Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Le Doux, Joseph; Committee Member: Bellamkonda, Ravi; Committee Member: Lyon, Andrew; Committee Member: Prausnitz, Mark; Committee Member: Spencer, Trent. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D. in Microbiology and Immunology)--Vanderbilt University, May 2009. / Title from title screen. Includes bibliographical references.

The study of retroviruses has been of pivotal significance to the field of biomedical science, where it has provided fundamental insights into the processes underlying both viral and non-viral carcinogenesis. Ovine pulmonary adenocarcinoma (OP A), a contagious lung cancer of sheep and goats, has emerged over the past three decades as an invaluable model of human epithelial cancers. It is one of the very few animal models of retrovirus induced neoplasia of epithelial tissues, whereas most other such animal models of human cancers pertain to the haematopoietic system. OP A represents a unique, naturally occurring, inducible, outbred animal model of peripheral lung carcinomas, and is caused by a betaretrovirus - jaagsiekte sheep retrovirus (JSRV) - that is receiving increasing attention in the fields of retrovirology and lung cancer research. JSRV exists in two highly homologous, yet molecularly distinct forms. The first is an exogenous form of the virus that is transmitted horizontally from one animal to another. This form is infectious and the direct cause of OP A. The other is an endogenous form, 15 to 20 proviral copies of which reside benignly in the genome of sheep and are transmitted vertically from one generation to the next. At the time this study commenced, no knowledge existed regarding the underlying pathogenic mechanism by which JSRV causes OPA. Even though the nucleotide sequence of exogenous JSRV had been elucidated seven years earlier, only limited sequence information was available on endogenous JSRVs. With a view towards identifying genetic regions or elements within exogenous JSRV that could potentially be implicated in its pathogenic function, this study began with the cloning of the first three full-length endogenous JSRV loci ever isolated from sheep. The DNA sequences of these full-length endogenous JSRV loci were determined and comprehensively analysed. Comparison with exogenous JSRV isolates revealed that the two forms of the virus are highly homologous, yet can be consistently distinguished in three short regions within the coding genes. Two of these reside in the gag gene, and one at the end of the env gene. These regions were named the variable regions (VRs) of sheep betaretroviruses. The JSRV VR3 in env was linked by our collaborators to the virus's ability to transform cells in tissue culture. The effects and biological significance of VRI and VR2 in gag are subtler and more difficult to determine. After identifying these regions, it became the objective of this study to develop relevant molecular tools that could be used to discern the significance of these variable regions in vivo, and to characterise these tools in vitro to assess their suitability for in vivo studies. The development of these tools entailed the design of a novel strategy that was implemented to precisely substitute the endogenous VRI and VR2 (individually and in combination) into an infectious molecular clone of exogenous JSRV. These chimeric constructs were shown to support retroviral particle release into the supernatant of transiently transfected cells in tissue culture. These particles were confirmed by independent experiments to have arisen specifically from transfection with the chimeric clones. Finally, the particles were shown to be capable of infecting cultured cells and of productively integrating their genomes into those of their host cells, rendering these particles fully competent retroviruses that can be used in the context of in vivo studies to determine the biological significance of VRI and VR2. This study has made a significant contribution to the further development of the OP A / JSRV model system of human epithelial lung cancers. It has also led to the design of a molecular substitution strategy that can be adapted to introduce any genetic region into a cloned DNA construct, regardless of the degree - or lack of interrelation - of the two DNA sequences, thereby creating a highly versatile molecular biological tool. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2007.

It is known that many types of leukemias and lymphomas are of viral origin. A new strain of immunologically deficient mice, the BALB/c x C57B1/6 beige nude mice, has been observed to develop spontaneous lymphomas of unknown origin at a high frequency. It is possible the tumors originate from a retroviral infection, which we attempted to show by detection of viral reverse transcriptase (RT) activity. We measured the (RT) activity in the supernatants of cocultures from the spleen and lymph node tissues of the beige nude animals by two methods, tritiated thymidine triphosphate incorporation in a standard RT assay, and the commercially available RT-DetectTM (DuPont) method. Of all supernatants tested, none showed a significant amount of RT activity compared with a cell line that was known to be actively producing the retrovirus MuLV. Upon electron microscopic analysis of the tumor-like cells grown in coculture, no viral particles were observed. Flow cytometric analysis of the tumor-like cells showed two general phenotypes; one predominately of a helper T cell type, and the other of a less differentiated immature thymocyte type. / Department of Biology

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.

The most common gene transfer vehicle used in gene therapy protocols are mammalian virus vectors. Specifically, retroviruses are one of the most common viral vectors used since they are able to permanently integrate their transgene into the host cell genome, providing, in principal, to a long-term cure. The potential applications of gene therapies are vast, ranging from monogenic disorders such as cystic fibrosis to complex gene disorders such as cancer. However, the application of such therapies in clinical settings has been limited partially because of inefficient gene delivery of the therapeutic gene to diseased cells. Furthermore, safety concerns of accidently altering the genetic expression in healthy bystander cells or nearby tissue has increased the interest in creating targeted viral vectors which infect only the diseased cells without infecting others. Thus, the success of gene therapy will depend on identifying and understanding the parameters critical for virus entry into cells, including factors that facilitate virus absorption onto the cell surface, virus binding, and fusion. The objective of this thesis was to understand the role of lipids in binding and infection, and to investigate the use of lipid-based conjugates to alter the surface of virus particles.

Understanding the host response immediately following mucosal HIV-1 infection will be pivotal in determining whether the immune response induced by a vaccine will successfully sense and control viral replication. In order for effective vaccine strategies and modalities to be developed, these earliest immunological events must be fully assessed in a non-biased manner. Nonhuman primates (NHP), specifically Rhesus macaques (RM), serve as a model to investigate the immunological landscape immediately post-challenge and to define the spatiotemporal path of simian immunodeficiency virus (SIV). SIV infection of RM serves as a model of human HIV infection as it recapitulates many of the virological, immunological, and pathological features of HIV infection in the human host. In this thesis I will test the hypothesis whether transcriptional analysis will allow a sensitive measure of the early innate immune responses that accompany detection of the SIV virus in the periphery. I have determined that an early inflammatory profile arises early in tissues proximal to the challenge site that precedes widespread immune activation and the systemic antiviral interferon response. This study defines in detail the spatiotemporal relationship between virus and host immune response and may be a valuable resource in guiding future vaccine design strategies. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection