MS, University of Cincinnati, 2024, Medicine: Immunology

Human Immunodeficiency virus (HIV-1) was discovered in 1983 from the lymph node sample of a patient at the Pasteur Institute and subsequently was found to be strongly linked to onset of AIDS. HIV-1 and its pathogenesis has been extensively studied now for decades. Many crucial components of the virus such as the viral replication machinery, proteins involved in replication and assembly, fusion of viral and host cells during infection, and many other aspects of the viral lifecycle have been defined. The HIV-1 envelope glycoprotein (Env) is an essential component of the virus, present on the lipid envelope of the virus and involved in cell membrane fusion and entry. An aspect of HIV-1 Env biology that is not well understood is how it interacts with host cell machinery to reach the site of particle assembly on the plasma membrane. This study investigates the involvement of tubular recycling endosome (TRE), a component of the cellular recycling machinery that is involved in transporting internalized cargo, in the trafficking of Env following endocytosis from the plasma membrane. In particular, this study addresses the interactions of primary isolates of HIV-1 Clade B Env with the TRE, using immunofluorescence staining and imaging of TRE markers. This study also focuses on the role of Phosphatidic Acid (PA) in modulating Env interaction with the TRE, and its regulation through phospholipase D (PLD). The effect of disruption of PA production through inhibition of PLD on Env-TRE colocalization was critically assessed using live cell and pulse-chase imaging along with monitoring of TRE markers. The effects of inhibition on infectivity and particle incorporation were evaluated after PLD inhibition. This study confirmed that primary isolate Env associates strongly with the TRE, and that inhibition of TRE formation through PLD inhibition disrupts Env trafficking. Findings here build on those with laboratory isolates of HIV-1 and provide a new potential therapeutic target to in (open full item for complete abstract)

Committee: Paul Spearman M.D. (Committee Chair); Claire Chougnet Ph.D. (Committee Member); Ian Lewkowich Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy, The Ohio State University, 2024, Comparative Biomedical Sciences

More than 80 million people have been infected with human immunodeficiency virus type-1 (HIV-1), with approximately 38 million people currently living with the virus, according to UNAIDS (2021). Although antiretroviral therapy (ART) is available, the persistence of the virus within the viral reservoir remains a significant obstacle in developing more effective HIV-1 treatments. Recent advancements in technology have allowed studies to reveal that RNA modifications in HIV-1 play key roles in the biological processes, offering deeper insights into the disease. HIV-1 exploits all aspects of RNA, a versatile macromolecule that undergoes various post-transcriptional modifications. Despite recent studies highlighting the importance of chemical modifications on RNA, the evolutionary advantages of these modifications for HIV-1 are still not well understood. Most research has only provided low-resolution, population-averaged data on these modifications, often overlooking site-specific details and intra-RNA variability. In this study, we introduce multiplex reverse-transcription (mRT) and read-level binary-classification (m6Arp) techniques that enable full-length, single-molecule analysis of HIV-1 RNAs using nanopore direct RNA sequencing (DRS). Our analysis uncovered an unexpectedly simple modification landscape for HIV-1, identifying three dominant N6-methyladenosine (m6A) modifications near the 3' end. These m6As play critical roles in maintaining proper RNA splicing and translation and are much more densely present in mRNAs compared to genomic RNAs. HIV-1 produces various RNA subspecies with distinct patterns of m6As, demonstrating that each of the three m6As can independently regulate RNA splicing. The functional redundancy and intra-RNA diversity of these m6As help the virus reduce the impact of mutations that may eliminate a major m6A modification. This single-RNA-level study unveils novel RNA strategies employed by HIV-1 that contribute to the virus's stability and ad (open full item for complete abstract)

Committee: Sanggu Kim (Advisor); Michael Oglesbee (Committee Member); Karin Musier-Forsyth (Committee Member); Jian Zhu (Committee Member) Subjects: Virology

Doctor of Philosophy, The Ohio State University, 2017, Biochemistry Program, Ohio State

