Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0860. Adviser: Gail Scherba. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Marek's Disease Virus (MDV) induces a wide range of neurological syndromes in susceptible hosts; however, the mechanisms behind the MDV-induced neuropathology are still poorly understood. The immediate-early 14kDa phosphoprotein, pp14, is associated with the neurovirulence phenotype of the virus. Yeast-two-hybrid screening identified the ER-bound transcription regulator, human CREB3 (cAMP Response Element-Binding protein), as an interacting partner of pp14, and fluorescence colocalisation between pp14 and chicken CREB3 (chCREB3) in MDV infected cells suggested an interaction between these proteins. The primary focus of this DPhil project was to further investigate this putative interaction using in vitro studies, with a view to determining if the interaction is linked to the neurovirulence of MDV. This investigation, which employed a combination of biochemical, cellular, and functional assays, found no conclusive evidence in support of the predicted interaction. In addition, this project aimed to gain structural and functional insights into the MDV neurovirulence factor pp14 and the host transcription factor, chCREB3. Biophysical characterisation of recombinant pp14B identifies pp14 as a molten globule. The results reveal the protein, while possessing substantial secondary structure, is largely disordered lacking a stable tertiary structure. Multiple lines of evidence from this study also indicate pp14 is a putative zinc-binding protein. Moreover, phosphorylation analysis of recombinant pp14B, extracted from DF1 cells, by mass spectrometry provides conclusive evidence for the presence of two phosphorylation sites in the shared C-terminal region of pp14 - serines 72 and 76 of pp14B. Structural flexibility, through a lack of a definite ordered tertiary structure, and functional features that can induce structural modifications indicate pp14 might interact with a number of binding partners and therefore could play multiple roles during MDV infection - a strong possibility due to the expression of the protein in all the different stages of virus infection. Furthermore, this thesis presents the crystal structure of the homodimeric chCREB3 bZIP. The chCREB3 bZIP possesses a structured DNA binding region even in the absence of DNA, a feature that could potentially enhance both the DNA-binding specificity and affinity of chCREB3. Significantly, chCREB3 has a covalent intermolecular disulphide bond in the hydrophobic core of the bZIP, which may play a role in promoting stability. Moreover, sequence alignment of bZIP sequences from chicken, human and mouse reveals only members of the CREB3 subfamily possess this cysteine residue, indicating it could act as a redoxsensor. These results indicate members of the CREB3 subfamily, by possessing a putative redox-sensitive cysteine with the capacity to form an intermolecular disulphide bond, may be activated in response to oxidative stress.

<p> The maintenance of cell surface proteins is critical to the ability of a cell to sense and respond to information in its environment. As such, modulation of cell surface composition and receptor trafficking is a potentially important target of control in virus infection. Sorting endosomes (SEs) are control stations regulating the recycling or degradation of internalized plasma membrane proteins. Here we report that human cytomegalovirus (HCMV), a ubiquitous beta herpesvirus, alters the fate of internalized clathrin-independent endocytosis (CIE) cargo proteins, retaining them in virally reprogrammed SEs. We show that the small G protein ARF6, a regulator of CIE trafficking, is highly associated with SE membranes, relative to uninfected cells. This finding suggests that ARF6 and CIE cargo egress from the SE is diminished by infection. Over expression of the ubiquitin specific protease (USP) 6, also known as TRE17, was sufficient to restore ARF6 and some ARF6 cargo trafficking to the cell surface in infected cells. The USP-activity of TRE17 is required to rescue both ARF6 and associated cargo from SE retention in infection. Intriguingly, TRE17 expression does not affect all CIE cargos retained at SEs in infection. Although TRE17 mediates the trafficking of internalized major histocompatibility complex type I (MHCI) to the cell surface in uninfected cells, MHCI is insensitive to TRE17-mediated trafficking in the context of HCMV infection. These findings demonstrate a reprogramming of endocytic trafficking by HCMV infection and suggests that HCMV hijacks the normal sorting machinery and selectively sorts specific cargos into endocytic micro-domains that are subject to alternate sorting fates.</p><p>

