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Production of infectious retrovirus particles is a complex and poorly-understood process with multiple steps that are often linked to one another. Our aim in this study was to gain better understanding of the path the murine leukemia virus (MLV) structural protein Gag follows to assemble into immature capsid structures, the process of which is central to retroviral assembly and release. Extensive studies of human immunodeficiency virus type 1 (HIV-1) assembly have led to the development of a model proposing that the assembly of immature HIV-1 capsids proceeds sequentially through multiple intermediates, in association with an RNA granule containing some well-conserved cellular factors, such as ATP-binding cassette subfamily E member 1 (ABCE1) and DEAD-box helicase 6 (DDX6). In this work, we provided evidence suggesting that MLV Gag associates with endogenous ABCE1 in human cells expressing assembly-competent MLV, and can be found in at least three high-molecular weight complexes with sedimentation properties highly resembling the HIV-1 assembly intermediates. Furthermore, we assessed the Gag proteins of select assembly-defective MLV mutants in terms of their expression levels, ability to form viral particles, involvement in intracellular complexes, membrane association, and ABCE1 interaction. Our findings were not only consistent with a model of MLV assembly through host-mediated intermediates, but also provided novel information about the effects of various MLV Gag mutations that are associated with defects in particle production.

Methotrexate, a folic acid antagonist, has been used in the clinical treatment of a wide variety of malignant neoplasms for over 20 years, either as a single agent or in combination with other antineoplastic agents. It is a cell cycle specific inhibitor and kills cells only in the S phase of growth. MTX is a potent inhibitor of the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3.), which catalyses the NADPH dependent reduction of dihydrofolic acid and folic acid to tetrahydrofolic acid: the metabolically active coenzyme form of folic acid essential in the biosynthesis of dTMP from dUMP by thymidylate synthetase. Inhibition of DHFR therefore leads to the inhibition of DNA synthesis and cell death. Methotrexate has many favourable properties; for instance, it interacts directly with intracellular sites without the need for prior metabolic transformation. It can be administered in large doses because toxicity to normal cells can be minimized by the administration of folinic acid (N5 formyl tetrahydrofolic acid) shortly after the administration of MTX. However, the effectiveness of MTX is inevitably compromised by the emergence of drug resistance, which can be either intrinsic, i.e. the tumour cells are resistant to MTX at the outset, or the tumour cells acquire resistance after exposure to MTX. An understanding of the mechanisms of resistance to MTX is therefore very important if treatment with this potent antineoplastic agent is to be improved. Three mechanisms of resistance to MTX have been determined from studies with experimental tumour systems: impaired uptake of MTX; increased levels of dihydrofolate reductase; and appearance of altered dihydrofolate reductase with a lower affinity for MTX. Impaired uptake of MTX and increased levels of OHFR can both theoretically be overcome by sustaining increased concentrations of free intracellular MTX. This can be achieved by exposing the cells to higher concentrations of MTX, and many chemotherapeutic regimens now use 'high-dose' MTX which can achieve plasma concentrations of MTX as high as 10⁻³M. However, resistance to MTX is still a major clinical problem and the use of 'high-dose' MTX has not significantly increased the therapeutic index of MTX treatment. Appearance of DHFR with a lower affinity for MTX suggests as an alternative the synthesis of an agent which would be a potent inhibitor of the altered enzyme, and requires the detailed characterization of the properties of this enzyme. If the altered enzyme retains some affinity for MTX, the administration of MTX and the more potent agent would result in better growth inhibition of the resistant tumour. In this thesis, a mouse leukemia cell line (L5178Y) grown in suspension culture was used to isolate two MTX-resistant cell lines and these were used to study the mechanisms leading to MTX resistance. Both resistant cell lines exhibited impaired MTX uptake when exposed to 10⁻⁶M MTX but not when exposed to 10⁻⁴M MTX, Both lines also had elevated DHFR levels (7 to 9 fold). A variant form of DHFR present in small amounts in both cell lines was isolated by MTX-sepharose affinity chromatography. The altered DHFR differed from the major form of reductase present in these cells in its markedly lower affinity (100,000 fold) for MTX. The two forms of the enzyme were purified from the most resistant cell line and their properties compared. They were found to differ moderately in their Km for substrates, however, the Ki of MTX differed by a factor of 100,000 for the two forms. In addition there were marked differences in their heat stability, isoelectric points and sensitivity to p-chloromercuriphenyl-sulphonate, and a minor difference in their molecular weights. It is concluded that the presence of a highly resistant form of DHFR in these cell lines represents an important mechanism in conferring a high degree of resistance to these cells. The importance of this form of DHFR in MTX resistance is discussed in relation to impaired transport and elevated DHFR levels. Experiments to determine the amino acid sequence of the altered enzyme are underway and once determined should facilitate the synthesis of specific inhibitors of its activity. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 169 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains iv, 66, [6] p. : ill. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 233-247). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 233-247). Also available on the Internet.

Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains x, 231 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 188-203).

Ribonuclease HI (RNase HI), a ubiquitous, non-sequence-specific endonuclease, cleaves the RNA strand in RNA/DNA hybrids. The enzyme has roles in replication, genome maintenance, and is the C-terminal domain of retroviral multi-domain reverse transcriptase (RT) proteins. Murine Leukemia Virus (MLV) and Human Immunodeficiency Virus (HIV) are two such retroviruses and their RNase HI (RNHI) domains are necessary for viral replication, making it an attractive drug target. RNase HI has a “handle region”, an extended loop with a large cluster of positive residues, that is critical for substrate recognition. MLV-RNHI is active in isolation and contains a handle region, but, HIV-RNHI is inactive in isolation and does not contain a handle region. HIV-RT, however, has a region in its polymerase domain (positive charge cluster and aromatic cluster) that makes contact with the RNHI domain that may be serving as a “pseudo” handle region; additionally, insertion of a handle region into isolated HIVRNHI restores its activity. Overall, a breadth of information exists on this region’s dynamics, but important gaps remain unfilled; gaps that may potentially lead to creating effective drugs to treat the above-mentioned viruses. Solution-state nuclear magnetic resonance (NMR) spectroscopy combined with Molecular Dynamic (MD) simulations suggest a model in which the extended handle region domain of the mesophilic Escherichia coli RNHI (EcRNHI) populates "open" (substrate-bindingcompetent) and "closed" (substrate-binding incompetent) states, while the thermophilic Thermus thermophilus RNHI (TtRNHI) mainly populates the closed state at 300 K. In addition, an in silico designed mutant Val98Ala (V98A) EcRNHI was predicted to populate primarily the closed state. Understanding the structural features and internal motions that lead RNase HI to adopt these various conformers is of central importance to better understanding RNase HI’s role in retroviral infection. To formulate a comprehensive model on handle region dynamics, an integrative approach of NMR spectroscopy, X-ray crystallography, and MD simulations is employed. The sensitivity to internal conformational dynamics at multiple time scales of NMR spectroscopy, molecular range and resolution of X-ray crystallography, and structural interpretations of dynamic processes by MD simulations create a synergistic trio capable of tackling this issue. First, the in silico 2-state Kinetic model is validated through NMR observables that correlate with the respective conformers, thus serving as experimental analogs. The NMR parameters also correlate with the Michaelis constants (KM) for RNHI homologs and help to confirm the in silico predictions of V98A EcRNHI. This study shows the important role of the handle region in modulation of substrate recognition. It also illustrates the power of NMR spectroscopy in dissecting the conformational preferences underlying enzyme function. Next, a deeper dive is taken into handle region dynamics, specifically focusing on residue 88 and the impact its identity has on this region. Its sidechain interactions are shown to directly correlate with handle region conformations and helps to amend the originally proposed in silico 2-state Kinetic model. Lastly, looking at RNHI handle region dynamics through an evolutionary lens opens the door to uncovering novel mutations that have been previously overlooked or not identified. Through a phylogenetic analysis, researchers have reconstructed seven ancestral RNHI mutants and three of them have been expressed here. The sequence identity of these three ancestral mutants range from 60-87% to extant homologs and this is reflected by similar peak positions in their 15N HSQC spectra. Requisite experiments to assign the NMR backbone have been completed.

Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains xi, 185 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.