The hallmark of a retrovirus is the necessity to reverse transcribe the single-stranded genomic RNA (gRNA) into a double-stranded DNA that is incorporated into the host cell genome. After integration, host transcription and translation machinery produces viral RNA and proteins, including one main component of the viral lifecycle, Gag. The HIV-1 Gag polyprotein, is composed of 3 primary domains: matrix (MA), capsid (CA), and nucleocapsid (NC), which play critical roles in almost every aspect of the retroviral lifecycle, including reverse transcription, selection of gRNA and assembly of new virions. HIV-1 MA targets assembly to the phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2)-enriched plasma membrane (PM) through residues in its highly basic region (HBR) and anchors multimers of Gag to the PM with a myristic acid moiety. CA is able to form protein-protein interactions that facilitate Gag assembly and form the capsid core of the virion. Finally, NC selects gRNA from the large cytoplasmic pool of host RNAs. Intriguingly, host tRNAs and host translational machinery have been found to regulate several aspects of the HIV-1 lifecycle examined here. Along with viral proteins and gRNA, several host factors are selectively packaged into HIV-1 virions. Human tRNALys3 acts as the primer for reverse transcription in HIV-1, during which the 3' 18-nt are unwound and annealed to a complementary sequence in the 5'UTR of the genome known as the primer binding site (PBS). A stem-loop directly 3' to the PBS mimics tRNALys and efficiently binds the aminoacyl-tRNA synthetase responsible for aminoacylating tRNALys isoacceptors, lysyl-tRNA synthetase (LysRS). LysRS is known to interact with and be packaged by HIV-1 Gag, facilitating the incorporation of tRNALys3 and correct placement of the tRNA onto the PBS. Under homeostatic conditions, LysRS is sequestered in a multi-aminoacyl-tRNA synthetase complex (MSC). Here, we show that HIV-1 infection results in a free pool of uncharged (open full item for complete abstract)

Committee: Karin Musier-Forsyth (Advisor); David Bisaro (Committee Member); Michael Ibba (Committee Member); Li Wu (Committee Member) Subjects: Biochemistry; Virology

Doctor of Philosophy, Case Western Reserve University, 2017, Molecular Virology

The sequence of events from HIV-1 entry through transcription of proviral DNA is not well understood. While many studies have attempted to investigate these post-fusion events, the unstable nature of intracellular HIV-1 particles has made biochemical analyses difficult. Developments in imaging techniques have given a glimpse of intracellular HIV-1 particles during active infection, but few of these attempts have connected both the early and late events of HIV-1 infection in primary human target cells. Even fewer of these attempts effectively visualized actively transcribing viruses that contribute to productive infection. Here, we developed a method to visualize HIV DNA reverse transcription (RT) products by incorporation and fluorescent labeling of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) in infected monocyte-derived macrophages (MDM). We observed distinct EdU puncta in the cytoplasm and nuclei of infected cells which increased in number following depletion of the dNTPase SAMHD1. Interestingly, we found HIV-1 capsid associated with HIV-1 DNA in the cytoplasm and nuclei of infected MDM. Our assay also represented improvements upon current qPCR techniques in measuring the efficiency of nuclear import of two CA mutants, N74D and Q63A/67A, and showed marked sensitivity in detecting intranuclear HIV-1 genomes in productively and non-productively infected MDM. Because we were able to track nuclear HIV-1 DNA through productive infection of MDM, we were interested in whether we could identify actively transcribing proviruses. Using RNA fluorescence in situ hybridization (FISH) we were for the first time able to image actively transcribing proviruses through colocalization of both HIV-1 DNA and HIV-1 RNA. These transcribing proviruses were further confirmed through colocalization of nuclear HIV-1 DNA and RNA with specific HIV-1 transcription factors. Modification of our newly developed RNA FISH technique to monitor HIV-1 transcription in productively infected (open full item for complete abstract)

