Cell Biology & Molecular Genetics Theses and Dissertations
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Item A Comparative Analysis of the Binding Affinity of HIV-1 Reverse Transcriptase to DNA vs. RNA Substrates(2010) Olimpo, Jeffrey T.; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA-DNA versus DNA-DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3´ recessed DNA base) are required for stable binding to RNA-DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA-DNA. Replacing 2´- hydroxyls on pivotal RNA bases with 2´-O-methyls did not affect stability, indicating that interactions between hydroxyls and RT amino acids do not stabilize binding. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA-DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA-DNA and DNA-DNA, respectively, allow the former to conform more easily to HIV-RT's binding cleft, leading to more stable binding. Biologically, this may aid in degradation of RNA fragments that remain after DNA synthesis.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 Functions of the Tobacco mosaic virus helicase domain: regulating formation of the virus replication complex and altering the activity of a host-encoded transcription factor(2008-04-23) Wang, Xiao; Culver, James N; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tobacco mosaic virus (TMV)-encoded 126-kDa and 183-kDa replicases are multidomain and multifunctional proteins. The helicase domain shared by both replicases has been shown to perform multiple tasks during the virus life cycle. In vitro structural and functional analyses demonstrated that monomers and dimers of the TMV helicase domain were the active forms for ATP hydrolysis. However, self-interaction of the helicase polypeptides resulted in the formation of higher-order structures that likely serve as structural scaffolds for the assembly of virus replication complexes (VRCs). Mutagenesis studies of the TMV helicase motifs showed that conserved amino acid residues played important roles in protein ATPase and/or RNA binding activities. A close correlation between ATPase activity of the helicase domain and assembly of wild-type VRC-like vesicles by the 126-kDa replicase further suggests that ATPase activity of the TMV helicase domain may modulate proper VRC assembly. In addition to helicase self-interaction, a novel virus-host interaction involving ATAF2, a NAC domain transcription factor was identified. Members within the NAC domain family are involved in plant developmental processes and stress/defense responses. In this study, transgenic plants overexpressing ATAF2 showed a strong developmental phenotype. Inoculation of TMV in these transgenic plants resulted in reduced virus accumulations. Additionally, transcriptional induction of ATAF2 occurred in response to TMV infection and salicylic acid treatment. Combined, these results suggest that ATAF2 is involved in a host defense response. One interesting finding was that in susceptible hosts, virus-directed induction of ATAF2 and PR1, a well-defined pathogenesis-related (PR) marker gene for host defense system, occurred only in locally-infected but not in systemically-infected tissues. Dynamic changes in the expression of host defense genes suggest that viruses have evolved certain mechanisms to actively modulate host gene expression. Interaction between the TMV helicase domain and ATAF2 may provide one way to suppress the ATAF2-mediated host defense signaling pathway. Combined these studies investigated the importance of the TMV helicase domain in VRC formation and in manipulating the host defense system. The results confirmed the functional versatility of the TMV helicase domain in establishing a successful virus life cycle.Item Characterization of the TMV replicase proteins: localization and interactions with Rab GDI proteins(2008-04-22) Kramer, Sabrina Renee; Culver, Jame; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tobacco mosaic virus (TMV) is a model positive-strand RNA virus. TMV encodes two replicase proteins, both of which contain methyltransferase and helicase domains; the 183 kDa protein contains an additional RNA-dependent RNA polymerase domain. Using this virus, virus-host interactions important in the initial establishment of infection and formation of replicase complexes were investigated. Specifically, on the virus side, replicase proteins were examined for regions that may contribute to its localization to the endoplasmic reticulum (ER) during TMV infection. An ER localization domain was identified in a region between amino acids 599 and 701. Alanine substitutions were introduced into this region and examined for their effects on the virus. Several possible hypotheses are discussed as to how this domain may function during infection. Concerning the host, an interaction with a host protein, a Rab GDP Dissociation Inhibitor (Rab GDI), was examined. This interaction occurred with tomato and tobacco Rab GDIs as well as with the originally identified Arabidopsis thaliana Rab GDI (AtGDI2). Silencing of Rab GDI transcripts enhanced the number of infection sites in TMV:GFP-infected plants, but did not alter viral movement or overall accumulation, indicating a possible role in initial establishment of infection. Rab GDI-silenced Nicotiana benthamiana plants showed cellular morphologies similar to those of TMV-infected cells. Moreover, TMV infection results in Rab GDI proteins localizing to structures associated with viral replication. Taken together these data indicate a role for Rab GDI proteins in the initial establishment of infection. Two models of how Rab GDI proteins may contribute to TMV infection are discussed. These studies examine parts of the viral life cycle that are not very well understood, in particular the initiation and establishment of infection. Although vesicle trafficking has been shown to be important for several different pathogens, this is the first time that a Rab GDI protein has been identified as participating in viral replication. Understanding initiation of infection and susceptibility of a host to a pathogen are vital to elucidating pathogen-host interactions and developing disease resistance strategies.Item Regulation of infected cell fusion by the vaccinia virus A56 and K2 proteins(2008-02-29) Wagenaar, Tim; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Poxviruses are a group of large double-stranded DNA virus that replicate in the cytoplasm of the cell. The Orthopoxvirus genus includes variola virus, the etiological agent of smallpox, and vaccinia virus (VACV), the prototypical member of the genus. Cells infected with VACV display very little cell-cell fusion, however VACV mutants deleted for either the A56R or K2L gene display extensive cell-cell fusion. A56 and K2 interact with one another (A56/K2) and expression of both proteins is important for preventing cell fusion. VACV entry and fusion requires a multiprotein entry fusion complex (EFC) composed of at least eight proteins. In the absence of a functional EFC infected cell fusion does not occur even when the viruses lack either A56 or K2. A panel of recombinant VACVs was used to define protein interaction important for regulation of cell fusion. Affinity purification of A56, K2 and the EFC revealed an interaction between A56/K2 and the EFC. This interaction required expression of both A56 and K2 as A56 did not bind the EFC in the absence of K2 and vice versa. Interestingly, the ability to bind the EFC correlated with the inhibition of infected cell fusion by A56 and K2. Although the EFC contains eight proteins, only two entry proteins, A16 and G9, were important for binding A56/K2. Individually, A16 and G9 did not bind A56/K2; instead both A16 and G9 were needed for efficient interaction with A56/K2. A16 and G9 copurified with one another when expressed by transfection in uninfected cells, confirming that the two proteins bind to one another suggesting they directly interact within the EFC. To support a biological role for A56/K2 binding the EFC, cells expressing A56 and K2 were tested for infectivity as well as their ability to undergo cell-cell fusion. In both cases, cells expressing A56 and K2, but not individual expression of A56 or K2, showed reduced cell-cell fusion and virus entry. Collectively, these data support a model by which A56/K2 regulate infected cell fusion through an interaction with the viral EFC.Item An Internal tRNA-Like Structure Regulates the Life Cycle of a Plus-Sense RNA Virus(2007-12-12) McCormack, III, John Crisler; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Turnip crinkle virus (TCV) is a 4054 b plus-sense RNA virus that belongs to the genus Carmovirus in the Family Tombusviridae. The 3' terminal 200 b of TCV are predicted to fold into 5 hairpins labeled in the 3' to 5' direction as the promoter (Pr), hairpin 5 (H5), hairpin 4b (H4b), hairpin 4a (H4a), and hairpin 4 (H4), using 3' UTR phylogenetic comparisons with other carmoviruses and the RNA structural prediction program, mfold. H5 was found to be a highly-conserved structure containing a large symmetrical loop (LSL) that formed a tertiary interaction between the 3' side of the LSL and the 3' terminal nucleotides using compensatory mutational analysis in vivo. In plants, LSL mutations resulted in a mutation frequency that was increased by as much as 12-fold without inducing error catastrophe. The original mutations frequently reverted and led to second site alterations biased for uridylate to cytidylate and adenylate to guanylate changes. These results suggest that H5 may function as a chaperone to properly fold the RdRp. The TCV 5' UTR, which binds 40S ribosomal subunits, contains two short segments exhibiting IRES activity that function synergistically with the 3' terminal region to enhance cap-independent translation in vivo. In the TCV 3' UTR, H4a, H4b, H5, and flanking sequences, form an internal tRNA-like structure (iTLS) that binds 60S ribosomal subunits and the P-site of salt washed 80S ribosomes. The iTLS may therefore mediate assembly of 80S ribosomes, which are then transported to the 5' end for translation of virally-encoded proteins. Phylogenetic comparisons of carmovirus 3' UTRs revealed that Cardamine chlorotic fleck virus (CCFV) and Japanese iris necrotic ring virus (JINRV) are capable of forming the 5 elemental features comprising the iTLS. Ribosome binding and plant cell culture assays showed that only the CCFV iTLS bound 80S ribosomes and could functionally replace the TCV iTLS. These results suggest that closely-related members of the same viral genus may utilize different strategies for cap-independent translation.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 MOLECULAR CHARACTERIZATION OF INTERACTIONS BETWEEN TMV REPLICASE PROTEIN AND AUXIN RESPONSIVE PROTEINS: IMPLICATIONS IN DISEASE DEVELOPMENT(2006-11-25) Padmanabhan, Meenu Sreedevi; Culver, James; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tobacco Mosaic Virus and Arabidopsis thaliana serve as ideal model systems to study the molecular aspects of virus - host interactions. Using this system, an interaction between the helicase domain within TMV replicase protein and an auxin responsive protein, IAA26 was identified. IAA26 is a member of the Aux/IAA family of transcription factors that function as repressors in signaling pathways controlled by the phytohormone auxin. Characterization of the interaction was carried out utilizing a helicase mutant defective in its interaction with IAA26 and by creating transgenic plants silenced for IAA26 expression. These studies suggest that the interaction was not essential for either viral replication or movement but promoted the development of disease symptoms. Cellular co-localization studies revealed that in TMV infected tissue, the nuclear localization and stability of IAA26 was compromised and the protein was relocalized to distinct cytoplasmic vesicles in association with the viral replicase. In keeping with its role as a transcription factor, the alterations in IAA26 function should lead to misregulation of downstream auxin responsive genes and this is supported by the fact that ~ 30% of the Arabidopsis genes displaying transcriptional alterations to TMV could be linked to the auxin signaling pathway. Aux/IAA family members share significant sequence and functional homology, and an additional interaction screen identified two more Arabidopsis Aux/IAA proteins, IAA27 and IAA18 and a putative tomato Aux/IAA protein, LeIAA26 that could interact with TMV helicase. The nuclear localization of these three proteins was disrupted by TMV and alterations in LeIAA26 levels induced virus infection-like symptoms in tomato. Additionally, transgenic plants over-expressing a proteolysis resistant mutant of IAA26 showed abnormal developmental phenotype, the severity of which was abrogated during TMV infection which blocked nuclear accumulation of the protein. Taken together, these findings suggest that TMV induced disease symptoms can partially be explained by the ability of the virus to disrupt the functioning of interacting Aux/IAA proteins within susceptible hosts. The significance of such interactions is yet to be determined but it appears that they may be advantageous to the virus while infecting tissues that are in a developmentally static stage.