The Urabe AM9 mumps vaccine was removed from use in many countries due to an unacceptable incidence of post-vaccination disease. The vaccine was found to be a mixture of closely related viruses, presumably with differing degrees of attenuation. One virus isolated from the vaccine has been shown to be fully attenuated in a rodent model. This virus, Gw7, is characterized by growth in Vero cells (titers 2x106 pfu/ml) but extremely limited growth in human cell lines (&lt;104 pfu/ml), while a virulent Urabe virus, 1004, from a patient with post-vaccination meningitis, grows well (>2x107 pfu/ml) in all the cell lines tested to date. Initial sequencing of the genome of the two viruses has identified genetic differences in 6 of the 7 genes, including the two proteins of the replication complex, the polymerase, L (Large protein), and in the P protein (phosphoprotein). The hypothesis was that the amino acid differences observed in L and P play a role in the differences in attenuation in vivo and growth differences in tissue culture cells. The first objective was to confirm the sequence differences in the L and P genes of the viruses and the sequencing results confirmed all the reported differences in the L gene between 1004 and Gw7 plus identified another sequence difference in the L gene which had not been reported in the original sequencing; it also showed that there was no sequence difference in the P gene of the two viruses. The next objective was to compare the polymerase activity of the two viruses. Two methods have been employed to measure the polymerase activity of the viruses; In vivo incorporation of 3H UTP in the presence of Actinomycin D, and transcription of luciferase reporter gene from the Urabe mini-genome construct. For the first assay, the polymerase activity was examined under two different conditions; and in one case, a higher polymerase activity was observed for 1004, but this was not confirmed in the second set of experiments. To compare the polymerase activities of the two viruses with the help of a mini-genome system, the Urabe mini-genome was constructed and the polymerase activity was determined in two ways; using the polymerase complex (L, P and NP proteins) of each virus or the whole viral particles to drive the luciferase expression from the mini-genome. In either case, a significant difference in the polymerase activity of the two viruses was not detected. Thus, it can be concluded that no inherent difference in the polymerase activity between 1004 and Gw7 was observed by the performed experiments in this study.

The present study examines the interaction between host RNA polymerase II (RNAP II) and potato spindle tuber viroid (PSTVd), with the goal of locating and characterizing a putative RNAP II promoter within the viroid's RNA genome. By using a co-immunoprecipitation approach coupled with deletion and mutational analysis, RNAP II was shown to specifically bind the left terminal hairpin loop of PSTVd(+) RNA. The interaction with RNAP II appears to be dependent on PSTVd secondary structure features, rather than a particular sequence. These findings provide direct evidence of association between RNAP II and PSTVd RNA, and render a unique example of a possible RNA promoter for RNAP II. The second part of the study examines the evolutionary relationship between viroids and the hepatitis delta virus (HDV), as these pathogens share key structural and functional characteristics. We conclude, based on infection experiments, that HDV and viroids share common strategies and host factors to fulfill their respective life-cycles. We found that both HDV and an HDV mutant lacking the HDAg protein-coding region (miniHDV) can replicate in a plant host. However, miniHDV and PSTVd can replicate in human cells only in the presence of the small delta antigen (HDAg-S). Together, these results provide support for the hypothesis that HDV evolved from a viroid-like element through the capture of a cellular transcript necessary for its adaptation to a human host.

<p> A defining characteristic of alphaherpesviruses is the establishment of lifelong latency in host sensory ganglia with occasional reactivation causing recurrent lytic infections. Much remains unknown regarding the cellular and viral mechanisms involved in HSV exit from latency. We hypothesize that VP16 recruits chromatin-remodeling enzymes to immediate early gene promoters on compact-latent chromatin as a necessary step for reactivation. In order to test this hypothesis, a robust in vitro assay in which HSV latency can be established in neurons was required. In this dissertation, I explored the use of a human sensory neuron cell line as a novel in vitro model of HSV-1 latency and reactivation. HD10.6 cells were derived from embryonic human dorsal root ganglia and immortalized by a tetracycline-regulated v-myc oncogene. HD10.6 cells mature to express a sensory neuron-associated phenotype when treated with doxycycline which suppresses proliferation mediated by the v-myc oncogene. Infection at a low MOI in the presence of acyclovir results in a quiescent infection resembling latency in matured cells. HD10.6 cells provide a novel context in which to study the host and viral mechanisms of HSV-1 latency establishment, maintenance, and reactivation.</p><p>

The Herpesviridae family of viruses includes a number of human pathogens of clinical importance. Like other herpesviruses, cytomegaloviruses require a heterodimeric nuclear egress complex (NEC) consisting of a membrane-bound protein and a soluble nucleoplasmic protein, termed in murine cytomegalovirus (MCMV) M50 and M53, respectively. Genetic, electron microscopic, and immunocytochemical studies have revealed the importance of this complex for viral replication, most predominantly in facilitating egress of viral nucleocapsids across the nuclear membrane. Despite the significance of the NEC to the herpesvirus life cycle, there is a dearth of structural information regarding the components of the complex. We present here an NMR-determined solution-state structure of the conserved, structured, soluble portion of M50 (residues 1-168), which exhibits novel structural character. We mapped the binding site of a highly conserved minimal binding domain of the M53 homologue from human cytomegalovirus (HCMV; UL53) required for heterodimerization onto the structure and identified specific residues in a groove within the M50 protein fold that interact with the UL53 peptide. This site was verified biophysically and biologically: single amino acid substitutions of the corresponding residues of the homologous protein from HCMV (UL50) resulted in decreased UL53 binding in vitro, as measured by isothermal titration calorimetry, and substitutions that had the greatest effect on binding affinity caused disruption of UL50-UL53 co-localization and lethal defects in the context of HCMV infection. We then compared the effect of binding UL53 peptide with binding of the larger natural binding partner, M53 (residues 103-333) via NMR, with the results suggesting that conformational changes most likely occur on a fold-wide level in the context of the full complex. We suggest that these findings combined with the clinical relevance, the virus-specific aspects of nuclear egress, and the novelty of the structure make the HCMV NEC an attractive potential drug target. To this end, we used in silico screening to identify possible small molecule inhibitors and have begun validating top screen hits biophysically and biologically.