Committee: David McDonald (Advisor); John Tilton (Committee Chair); Jacek Skowronski (Committee Member); George Dubyak (Committee Member) Subjects: Molecular Biology; Virology

MS, Kent State University, 2013, College of Education, Health and Human Services / School of Health Sciences

The purpose of this study was to assess the nutrition education received and desired by HIV-positive individuals, and the relationship of nutrition education received, quality of life, and nutrition-related symptomologies. HIV-positive individuals (n=76) were recruited at an HIV clinic, support group, and community center to complete a questionnaire. The questionnaire evaluated participant knowledge of their HIV-related and nutrition-related laboratory values, their quality of life, and present symptoms. Pearson’s correlation coefficients were calculated to assess the relationship between quality of life sum scores and symptomology scores, as well as the relationship between nutrition topics discussed and topics desired by participants. A regression was used to compare which factors were influenced by symptoms. The data was compiled and analyzed using social sciences software (SPSS, version 13.0). There was a significant, positive correlation between symptoms present and worsening of quality of life scores (p = 0.05). In addition, there was a significant, negative correlation found between nutrition topics discussed and desired (p < 0.05). Furthermore, the regression was significant with a moderate effect size and low beta weights with quality of life being the only significant factor (p < 0.05). Thus, the conclusions drawn from the regression alone was limited. Regardless, participants with more symptoms had poorer quality of life, and those with a high desire of nutrition education received little information. Advocacy is needed to limit this disconnect and enhance the disease management of the HIV-positive community.

Committee: Natalie Caine-Bish (Advisor); Nancy Burzminski (Committee Member); Dianne Kerr (Committee Member) Subjects: Health Education; Health Sciences; Immunology; Nutrition

MS, University of Cincinnati, 2012, Arts and Sciences: Chemistry

Human immunodeficiency virus type 1 (HIV-1) initiates replication of its RNA genome by priming from the 3¿¿¿¿ end of a host cell tRNA complementary to the primer binding site on the viral genome. The virus packages its primer, human lysyl tRNA during virus assembly incorporating human lysyl-tRNA synthetase (hKRS) into virions using viral Gag and Gag-Pol proteins. Here, we report our attempts to study the tRNA binding properties, and in particular KD and stoichiometry of binding of the domains of Human lysyl-tRNA synthetase (hKRS) by UV/Vis and circular dichroism (CD). hKRS consists of three domains, which can function independently, a tandem domain, or as the full length enzyme. Cognate lysyl tRNA and non-cognate phenylalanine tRNA were used for an attempt to determine KD and stoichiometry upon binding to hKRS. We were able to obtain some conclusive results pertaining to KD and stoichiometry for the domains of hKRS. For example, significant binding has been observed between yFtRNA and 2D domain as determined both by CD and UV/Vis. With regards to stoichiometry, a yFtRNA:rrACB C_S titration was performed and a 1:1 stoichiometric ratio was estimated by CD. Also, a KD value of 18 uM for the yFtRNA:2D native complex was obtained by UV/Vis. Finally, an online computerized algorithm named DICHROWEB was used to show that that the secondary structure of various truncated variants of the ACB domain were less disordered in solution than the full length ACB. With respect to all constructs, each domain has at least 50% random coil on average and the trend of greatest to least random coil goes as follows modnhKRS>2D protein >rrACBcs> ghKF_ACB> flhKRS>catalytic>GED_ACB. However, most of our attempts in determining KD and stoichiometry were unsuccessful. Upon further investigation of the various domains of hKRS', we were not able to determine qualitative binding between yFtRNA and modnhKRS, ACB, or flhKRS as well as hktRNA to 2D domain by CD because of improper procedures and anal (open full item for complete abstract)

Committee: Pearl Tsang PhD (Committee Chair); Apryll Stalcup PhD (Committee Member) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2008, Integrated Biomedical Sciences

There is a growing population of human immunodeficiency virus (HIV) infected drug abusers and epidemiological evidence suggests that the psychostimulant drug methamphetamine (METH) is a risk factor for HIV dementia. However, the mechanism by which this occurs is not well defined. The feline immunodeficiency virus (FIV) is a disease in cats that includes encephalitis and neurological symptoms resembling HIV dementia in humans. Astrocytes maintain homeostasis in the brain and may participate in the FIV-induced neurotoxicity. Our findings show that METH had short lived proliferative effects on cell growth of G355-5 cells, a feline astrocyte cell line, with a peak at 24 h. During this time period, METH promoted cell-mediated FIV infection of astrocytes. These results indicate that METH may increase FIV infection by one or both of the following mechanisms: cell proliferation and/or expression of lentiviral entry receptors. Recent studies show that both OX40 and CXCR4 are important for FIV entry into cells. FIV infected peripheral blood mononuclear cells (PBMC) are needed to mediate FIV infection of G355-5 cells. G355-5 cells exposed to METH however, increased astrocytic CXCR4 RNA expression at early time points . The presence of METH also increased cell surface CXCR4 receptor expression at 12 and 24 h. The increase in RNA expression, in addition to the increase in total CXCR4 protein strongly suggests CXCR4 protein synthesis. These results indicate that an increase in proliferation and upregulation of CXCR4 receptors on G355-5 cells may contribute to METH-induced susceptibility of astrocytes to FIV infection. These results lead us to further study the role of METH on the modulation of CXCR4 receptors in primary rat astrocytes. This animal model more closely mimics the human disease. In this group of studies, we confirmed that METH increased expression CXCR4 RNA and surface protein expression. Furthermore, studies with pharmacological inhibitors indicated that the METH mo (open full item for complete abstract)

Committee: Caroline Whitacre PhD (Advisor); Virginia Sanders PhD (Committee Member); John Sheridan PhD (Committee Member); Lawrence Mathes PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2007, Molecular, Cellular, and Developmental Biology

Efficient translation of mRNA is an essential step in expression of all genes. Retroviruses encode obstacles to efficient viral mRNA translation. Study of retrovirus mRNA translational control is relevant to the processes of immunodeficiency, progression to neoplasia, and safe and efficacious gene transfer vector by this diverse class of RNA virus. This dissertation investigated fundamental mechanisms that control translation of retroviral mRNA and applied our findings to improve translation of transgene mRNA in lentiviral vectors. Chapter 2 unraveled a dynamic interface between HIV-1-induced cell cycle arrest and host translation suppression. Metabolic labeling experiments demonstrated that HIV-1-induced cell cycle arrest attributable to Vpr and Vif accessory proteins significantly limits the translation capacity of infected lymphocytes. Kinetic and ribosomal profile analysis determined that HIV-1 gag mRNA translation is resistant to this potential block to virus production. Cytosolic fractionation experiments demonstrated that gag RNA/ribosome complexes are translated on membrane-bound ribosomes and that amino-terminal myristate of Gag provides an ER partitioning signal. These results overturn the common notion that retrovirus translation is confined to soluble polyribosomes. HIV-1 mRNA partitioning to ER ribosomes represents a novel co-adaptation strategy to promote synthesis of viral structural protein. Chapter 3 developed a sucrose gradient fractionation method to interrogate the translation activity of the entire HIV transcriptome in lymphocytes. The assay verified that alternatively spliced transcripts are associated with polyribosomes. Chapter 4 applied for the first-time the post-transcriptional control element (PCE) of spleen necrosis virus (SNV) to facilitate translation of vector transgene mRNA. Coordinate enhancement of transgene transcription and translation in a lentiviral vector was achieved by combination of SNV PCE with a CMV transcriptional enhanc (open full item for complete abstract)

