Retroviruses are capable of infecting diverse vertebrates, and successful infection requires intimate interaction between virus and the host cell. During an infection, retroviral particles must bind specifically to cell surface receptors on the target cell, cross the plasma membrane, reverse-transcribe their RNA genome into double stranded DNA, find their way to the nucleus, enter the nucleus and integrate its DNA into host chromosomes. Following integration, expression of viral mRNA ensues, followed by viral mRNA export into the cytoplasm, translation of viral mRNA into proteins, and assembly of new virions that will egress from the host cell. We now appreciate that at many steps of this complex process, the virus must hijack the cellular machinery to replicate. At the same time, the host cell mobilizes a variety of cellular defense mechanisms to suppress viral infection. This thesis investigates various aspects of virus-host interactions. I will first describe the involvement of cellular transcriptional repressor protein ErbB3 binding protein 1 (EBP1) in facilitating transcriptional shutdown of Moloney murine leukemia virus (MLV) gene expression in mouse embryonic cells. Next, I describe a novel means of regulating the activity of Yin Yang 1 (YY1), a cellular transcription factor regulating retroviral gene expression, through post-translational modifications. I show that YY1 is a target of tyrosine phosphorylation by Src family kinases. Phosphorylation of YY1 impairs its ability to bind DNA and RNA, thereby downregulating its activity as a transcription factor on retroviral and cellular promoters. Apart from studying retroviral gene expression, I have also investigated intrinsic cellular defenses against retroviral infection. This is exemplified by our finding that mouse cells are intrinsically resistant to infection by betaretroviruses such as Mason-Pfizer monkey virus (M-PMV). The block against M-PMV occurs after reverse transcription and prior to viral nuclear entry. Finally, I will present ongoing work examining the fate of viral DNAs following infection, focusing on the kinetics of its association with cellular core histones and viral structural proteins. Together, this work provides critical insights into numerous aspects of the virus-host interactions.

Marburg virus (MARV)–along with Ebola Virus–comprises Filoviridae, a family of virus which causes the life-threatening hemorrhagic fever in human and non-human primates for which there is no clinically approved vaccine. For this reason, this virus can potentially lend itself to pandemic and weapons of bioterrorism. Strikingly, this virus yields asymptomatic responses in its recently discovered host Rousettus aegyptiacus. Understanding of the interaction between MARV and different animal hosts will enable the improved understanding of filovirus immunology and the development of effective therapeutic agents. Although cell lines and primary cells have been used to investigate gene expression analysis of this virus, the transcriptomic view of MARV infection on the tissue samples of animal hosts has been an uncharted territory. The comprehensive analysis of transcriptome in hosts and spillover hosts will shed light on the immune responses on a molecular level and potentially allow the comparative analysis to understand the phenotypical differences. However, there have been gaps in resources necessary to carry the transcriptome research for MARV. For example, MARV host Rousettus aegyptiacus genome and transcriptome had not been available. Furthermore, the statistical machinery necessary to analyze multi-tissue/multi-time data was not available. In this dissertation, I introduce the two items that fill these gaps and show the application of the tools I built for novel biological discovery. In particular, I have built 1) the comprehensive de novo transcriptome reference of Rousettus aegyptiacus and 2) the Multilevel Analysis of Gene Expression (MAGE) pipeline to analyze the RNA-seq data with the complex experimental design. I show the application of MAGE in multi-time, multi-tissue transcriptome data of Macaca mulata infected with MARV. In this study, 15 rhesus macaques were sequentially sacrificed via aerosol exposure to MARV Angola over the course of 9 days, and 3 types of lymph node tissues (tracheobronchial, mesenteric, and inguinal) were extracted from each sample and sequenced for gene expression analysis. With MAGE pipeline, I discovered that the posterior median log2FC of genes separates the samples based on day post infection and viral load. I discovered the set of genes such as CD40LG and TMEM197 with interesting trends over time and how similar and different pathways have been influenced in three lymph nodes. I also identified the biologically meaningful clusters of genes based on the topology-based clustering algorithm known as Mapper. Using the MAGE posterior samples, I also determined the genes that are preferentially expressed in tracheobronchial lymph nodes. In addition to new analysis tools and biological findings, I built the gene expression exploration tool for biologists to examine differential gene expression over time in various immune-related pathways and contributing members of the pathways. In conclusion, I have contributed to the two important components in the transcriptome analysis in MARV research and discovered novel biological insights. The MAGE pipeline is modular and extensible and will be useful for the transcriptome research with the complex experimental designs which are becoming increasingly prevalent with the decrease in the cost of sequencing.

Thesis: Ph. D., Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Department of Civil and Environmental Engineering; and the Woods Hole Oceanographic Institution), 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Viral predation on bacteria in the ocean liberates carbon from the particulate fraction, where it is accessible to higher trophic levels, and redirects it to the dissolved fraction, where it supports microbial growth. Although viruses are highly abundant in the ocean little is known about how their interactions with bacteria are structured. This challenge arises because the diversity of both bacteria and viruses is exceedingly high and interactions between them are mediated by specific molecular interactions. This thesis uses heterotrophic bacteria of the genus Vibrio as a model to quantify virus-host interactions in light of host population structure and ecology. The methods developed in this thesis include streamlining of standard bacteriophage protocols, such as the agar overlay, and facilitate higher throughput in the isolation and characterization of novel environmental virus-host systems. Here, >1300 newly isolated Vibrio are assayed for infection by viral predators and susceptibility is found to be common, though total concentrations of predators are highly skewed, with most present at low abundance. The largest phylogenetically-resolved host range cross test available to date is conducted, using 260 viruses and 277 bacterial strains, and highly-specific viruses are found to be prevalent, with nearly half infecting only a single host in the panel. Observations of blocks of multiple viruses with nearly identical infection profiles infecting sets of highly-similar hosts suggest that increases in abundance of particular lineages of bacteria may be important in supporting the replication of highly specific viruses. The identification of highly similar virus genomes deriving from different sampling time points also suggests that interactions for some groups of viruses and hosts may be stable and persisting. Genome sequencing reveals that members of the largest broad host-range viral group recovered in the collection have sequence homology to non-tailed viruses, which have been shown to be dominant in the surface oceans but are underrepresented in culture collections. By integrating host population structure with sequencing of over 250 viral genomes it is found that viral groups are genomically cohesive and that closely-related and co-occurring populations of bacteria are subject to distinct regimes of viral predation. / by Anne Kathryn Marie Kauffman. / Ph. D.

