Browsing by Subject "Retrovirus"
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Item Cloning and characterization of MRI, a modulator of retroviral infection.(2009-12) Agarwal, SumitAlthough significant progress has been made in elucidating the life cycle of HIV and identifying host cell proteins which interact with HIV, a much greater understanding of these processes is necessary in order to eradicate this disease. The studies presented in this dissertation further examine these processes and identifies a new host cell protein, MRI, which appears to be involved in the poorly understood step of viral uncoating.The first part of my project involved the creation, characterization, and utilization of a novel toxic retroviral vector encoding barnase, a non-specific protease derived from Bacillus amyloliquefaciens. In conjunction with an inhibitor of barnase (barstar), I showed that barnase-based retroviral vectors were capable of inducing cell death in a wide variety of cell types and that the presence of barstar was essential for the creation of high titer retroviral vectors. The second part of my project was to was to create a heavily mutagenized hamster cell line, V79-4, and repeatedly challenge this cell line with the toxic retroviral vector in order to isolate clonal populations of cells resistant to retrovirally mediated transduction. Ultimately, I isolated two cell lines, 31-2 and 67-1, which we characterized further. 31-2 was determined to be least five fold resistant to both MLV- and lenti-viral at a stage in viral replication post reverse transcription. 67-1 was minimally ten fold resistant to both MLV- and lenti-viral infection at a stage between viral entry and uncoating. We demonstrated that the mutation in 67-1 implicated the proteasome and identified a gene we designated MRI that reversed the inhibition of infection in the mutated 67-1 line. The implication of MRI in the retroviral lifecycle identifies a new protein that could facilitate the creation of novel host cell based therapy for HIV.Item Compounds derived from birch trees that inhibit HIV-1 replication(2011-02) Dorr, Casey R.Human Immunodeficiency Virus type-1 (HIV-1) replication is introduced in Chapter 1 with an emphasis on the late phase of viral replication. Literature about the production of infectious HIV-1 is reviewed in Chapter 1 while focusing on targets for suppressive HIV-1 therapy. Chapter 2 describes the discovery of triterpene compounds derived from birch trees that inhibit HIV-1 replication reformatted from Bioorganic and Medicinal Chemistry Letters. Chapter 3 investigates the anti-HIV-1 mechanisms of the triterpenes described in Chapter 2. Virus release assays conclude that the triterpene compounds target and prevents cleavage of the HIV-1 Gag product CA-SP1. Virus release assays and transmission electron microscopy indicate that the triterpene compounds SY33 and Bevirimat have a secondary mechanism of action by causing 55 kDa Gag to accumulate in cells. The Gag accumulation in cells was observed in both wild-type HIV-1 and the mutant SP1-A1V. The SP1-A1V mutant causes a decrease in susceptibility, by viral replication and CA-SP1 processing, to SY33 and Bevirimat. An Epilogue describes recommended future experiments. Appendix A describes genotypic results of an experiment to select for HIV-1 resistant to SY33 and Bevirimat using wild type and SP1-A1V as the founder viruses. The data in Appendix A suggest SP1-A1V causes reduced susceptibility to SY33 and Bevirimat. Appendix B describes the initial discovery of fatty acid derivatives with anti-HIV-1 activity.Item Studies On The Antiretroviral Mechanism Of Action Of Clofarabine(2014-07) Beach, LaurenSince the beginning of the AIDS epidemic over thirty years ago, human immunodeficiency virus type 1 (HIV-1) has infected seventy-five million people and has claimed the lives of over thirty-six million people worldwide, making HIV/AIDS one of the most devastating global infectious disease epidemics in history. To date, no preventative vaccine or curative treatment exists for HIV-1 infection. The availability of drugs to treat HIV-1 infection has led to drug resistance, which limits the utility of antiviral therapy. This has provided the basis for the continual need for identifying new targets for antiviral drugs. This dissertation investigated the antiretroviral activity and mechanism of action for clofarabine, a purine nucleoside antimetabolite. Clofarabine was demonstrated to exert antiretroviral activity against both HIV-1 and human immunodeficiency virus type 2 (HIV-2). Studies directed at elucidating the antiretroviral mechanism of action support a model in which clofarabine acts as an inhibitor of ribonucleotide reductase, leading to imbalances in cellular dNTP pools, which reduces viral infectivity through an increase in the HIV-1 mutation rate.Item Studies on the Assembly and Morphology of Human T-Cell Leukemia Virus Type 1(2019-08) Maldonado-Ortiz, JoséThe group-specific antigen (Gag) polyprotein is an essential retrovirus structural protein required for the assembly and release of virus particles. Present knowledge of Gag biology has been limited to a few retroviruses. Furthermore, current understanding of the diversity in the nature of Gag structure and function in virus particle assembly is limited. Human T-cell leukemia virus type 1 (HTLV-1) is a deltaretrovirus that causes an adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated-myelopathy/tropical spastic paraparesis (HAM/TSP), and other neurotropic conditions. HTLV-1 has infected approximately 15 million individuals worldwide. A general knowledge gap exists regarding the details of HTLV-1 replication, including particle assembly. To address this, and to test the overarching hypothesis that HTLV-1 particle assembly is distinct from that of other retroviruses, this dissertation focused on investigating three key aspects of HTLV-1 immature and mature particle morphology. First, an analysis of the morphology and Gag stoichiometry of HTLV-1-like particles and authentic, mature HTLV-1 particles by using cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM) was conducted. HTLV-1-like particles mimicked the morphology of immature authentic HTLV-1 virions. Importantly, it was observed for the first time that the morphology of these virus-like particles (VLPs) has the unique local feature of a flat Gag lattice that does not follow the curvature of the viral membrane, resulting in an enlarged distance between the Gag lattice and the viral membrane. Measurement of the average size and mass of VLPs and authentic HTLV-1 particles suggested a consistent range of size and Gag copy numbers in these two groups of particles. The unique local flat Gag lattice morphological feature observed suggests that HTLV-1 Gag could be arranged in a lattice structure that is distinct from that of other retroviruses characterized to date. Second, the effects of Gag proteins labeled at the carboxy terminus with a fluorophore protein were analyzed for their influence on particle morphology. In particular, a HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus was used as a surrogate for the HTLV-1 Gag-Pro to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle morphology and particles were analyzed by cryo-TEM. STEM and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. Ratios of 3:1 (Gag:Gag-YFP) or greater were found to result in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. This information is useful for the quantitative analysis of Gag-Gag interactions that occur during virus particle assembly and in released immature particles. Third, cryo-electron tomography (cryo-ET) was used to analyze mature HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the viral membrane. Most of the particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity. Taken together, the findings of this dissertation provide new insights into the nature of immature and mature HTLV-1 assembly and morphology and provide foundational knowledge towards an advanced understanding of the HTLV particle assembly pathway.