Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item Mutational analysis of Human Immunodeficiency Virus type-1 nucleocapsid protein to evaluate its nucleic acid chaperone activity(2006-09-15) NARAYANAN, NIRUPAMA; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The highly basic 55 amino acid nucleocapsid protein (NC) that coats the HIV-1 genome has two zinc fingers that differ by five amino acids (strain pNL4-3). Previous work showed that NC's first finger (N-terminal) is primarily responsible for unwinding secondary structures (helix destabilizing activity), while the second (C-terminal) plays an accessory role. The amino acid differences between the fingers are (finger one to finger two): phenylalanine to tryptophan (F to W), asparagine to lysine (N to K), isoleucine to glutamine (I to Q), alanine to methionine (A to M), and asparagine to aspartic acid (N to D) at positions 16, 17, 24, 25, and 27 of finger one, respectively. To determine at an amino acid level the reason for the apparent distinction between the fingers, five point mutants were designed with amino acid residues in finger one incrementally replaced by those at corresponding locations in finger two. Each mutant was analyzed in annealing assays with unstructured and structured substrates. Three groupings emerged: (1) those similar to wild type (wt) levels (N17K, A25M), (2) those with diminished activity (I24Q, N27D), and (3) mutant F16W which had substantially greater helix destabilizing activity than wt NC. All mutants retained wt levels of the condensation/aggregation activity of NC. Unlike I24Q and others, N27D was defective in DNA binding. Only I24Q and N27D showed reduced strand transfer in in vitro recombination assays. Double and triple mutants F16W/I24Q, F16W/N27D, and F16W/I24Q/N27D all showed defects in DNA binding, strand transfer, and helix destabilization, suggesting that the I24Q and N27D mutations have a "dominant negative" effect and abolish the positive influence of F16W. Results show that amino acid differences at positions 24 and 27 contribute significantly to finger one's helix destabilizing activity and hence NC's chaperone activity. Preliminary results from in vivo experiments indicated that virus with the N27D mutation is infectious at near wt NC levels. This suggests that aggregation activity may be more important than helix destabilizing for viral viability. Results from two other forms of HIV-1 NC (NCp9 and NCp15) and NC proteins from Simian Immunodeficiency Virus and Murine Leukemia Virus are also reported.Item In Vitro Synthesis of Long Reverse Transcription Products from Genomic RNA of Human Immunodeficiency Virus(2006-04-25) Anthony, Reshma Merin; DeStefano, Jeffrey J; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The retroviral reverse transcription reaction normally occurs in capsid-like structures in the cytoplasm of infected cells. Reverse transcription can also be carried out in vitro in totally reconstituted reactions with purified enzymes and model RNA templates. However, in this case fully synthesized DNAs are rarely generated from genomic RNA. This could be because the capsid creates an extremely concentrated and specific environment that cannot be completely reproduced in vitro. An in vitro system that closely mimics replication and that can be easily manipulated would enhance our understanding of the replication process. In this thesis report, in vitro reaction conditions that allowed efficient synthesis of DNA products up to 4 kb from genomic RNA segments of Human Immunodeficiency Virus (HIV) were generated. The reactions required high amounts of HIV reverse transcriptase enzyme (RT) and nucleocapsid protein (NC) sufficient to completely coat the RNA template in the reaction. Synthesis of long DNA products required the formation of high molecular weight aggregates with nucleic acids, RT and NC. Removal of the dimerization region did not affect synthesis of long DNA products in vitro. Processivity of RT does not play a role in the synthesis of long DNA products. NC finger mutants lacking either finger or with the finger positions switched were all effective in synthesizing long DNA products suggesting that the aggregation/condensation activity but not the unwinding activity of NC is required for the synthesis of long DNAs in vitro. These results taken together, we propose that high molecular weight aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC into a smaller area, thereby mimicking the role of the capsid environment within the host cell. In addition, strand transfer assays indicate that strand transfer is the molecular mechanism involved in the synthesis of long DNAs and the rate of transfer (cross-over events per nucleotide synthesized) is higher than that found in tissue culture-based recombination assays. An in vitro system that closely mimics what occurs in the cell could be used to screen inhibitors on RT, NC and recombination.