Dissertation (Ph.D.)--Auburn University, 2009. / Abstract. Includes bibliographic references.

Ribonuclease T1 oligonucleotide fingerprint (ONF) analysis was used to study genomic stability of La Crosse virus (Bunyaviridae) during vertical and horizontal transmission in the laboratory. No RNA genomic changes were detected in vertebrate cell culture-propagated virus isolated (following oral ingestion and replication) from the natural mosquito host, Aedes triseriatus. Genomic changes were not detected during transovarial passage of virus through two generations of mosquitoes or in virus isolated from suckling mice infected by transovarially infected mosquitoes. These results demonstrate that the La Crosse virus genome can remain relatively stable during transovarial transmission (TOT) in the insect host and during transfer between insect and vertebrate hosts. ONF analysis was used to demonstrate TOT of reassortant California serogroup bunyaviruses in Aedes treiseriatus. Mosquitoes were simultaneously inoculated with temperature sensitive mutants of La Crosse and Snowshoe hare viruses able to replicate at 33°C but not at 40°C. Putative reassortants, selected by replication at 40°C, were isolated from progeny mosquitoes and mice infected by these mosquitoes. ONF analysis confirmed that they were reassortants. Approximately 75% of the M segment and 25% of the L segment nucleotide sequences of Inkoo virus (Bunyaviridae) were determined by Sanger dideoxynucleotide sequencing of cDNA clones. Comparison of the M segment nucleotide and deduced amino acid sequences with those of four other bunyaviruses, representing two serogroups, revealed greater conservation of nucleotide than of amino acid sequence between serogroups. Areas of the sequences representing nonstructural protein(s) were less conserved than glycoprotein regions. The L segment nucleotide sequence begins with the known 3' end of the viral L segment and contains an open reading frame encoding the amino terminal 505 amino acids of the viral L protein. The amino acid sequence contains the glycine-aspartate-aspartate motif which is conserved in many RNA-dependent RNA polymerases. Comparison of the L segment sequences with those in the GEN Bank Data Base revealed no significant similarities with any other sequence.

Thesis (Ph. D.)--Duke University, 2008. / Includes bibliographical references.

Thesis (Ph.D.)--McMaster University, 2004. / Advisor: John P. Capone. Includes bibliographical references (p. 179-[208]). Available also online.

Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.

Results of the above studies thus suggested that immune dysregulation in SLE result in derangement of a spectrum of inflammatory mediators leading to possible multiple organs auto-inflammatory damages. However, the exact etio-pathogenic mechanism could not simply be explained by these phenomena. Infection has been invoked as an underlying etiology or trigger for the induction of autoimmune disease. Epstein-Barr virus (EBV) possesses multiple features that characterise its involvement in initiating or perpetuating SLE disease. Several research groups demonstrated that the peripheral blood EBV DNA load is significantly higher in SLE patients, yet cell-free viral DNA was also reported in other EBV-associated diseases such as nasopharyngeal carcinoma (NPC) and certain lymphomas, suggesting that relatively little is known about its biology and dynamic distribution in the blood circulation. In the second part of our study, we examined the cell-free and cell-associated distribution profile of EBV DNA load in SLE. Our data showed that the distribution of EBV DNA in the cell-free and cell-associated compartments exhibited a heterogeneous pattern in SLE patients. Contrary to the exclusive presence of circulating cell-free EBV DNA in NPC patients, both cell-free and cell-associated EBV DNA were detected in some SLE patients, while in others, no EBV DNA was measurable in either blood compartments. The level of cell-associated EBV viral load was significantly higher in SLE patients with active disease than those who presented with milder disease activity. This phenomenon indicated a possible association of EBV viral infection with the level of immune competence in SLE patients. It has been reported that EBV encodes proteins which shares significantly homology sequence with human IL-6, IL-8, IL-10, IL-12 and colony-stimulating factor (CSF)-1. This proposition brought our attention to the immune perturbation by EBV on the cytokine balance, possibly constitute in part, to the immune dysregulation and Th1 and Th2 dichotomy in SLE exacerbation. (Abstract shortened by UMI.) / The first section of this research study aimed to explore the messengers that influence Th1/Th2 cells differentiation, development, effector functions and hence their plausible contribution in SLE immunopathogenesis. We focused on studying the expression of cytokine and chemokine milieu that directs the traffic of T lymphocytes; co-stimulatory molecules in the activation of T lymphocytes; transcription factors T-bet and GATA-3 in regulating the differentiation of Th1 and Th2 cell lineage. We also investigated the involvement of the lymphocyte subpopulation, Th17 in the auto-inflammatory axis of SLE exacerbation. / Lit, Choi Wan. / Advisers: Christopher W.K. Lam; Y.M. Dennis Lo. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3358. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 203-235). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Thesis (Ph.D.) - University of Queensland, 2004. / Program shared between two schools. Includes bibliography.