Committee: Kathleen Boris-Lawrie (Advisor) Subjects: Biology, Molecular

Doctor of Philosophy, Case Western Reserve University, 2008, Pathology

Human immunodeficiency virus (HIV-1) is estimated to have newly infected 2.5 million people worldwide in 2007 (UNAIDS 2007) and current treatments only delay the course of this disease. HIV is primarily transmitted through mucosal surfaces and initially propagates in the lamina propria below the polarized epithelial mucosa. IgA, produced in the lamina propria and transcytosed across the mucosal epithelium, is the first line of defense against HIV. This study was initiated to determine if IgA against the more invariant internal HIV proteins are capable of mediating intraepithelial viral neutralization. Polarized primate epithelial cells expressing the polymeric immunoglobulin receptor (pIgR) were transfected with proviral DNA, and IgA was added to the basolateral side. Transcytosing IgA against the HIV proteins Gag and RT significantly inhibited HIV replication in a concentration dependent manner. Consistent with intracellular neutralization, colocalization of the internal proteins and IgA was visualized with confocal microscopy. Thus, at least in the context of infections of polarized epithelia, antibody-mediated neutralization is not necessarily restricted to viral surface antigens. Another potential IgA defense mechanism against HIV is immune excretion, where IgA can bind and form immune complexes in the basolateral compartment (lamina propria) that can be transported and released apically (luminal). IgA against HIV envelope proteins was used to study the ability of polarized epithelial cells to excrete virus from the basolateral to apical surface via pIgR-mediated binding and transport of HIV-IgA immune complexes. Excretion was dependent on IgA concentration and exposure time. Combination of IgA against gp120 and gp41 showed a synergistic increase of excretion. Each IgA antibody demonstrated different levels of excretion ability, which correlated with the ability of the different IgAs to bind HIV and of the immune complexes to bind pIgR. Confocal microscopy showe (open full item for complete abstract)

Committee: Yung Huang (Advisor) Subjects: Health Sciences, Immunology

Doctor of Philosophy, Case Western Reserve University, 1994, Nursing

Laboratory and clinical investigations have demonstrated the effectiveness of cognitive interventions for immune enhancement and symptom reduction. Insufficient research using these interventions with HIV positive populations, and findings confounded by multiple treatments supported the need for this study. A 3 x 3 randomized block pre-test, post-test design compared the effects of two treatments, a guided imagery (n = 23) or progressive muscle relaxation (n = 22) audiotape used daily for six weeks, to controls (n = 24). The three illness classifications, based on CDC criteria, were seropositive asymptomatic, AIDS-like syndrome, and AIDS. Sixty-nine of 81 randomly assigned subjects completed the study. Dependent variables included depression, fatigue, CD4+ T lymphocyte count, CD4+:CD8+ T lymphocyte ratio, and CD16+ lymphocyte count. These were measured with the Center for Epidemiological Studies Depression Scale (depression), the Sleep and Rest subscale of the Sickness Impact Profile (fatigue), and flow cytometry (lymphocytes). Data were analyzed with a hierarchical multiple regression model, holding pretreatment effects constant while assessing the contribution of each treatm ent to the dependent variables. Findings supported hypothesized associations between CD4+ lymphocyte count and CD4+:CD8+ ratio (r = .84, p < .000), CD4+ and CD16+ lymphocyte counts ( r = .38, p = .001), and CD16+ lymphocyte count and CD4+:CD8+ ratio ( r = .18, p = .07). Observed associations between fatigue and CD4+ lymphocyte count ( r = -.22, p = .04), CD16+ lymphocyte count ( r = -.11, p = .18), CD4:CD8+ ratio (r = -.19, p = .06), and depression (r = .52, p < .000) also were as hypothesized. Not supported were hypothesized associations between depression and CD4+ lymphocyte count (r = .01, p = .46), CD16+ lymphocyte count (r = .02, p = .43), and CD4+:CD8+ ratio ( r = -.01, p = .48). Hypotheses of lower posttreatment fatigue (t = -2.06; p <.04) and depression (t = -1.69; p <.10) in the guide (open full item for complete abstract)

Committee: Patricia Brennan (Advisor) Subjects: