaccines. As the sole viral target for bNAbs, HIV envelope glycoprotein (Env) has been engineered as soluble trimers to recapitulate bNAbs responses via vaccination. However, Env-based immunogens thus far primarily induce vaccine-matched neutralizing antibody (nAb) responses. This thesis aims to understand the mechanisms restricting the neutralization breadth and to provide strategies for iterative improvements. First, we have established an antigen-specific single B cell sorting and monoclonal antibody (mAb) cloning platform for guinea pigs, a small animal model desirable in the field for initial immunogenicity analysis. This method allowed us to dissect the antibody responses at the clonal level with high accuracy and efficiency. Secondly, we have delineated the specificity of autologous neutralization elicited by the current generation HIV trimer mimicry, BG505 SOSIP.664. Our results reveal a prominent epitope in the C3/V4 region of the Env targeted by one nAb/B cell clonal lineage. We demonstrate that the nAb responses to this neutralization determinant are prevalent in trimer-vaccinated guinea pigs, rabbits, and non-human primates. In addition, this defined nAb response shares a high degree of similarity with the early nAb response in an HIV- infected pediatric patient, who later developed a bNAb response. This study offers insights into re-designing Env immunogens in the highly immunogenic region to broaden nAb responses. Lastly, we have engineered novel immunogens based on the Env sequence of a virus strain isolated from bNAb VRC01 donor, which can engage the VRC01 germline precursor in vitro. Sequential prime-boost immunizations in a VRC01-germline immunoglobulin (Ig) encoding genes knock-in mouse model with the designed immunogens induced focused VRC01-like serum antibody responses and clustered VRC01-class somatic mutations in the knock-in VRC01-germline Ig genes. In addition, the mAbs recovered from the immunized mice neutralize selected viruses containing the N276 glycan, a critical roadblock impeding the affinity maturation of VRC01-class bNAbs. Our findings demonstrate that, in the transgenic mouse model, our immunogens effectively activate bNAb precursor B cells and guide their affinity maturations required for bNAb function, which has important implications for HIV vaccine development.Item Orthologous Gene Swapping and Experimental Evolution Provide Novel Way to Study Essential Poxvirus Genes(2018) Stuart, Carey A; DeStefano, Jeffrey J; Moss, Bernard; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The transcriptional program of poxviruses is divided into early, intermediate and late phases enabled by a multisubunit DNA-dependent RNA polymerase and stage-specific transcription factors that recognize cognate promoters. Although promoter sequences are highly conserved among the different chordopoxvirus genera, the transcription factors exhibit considerable amino acid divergence that parallels the evolutionary distance of the host species. Thus, the large/small subunits of the intermediate transcription factors (ITFs) of salmon gill poxvirus, crocodilepox, canarypox, and myxoma have 23/29, 40/31, 51/38 and 58/65 % amino acid identity, respectively, to the vaccinia virus (VACV) orthologs. The purpose of the present study was to determine the functional interchangeability of the ITF subunits and their putative interactions with other elements of the transcriptional machinery. A quantitative readout of ITF function using firefly luciferase (Fluc) was obtained. The activity of the large subunit orthologs was greater than that of the small subunit orthologs, with both sets following the degree of sequence similarity in relation to VACV. The same pattern was obtained with both heterospecific (e.g., myxoma large and VACV small subunits) and homospecific (e.g., myxoma large and small subunits) pairings, suggesting inefficient interactions with other elements of the transcription system. When recombinant hybrid VACV expressing the Myxoma virus (MYXV) ortholog of the small subunit (A8) were blind passaged multiple times, their replicative abilities were enhanced. Complete genome sequencing of the virus populations revealed five mutations present in the two largest subunits of the viral RNA polymerase (RNAP) and two predicted expression-enhancing mutations around the translation initiation site of the MYXV A8 ortholog. Amplicon sequencing was used to quantify the frequency of each mutation in its respective population, which revealed that they increased as passaging occurred. This indicated a correlation with increased fitness, which then needed to be confirmed, so these mutations were all experimentally introduced into the original hybrid virus and demonstrated to enhance virus replication independently. These mutations were then characterized to determine their specific effects on the viral RNAP (vRNAP) and viral replication and transcription. This approach could have broader applications for studying essential genes in poxviruses and other viruses as well.