Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
Browse
7 results
Search Results
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 HIV Reverse Transcriptase Fidelity And Inhibition Are Modulated By Divalent Cations In A Concentration-Dependent Manner In Vitro(2016) Achuthan, Vasudevan; DeStefano, Jeffrey; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human immunodeficiency virus (HIV) rapidly evolves through generation and selection of mutants that can escape drug therapy. This process is fueled, in part, by the presumably highly error prone polymerase reverse transcriptase (RT). Fidelity of polymerases can be influenced by cation co-factors. Physiologically, magnesium (Mg2+) is used as a co-factor by RT to perform catalysis, however, alternative cations including manganese (Mn2+), cobalt (Co2+), and zinc (Zn2+) can also be used. I demonstrate here that fidelity and inhibition of HIV RT can be influenced differently, in vitro, by divalent cations depending on their concentration. The reported mutation frequency for purified HIV RT in vitro is typically in the 10-4 range (per nucleotide addition), making the enzyme several-fold less accurate than most polymerases. Paradoxically, results examining HIV replication in cells indicate an error frequency that is ~10 times lower than the error rate obtained in the test tube. Here, I reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results, in physiological concentrations of free Mg2+ (~0.25 mM). At low Mg2+, mutation rates were 5-10 times lower compared to high Mg2+ conditions (5-10 mM). Alternative divalent cations also have a concentration-dependent effect on RT fidelity. Presumed promutagenic cations Mn2+ and Co2+ decreases the fidelity of RT only at elevated concentrations, and Zn2+, when present in low concentration, increases the fidelity of HIV-1 RT by ~2.5 fold compared to Mg2+. HIV-1 and HIV-2 RT inhibition by nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in vitro is also affected by the Mg2+ concentration. NRTIs lacking 3'-OH group inhibited both enzymes less efficiently in low Mg2+ than in high Mg2+; whereas inhibition by the “translocation defective RT inhibitor”, which retains the 3ʹ-OH, was unaffected by Mg2+ concentration, suggesting that NRTIs with a 3ʹ-OH group may be more potent than other NRTIs. In contrast, NNRTIs were more effective in low vs. high Mg2+ conditions. Overall, the studies presented reveal strategies for designing novel RT inhibitors and strongly emphasize the need for studying HIV RT and RT inhibitors in physiologically relevant low Mg2+ conditions.Item MODULATION OF HIV-1 REVERSE TRANSCRIPTASE AND FAMILY A DNA POLYMERASE PRIMER-TEMPLATE BINDING(2014) Fenstermacher, Katherine Joan; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Polymerases are enzymes used by all cellular and viral organisms to replicate their genomes. The human immunodeficiency virus (HIV) polymerase, reverse transcriptase (RT), uses a single-stranded RNA template to create double-stranded DNA during the course of the viral life cycle. Successful reverse transcription relies on the speed of catalysis and the ability of the enzyme to stay bound to the template during synthesis. I demonstrate that both of these properties can be modulated by the presence of different divalent cations, fundamentally altering the behavior of HIV RT. In the presence of 2 mM Mg2+, the HIV RT primer-template complex has a half-life of 1.7±1.0 min, incorporating nucleotides at a maximum rate of 3.5 nucleotides (nt) per second (average speed 1.4±0.4 nt/sec). Substituting 2 mM Mg2+ with 400 μM Zn2+ dramatically slows the speed of catalysis (maximum 0.1 nt/sec, average 0.022±0.003 nt/sec) and promotes primer-template complexes that last hours (half-life of 220±60 min). These profound changes to the enzyme's function critically inhibit reverse transcription, even in the presence of optimal concentrations of Mg2+. In addition to the cation composition during reverse transcription, previous studies have demonstrated that the sequence of the primer-template substrate can also affect the duration of a RT-primer-template complex. In light of this discovery, I investigated the tendency of two Family A DNA polymerases, the Thermus aquaticus DNA polymerase (Taq pol) and the Klenow fragment from Escherichia coli DNA polymerase I (Klenow), to selectively and tightly bind primer-template complexes. Using Primer-Template Systematic Evolution of Ligands by Exponential Enrichment (PT SELEX), I determined that both Taq pol and Klenow tightly bind to sequences containing regions that match the initiation and melting domains of promoters for the structurally similar bacteriophage T7-like RNA polymerases. This suggests a shared sequence preference that might be present in all Family A DNA polymerases, derived from a common ancestor. I plan to exploit this primer-template binding preference to advance biotechnologies utilizing these enzymes.Item Nucleocapsid protein modulates the specificity of plus strand priming and recombination patterns in Human Immunodeficiency Virus(2008-11-30) Jacob, Deena Thankam; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Replication in HIV (human immunodeficiency virus) occurs through reverse transcription in which the genomic single stranded RNA is copied into double stranded DNA. This process involves two priming events namely those of the minus and plus strand DNAs. The tRNA primer required to initiate the minus strand is carried by the virus into the host cell, while the plus strand primer is generated from a region of the genomic RNA called the polypurine tract (PPT). Results in this dissertation indicate a new role for HIV nucleocapsid protein (NC) in modulating the specificity of plus strand priming. For HIV, the central and 3′ (PPTs) are the major sites of plus strand initiation and other primers are rarely used. Using reconstituted in vitro assays, results showed that NC greatly reduced the efficiency of extension of non-PPT RNA primers, but not PPT. Extension assays in presence of mutant NCs show that the helix destabilization activity of NC and its ability to block the association of RT to non-PPT primers are responsible for the preferential extension of PPT in presence of NC. The effect of varying NC and Mg2+ concentrations on recombination during reverse transcription was also analyzed in this thesis. NC strongly influences the efficiency of recombination as well as the location where crossovers occurred. In contrast Mg2+ had a smaller effect on crossover locations. Both NC and Mg2+ influenced the level of pausing by RT during synthesis on RNA templates although NC's effect was more profound. At high NC concentrations, pausing was nearly eliminated even in locations with high predicted secondary structure. The results suggest that RT pausing may be limited during virus replication.Item Programmed Ribosomal Frameshifting in SARS-CoV and HIV-1(2007-12-10) Neeriemer, Jessica; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Programmed ribosomal frameshifting controls the ratio of two protein products made in a variety of viruses and mammalian cells. This occurs when the ribosome is translating mRNA, pauses at secondary structure, slips back one base in the 5' direction, and continues translation in a new reading frame. A series of SARS-CoV pseudoknot mutants were generated to examine important features of frameshifting, and an antibiotic was tested for its effect on HIV and SARS-CoV frameshifting. Other mutants were made in the human CCR5 gene to determine whether frameshifting occurs. It was found that mRNA stability and unpaired adenosines influence frameshifting, and increasing concentrations of the antibiotic gentamicin increases frameshifting. Moreover, CCR5, the co-receptor for HIV, contains a working frameshifting signal. This study pinpoints several antiviral targets and important factors for HIV and SARS-CoV pathogenesis.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 Human Immunodeficiency Virus Nucleocapsid Protein: Analysis of the mechanism of strand exchange and the role of the zinc fingers in nucleic acid chaperone activity.(2004-06-15) Heath, Megan Joy; DeStefano, Jeffrey J; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The human immunodeficiency virus genome is coated by the nucleocapsid protein (NC). NC is a 55 amino acid highly basic protein. It has two zinc fingers that differ by five amino acids. NC contains nucleic acid chaperone activity that aids in the formation of highly stable nucleic acid structures by destabilizing and preventing the formation of weaker structures. This activity is important for genome dimerization and maturation, tRNA:primer binding site annealing, and many steps in reverse transcription. Annealing experiments were performed with four different RNA structures and complementary DNAs. NC enhanced annealing of all structures showing that NC enhances both unwinding of nucleic acid structure and hybridization of unstructured sequences. NC mutant proteins were used in annealing assays. 1.1 NC had two copies of the first zinc finger, 2.2 NC had two copies of the second zinc finger, and 2.1 NC had both zinc fingers with their positions switched. Experiments showed that all mutants could enhance the annealing of weakly structured nucleic acids but only 1.1 NC and 2.1 NC enhanced annealing of strongly structured nucleic acids. Results suggest that finger one is important for nucleic acid unwinding while finger two plays an accessory role in annealing. The mechanism of strand exchange, another important aspect of NC chaperone activity, was also investigated. Experiments were performed using RNA:DNA hybrids with either the DNA or RNA radioactively labeled. Hybrids were incubated with different types of RNA acceptor molecules to which the DNA could transfer. The transfer of the DNA or the displacement of the original donor RNA was monitored. Experiments showed that optimal enhancement of strand exchange by NC occurred with acceptors that had more than 22 nucleotides that could anneal to the single stranded region of the DNA. Also, experiments with acceptors that had point mutations showed that the region of the acceptor that binds to the single stranded region of the DNA should be completely complementary for optimal NC stimulation. These results indicate the annealing of the acceptor and DNA outside the donor:DNA hybrid region can be an important initiation step for NC enhanced strand exchange.