The brain has a tightly regulated environment that protects non-regenerating post-mitotic neurons and limits inflammation, which led to its description as a site of ‘immune privilege’. For example, viral and bacterial stimuli elicit a weaker immune response within the brain than following systemic application; injection of pathogenic stimuli into the brain leads to significantly less, if any, monocyte recruitment, T-cell priming and B-cell antibody production when compared to systemic applications over the same time period. However, the difference between the immune response of the brain and systemic circulation is not absolute, but rather relative, and applies to both the innate and adaptive immune systems. Innate immunity provides a rapid response to infections, which is often referred to as a first line of host defense, and it also enhances adaptive immune responses. Innate immune pathways are activated in the brain by local infections, during neurodegeneration, as part of neuropsychiatric disorders, and following systemic pathogenic stimuli. However, the types of innate immune pathways that are activated in the brain are unclear, including the types of cells that mount an innate immune response, and the ways in which particular cell types respond to the different innate immunity signaling molecules are also not well defined. Perhaps of most importance, however, is whether these responses are functionally effective and can limit virus spread in the brain. The brain is structurally complex, harboring extensive interactions with both the lymphatic and vascular architecture. Within the brain, the parenchyma, which includes the white and grey matter, is the area of immune privilege. We sought to test if the brain parenchyma has the cellular and molecular elements to initiate an innate immune response capable of limiting the spread of directly delivered virus. We injected VSV, a well-characterized transsynaptic tracer, or VSV-derived defective interfering particles, into the caudate-putamen and scored for an innate immune response and inhibition of virus spread. We report that the brain parenchyma has a functional type I interferon response that can limit VSV spread at both the inoculation site and between neurons. Furthermore, we characterize the response of the microglia, which are believed to be the brain’s immune cells, to VSV infection and demonstrate that infected microglia produce type I interferon and that the innate immune response is induced in un-infected microglia following infection. / Medical Sciences

Direct brain infection with VSV in mice, including the attenuated DeltaM51 strain, results in widespread viral replication, leading to irreversible and often fatal damage. When DeltaM51-VSV is administered i.v., a trace amount of the virus is found within the brain, but is eventually cleared leaving the animal physically undamaged. In response to a peripheral infection, it seems that the host is able to mount an antiviral state in the CNS. Here, I show that by "priming" the host with mutant VSV prior to direct injection of VSV into the CNS, the host is able to send "warnings" in the form of cellular messengers and/or chemical signals preparing the brain to protect itself by any means possible against VSV. I used in vivo imaging techniques coupled with direct labelling of brain tissue sections to assess the effects of priming and CNS protection. My experiments indicate that priming mice with the interferon inducing mutant VSV, DeltaM51-VSV, is in fact CNS protective. Priming limits VSV spread in the brain not by preventing infection of neurons, but by rapid induction of apoptosis in either infected cells or cells neighbouring areas of active infection. Further investigation into the factor(s) or mechanism(s) mediating viral clearance and CNS protection indicates that the innate immune system is able to control and clear DeltaM51-VSV from the CNS before an adaptive response is required.

Adenovirus (Ad) infection is accompanied by an early inflammatory response that is stimulated by the binding and/or entry of the Ad capsid to the cell. Several studies have linked NF-kappaB to the activation of pro-inflammatory cytokines and chemokines early in Ad infection, however, for many other viruses, another key transcription factor, Interferon Regulatory Factor 3 (IRF-3), also acts early in viral infection. We sought to determine if IRF-3 is activated upon Ad infection, and to examine its importance in establishing an anti-viral state in Ad-infected cells. Our data suggests that wtAd5 infection results in phosphorylation of IRF-3 on a novel amino acid residue, and this event is dependent on virus replication. Moreover, IRF-3 is an important transcription factor for induction of RANTES expression in wtAd5-infected cells. Taken together, these data suggest that IRF-3 activation in response to Ad DNA replication is important in establishing an anti-viral state within the infected cell.