UMD Theses and Dissertations
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Item Orthologous Gene Swapping and Experimental Evolution Provide Novel Way to Study Essential Poxvirus Genes(2018) Stuart, Carey A; DeStefano, Jeffrey J; Moss, Bernard; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The transcriptional program of poxviruses is divided into early, intermediate and late phases enabled by a multisubunit DNA-dependent RNA polymerase and stage-specific transcription factors that recognize cognate promoters. Although promoter sequences are highly conserved among the different chordopoxvirus genera, the transcription factors exhibit considerable amino acid divergence that parallels the evolutionary distance of the host species. Thus, the large/small subunits of the intermediate transcription factors (ITFs) of salmon gill poxvirus, crocodilepox, canarypox, and myxoma have 23/29, 40/31, 51/38 and 58/65 % amino acid identity, respectively, to the vaccinia virus (VACV) orthologs. The purpose of the present study was to determine the functional interchangeability of the ITF subunits and their putative interactions with other elements of the transcriptional machinery. A quantitative readout of ITF function using firefly luciferase (Fluc) was obtained. The activity of the large subunit orthologs was greater than that of the small subunit orthologs, with both sets following the degree of sequence similarity in relation to VACV. The same pattern was obtained with both heterospecific (e.g., myxoma large and VACV small subunits) and homospecific (e.g., myxoma large and small subunits) pairings, suggesting inefficient interactions with other elements of the transcription system. When recombinant hybrid VACV expressing the Myxoma virus (MYXV) ortholog of the small subunit (A8) were blind passaged multiple times, their replicative abilities were enhanced. Complete genome sequencing of the virus populations revealed five mutations present in the two largest subunits of the viral RNA polymerase (RNAP) and two predicted expression-enhancing mutations around the translation initiation site of the MYXV A8 ortholog. Amplicon sequencing was used to quantify the frequency of each mutation in its respective population, which revealed that they increased as passaging occurred. This indicated a correlation with increased fitness, which then needed to be confirmed, so these mutations were all experimentally introduced into the original hybrid virus and demonstrated to enhance virus replication independently. These mutations were then characterized to determine their specific effects on the viral RNAP (vRNAP) and viral replication and transcription. This approach could have broader applications for studying essential genes in poxviruses and other viruses as well.Item INVESTIGATION OF POXVIRUS HOST-RANGE AND GENE EXPRESSION IN MAMMALIAN CELLS.(2014) Mendez Rios, Jorge David; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Members of the Poxviridae family have been known as human pathogens for centuries. Their impact in society included several epidemics that decimated the population. In the last few centuries, Smallpox was of great concern that led to the development of our modern vaccines. The systematic study of Poxvirus host-range and immunogenicity provided the knowledge to translate those observations into practice. After the global vaccination campaign by the World Health Organization, Smallpox was the first infectious disease to be eradicated. Nevertheless, diseases such as Monkeypox, Molluscum contagiosum, new bioterrorist threads, and the use of poxviruses as vaccines or vectors provided the necessity to further understand the host-range from a molecular level. Here, we take advantage of the newly developed technologies such as 454 pyrosequencing and RNA-Seq to address previously unresolved questions for the field. First, we were able to identify the Erytrhomelagia-related poxvirus (ERPV) 25 years after its isolation in Hubei, China. Whole-genome sequencing and bioinformatics identified ERPV as an Ectromelia strain closely related to the Ectromelia Naval strain. Second, by using RNA-Seq, the first MOCV in vivo and in vitro transcriptome was generated. New tools have been developed to support future research and for this human pathogen. Finally, deep-sequencing and comparative genomes of several recombinant MVAs (rMVAs) in conjunction with classical virology allowed us to confirm several genes (O1, F5, C17, F11) association to plaque formation in mammalian cell lines. We also provided additional evidence that plaque formation and virus replication can be independent. More importantly, we identified a gene as the first gene outside MVA's deletion that explains its host-restriction. Replacement of this region with a cassette containing that gene derived from a replication-competent virus demonstrated to be sufficient to increase viral yield in all mammalian cell lines tested. Several research and clinical applications can be envisioned derived from this work.Item CHARACTERIZATION OF TWO HIGHLY CONSERVED POXVIRUS TRANSMEMBRANE PROTEINS OF UNKNOWN FUNCTION(2009) Sood, Cindy Leigh; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The vaccinia virus I5L open reading frame encodes a 79-amino-acid protein, with two predicted transmembrane domains, conserved among all sequenced members of the chordopoxvirus subfamily. No nonpoxvirus homologs or functional motifs have been recognized, and the role of the I5 protein remains unknown. I5 synthesis was dependent on viral DNA replication and occurred exclusively at late times, consistent with a consensus late promoter motif adjacent to the start of the open reading frame. I5 was present in preparations of purified virions and could be extracted with nonionic detergent, suggesting membrane insertion. Transmission electron microscopy of immunogold-labeled thawed cryosections of infected cells revealed the association of an epitope-tagged I5 with the membranes of immature and mature virions. Viable I5L deletion and frameshift mutants were constructed and found to replicate like wild-type virus in a variety of cell lines, indicating that the protein was dispensable for in vitro cultivation. However, mouse intranasal challenge experiments indicated that a mutant virus with a frameshift resulting in a stop codon near the N terminus of I5 was attenuated compared to control virus. The attenuation correlated with clearance of mutant viruses from the respiratory tract and with less progression and earlier resolution of pathological changes. We suggest that I5 is involved in an aspect of host defense that is evolutionarily conserved although a role in cell tropism should also be considered. The vaccinia virus A43R open reading frame encodes a 168-amino acid protein with a predicted N-terminal signal sequence and a C-terminal transmembrane domain. Although A43R is conserved in all sequenced members of the orthopoxvirus genus, no non-orthopoxvirus homolog or functional motif was recognized. Biochemical and confocal microscopic studies indicated that A43 is expressed at late times following viral DNA synthesis and is a type-1 membrane protein with two N-linked oligosaccharide chains. Neither mature nor enveloped virions contained appreciable amounts of A43, which was detected in Golgi and plasma membranes. Loss of A43R expression had no discernible effect on plaque size or virus replication in cell culture and little effect on virulence in a mouse intranasal infection model. Although the A43 mutant produced significantly smaller lesions in the skin of mice than the control, the amounts of virus recovered from the lesions were similar.Item Active and Passive Immunization Strategies for Protection of Mice and Monkeys Against Orthopoxvirus Infection(2006-10-18) Fogg, Christiana; Simon, Anne; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Poxviridae are large DNA viruses that replicate in the cytoplasm of vertebrates or invertebrates. The genus Orthopoxvirus includes variola virus, the cause of smallpox, and vaccinia virus (VACV), the prototypal family member used in the licensed smallpox vaccine. Interest in the development of an alternative smallpox vaccine emerged because of complications associated with recent vaccination efforts and the growing number of people excluded from vaccination. Antibody therapies are also of interest for Orthopoxvirus infection treatment instead of vaccinia immune globulin from human donors. Essential to these efforts are studies that elucidate aspects of the immune response required for protection against disease. Two infectious forms of virus exist, intracellular mature virus (IMV), which mediates spread between hosts, and extracellular virus (EV), which is required for efficient spread within a host. IMV and EV each possess an outer membrane with viral proteins targeted by the adaptive immune response. I have used soluble baculovirus-expressed forms of VACV proteins from the IMV and EV in order to understand the role of immunity to these particles during infection. Subcutaneous immunization of mice multiple times with the EV proteins A33 and B5 and the IMV protein L1 either individually or in combinations induced specific antibody responses and protected against weight loss and death caused by virus infection, especially following immunization with A33+B5+L1 or A33+L1. Similar patterns of protection were observed by passive immunization of mice with polyclonal or monoclonal antibodies against A33, B5, or L1 prior to or after intranasal challenge. A27 was investigated as an alternative IMV protein to L1, but proved less effective alone or in combination with A33. Potent and more rapid immune responses to the A33 and L1 proteins were stimulated by the use of the adjuvants QS-21, or alum mixed with CpG oligodeoxynucleotides. Protection against a lethal challenge was observed in a small study with monkeys that were immunized with A33, B5, and L1 and challenged with monkeypox. My data indicate protection against orthopoxviruses is seen in animal models so long as a good antibody response is made to both the IMV and EV forms.