Browsing by Author "Fred Fuller, Committee Member"

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    Characterization of Furin Protease Sensitive Site Processing and Its Effects on Sindbis Virus Assembly and Budding
    (2006-02-27) Nelson, Steevenson; Carla Mattos, Committee Member; Fred Fuller, Committee Member; Dennis Brown, Committee Chair; John Cavanagh, Committee Member
    Sindbis virus particles are composed of three structural proteins (C/E2/E1). The E1 glycoprotein is organized into a highly constrained, energy-rich conformation. Its hypothesized that this energy is utilized to drive events that deliver the viral genome to the cytoplasm of a host cell. The extraction of the E1 glycoprotein from virus membranes results in disulfide-bridge rearrangement and the collapse of the protein to a low-energy, non-native configuration. In a new approach to the production of membrane glycoproteins, furin protease recognition motifs were installed at various positions in the E1 glycoprotein ectodomain. Proteins containing the furin sensitive sites undergo normal folding and assembly in the endoplasmic reticulum and only experience the consequence of the mutation after transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane from which they are assembled. This processing also impacts the envelopment of the nucleocapsid in the modified plasma membrane. E2 has been shown to be responsible for host receptor recognition and thus plays a critical role in the virus lifecycle. To expand on our previously characterize E1 furin mutant study, we installed furin protease recognition motifs at various positions in the ectodomain of E2. Mutants were analyzed for production of truncated proteins and the effect of the mutations on virus assembly and budding was also characterized. Processing of the E2 mutants by the enzyme furin results in the release of the truncated proteins from the membrane in a fashion that is similar to the processing observed in the E1 furin sensitive mutants. This processing was also observed to impact envelopment of the nucleocapsid with virus protein modified plasma membrane at a step consistent with an early event in the envelopment process. Overall, this technique provides a unique method for studying the mechanism of virus assembly and protein structure without altering crucial early events in protein assembly, folding and maturation.
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    PKR, Myocarditis and the Cardiac Response to Reovirus Infection
    (2004-06-20) Stewart, Michael Jude; Scott Laster, Committee Member; Tim Petty, Committee Member; Fred Fuller, Committee Member; Barbara Sherry, Committee Chair
    Viral myocarditis is an important human disease associated with many viruses. Mechanistically, cardiac damage associated with viral myocarditis can be immune mediated and/or the result of direct cytopathic effect. Reovirus induced myocarditis is not immune mediated, and thus, provides an excellent model for the study of direct cytopathic effect in the heart. Previous work has demonstrated that reovirus myocarditic potential reflects induction and/or sensitivity to interferon (IFN). Specifically, nonmyocarditic reoviruses induce greater IFN-β and/or are more sensitive to the antiviral effects IFN than are myocarditic reoviruses. Importantly, IFN mediates its antiviral effects through the induction of interferon-stimulated genes (ISGs); ISGs function as the antiviral effector proteins that block replication in the host cell. The work presented in this dissertation further defines the cardiac response to reovirus infection. In chapter 2, we examined the double-stranded activated protein kinase PKR: the role of PKR in cardiac IFN induction and protection against reovirus-induced myocarditis. We demonstrate that PKR is critical to the robust induction of IFN-β in primary cardiac myocyte cultures (PCMCs). Additionally, we show that nonmyocarditic reoviruses become myocarditic in PKR-null mice, even though reovirus growth in PKR-null hearts is similar to that in wild type mouse hearts. Finally, we demonstrate that relative to wild type mice, reoviruses induce significantly greater morbidity in PKR-null mice. In the following chapter, we compare the IFN response in PCMCs: a non-replenished, critical cell type, to that in primary cardiac fibroblast cultures (PCFCs): a readily replenished cardiac cell type. By quantitative real-time PCR, we find that PCMCs express IFN-β and the ISGs IRF-7 and 561 constitutively at higher levels than PCFCs. We also identify constitutive IFN as a primary means of constitutive ISG expression. Additionally, we demonstrate that PCFCs, more so than PCMCs are more dependent on IFN-mediated protection against reovirus infection. The regulation of IFN-β expression is achieved primarily through the actions of Interferon Regulatory Factors (IRFs). In Appendix 1, we show contributions to a publication demonstrating that IRF-1 is dispensable for IFN-β induction in PCMCs, and yet critical for defense against reovirus-induced myocarditis in the mouse. Finally, a summary of this work is provided.
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    T regulatory Cell Suppression of CD8+ Lymphocyte Responses During FIV Infection.
    (2009-01-07) Fogle, Jonathan Edward; Fred Fuller, Committee Member; Gregg Dean, Committee Member; Ed Breitschwerdt, Committee Member; Mary B. Tompkins, Committee Chair
    BSTRACT FOGLE, JONATHAN EDWARD. T regulatory Cell Suppression of CD8+ Lymphocyte Responses During FIV Infection. (under the direction of Mary Tompkins.) The action of activated CD8+ lymphocytes is critical to the control and elimination of viral pathogens. Impaired CD8+ immune responses are well recognized in lentiviral infections; however, the mechanisms underlying CD8+ impairment are incompletely understood. Using the FIV model for human AIDS, we reported previously that CD4+CD25+ Treg cells in both the acute phase and long-term, asymptomatic phase of infection are constitutively activated and suppress CD4+CD25- T cell immune responses. Building upon these observations, we tested the hypothesis that CD4+CD25+ Treg cells suppress CD8+ responses to immune stimulation during both the acute and chronic, asymptomatic stages of FIV infection. SPF cats were infected with NCSU1 FIV. During the acute stage of infection, plasma viremia as well as PBMC and LN lymphocyte phenotype was assessed at regular intervals. Unfractionated lymph node, CD4+CD25+ depleted lymph node, and CD8+ / CD4+CD25+ co-cultures were assayed for IFNï § production via a feline specific ELISpot. During the chronic, asymptomatic phase of infection, IFNï § mRNA in CD8+ lymphocytes was assessed using real time RT-PCR following CD8+ co-culture with CD4+CD25+ lymphocytes. Our results demonstrated that the CD8+ nadir at 14 days corresponds to peak plasma viremia and is followed by an increase in CD8+ number to greater than pre-infection values. Ex-vivo depletion of CD4+CD25+ lymphocytes from lymph node suspensions significantly enhanced the production of IFNï § during the acute phase of infection. Furthermore, co-culture of CD8+ lymphocytes with CD4+CD25+ lymphocytes results in suppression of CD8+ IFNï § production during both the acute and chronic stages of infection. The same observations were not evident in uninfected cats evaluated in an identical manner. These results demonstrate the profound suppressive effect of CD4+CD25+ T regulatory cells on the CD8+ immune response during the acute and chronic stages of FIV infection. Although the mechanism of CD4+CD25+ T cell-mediated suppression is controversial, there is strong evidence to suggest that, at least in some models, it occurs via a TGFb / TGFbRII signaling pathway. We hypothesize that during the early acute stage of FIV lentiviral infection, TGFï ¢ is up-regulated on the plasma membrane of Treg cells (mTGFï ¢), which engages TGFï ¢RII on the surface of antigen activated CD8+ cells thus transducing a signal through the Smad pathway for G1 cell cycle arrest (anergy) and effectively aborting CD8+ T cell expansion and a sustained CD8+ immune response. The experiments that follow demonstrate up-regulation of mTGFï ¢ in the CD4+CD25+ subset and up-regulation of TGFï ¢RII in the CD8+ subset of FIV+ cats as assessed by FACS analysis. Furthermore, we demonstrate Smad 2 phosphorylation in CD8+ targets following CD4+CD25+ / CD8+ co-culture.