Herpes Simplex Virus-1 (HSV-1) infection has been shown to be very resistant to the effects of the cellular interferon response. However, in the absence of viral gene expression HSV-1 induces the expression of cellular interferon-stimulated genes, possibly through activation of the interferon-regulatory factor 3 (IRF-3) cascade. The induction of IRF-3 by HSV-1 infection has not been well characterized. Using an HSV-1 mutant deficient in the expression of viral genes, we show that HSV-1 can induce the cascade of IRF-3 activation by stimulating the phosphorylation of IRF-3 and the nuclear localization of the protein. We will show that activation of this pathway leads to the expression of interferon-stimulated genes and the production of a protective antiviral response. Further we will show that the expression of one viral gene, ICP0, is essential and sufficient to the inhibition of interferon-stimulated gene expression. An HSV-1 mutant deficient in expression of all immediate-early HSV-1 genes except ICP0 does not induce nuclear accumulation of IRF-3 and does not induce the expression of interferon-stimulated genes. This virus induces the mis-localization of the kinase (TBK-1) responsible for the phosphorylation of IRF-3. Cytoplasmically localized ICP0 expressed in wild-type infection stimulates the translocation of TBK-1 from the cytoplasm to the Golgi apparatus. This ICP0-mediated mis-localization, as well as a portion of that induced by the ICP0 expressing mutant, can be inhibited through the inhibition of the proteasome. Further, IRF-3 nuclear translocation and interferon-stimulated gene expression can be restored during infection with the ICP0-expressing mutant virus in the presence of proteasome inhibitors. This study explores the induction of the interferon regulatory factor-3 cellular antiviral response pathway, and presents a possible mechanism for the virus' resistance to the cellular antiviral response.

Extensive genetic variability at particular loci is observed among many bacteria because alleles confer higher fitness advantages under certain situations. Extensive diversity is observed at the Salmonella rfb locus, encoding enzymes responsible for synthesis of the O-antigen polysaccharide. Historically, diversity at the rfb locus was thought to be caused by selective pressures from the immune system and maintained by frequency dependent selection (FDS). This hypothesis works well for pathogens like Haemophilus influenzae and Neisseria meningitis, which alter their O-antigens during the course of an infection. In contrast, Salmonella does not alter its O-antigen. More importantly, Salmonella shows host-serovar specificity, whereby strains bearing certain O-antigens cause disease primarily in specific hosts; this is inconsistent with FDS. Alternatively, selective pressure may originate from the host intestinal environment itself, wherein diversifying selection (DS) mediated by protozoan predation allows for the continued maintenance of rfb diversity and the survival of Salmonella. To test if predation may be a selective pressure influencing O-antigen diversity, amoebae were isolated from separate intestinal environments and shown that these amoebae recognize antigenically diverse Salmonella with different efficiencies. More importantly, it was demonstrated that feeding preferences are upheld when Salmonella differ only by their O-antigen. Thus, protozoan predation may be the selective pressure influencing O-antigen diversity. For extensive genetic diversity to be maintained by DS, a particular O-antigen should confer a higher fitness in a certain environment. To test this hypothesis, amoebae were isolated from the intestines of fish, tadpoles, lizards, and turtles and their feeding preferences were determined. As expected, related amoeba from the same host share preferences. Strikingly, unrelated amoebae from the same intestinal environment also had significantly similar feeding preferences, and related amoebae isolated from different environments showed no similarity in prey choice. This demonstrates that amoebae from an environment share feeding preferences. In concert, O-antigen variability may result from selective pressures of predation and subsequently may be maintained by DS whereby a certain O-antigen confers a higher fitness advantage depending on its residing environment. This makes sense of the serovar-host specificity and the clonality of O-antigens among Salmonella that were not explained by previous hypotheses.

Antiretroviral drug treatments and vaccine strategies are hampered by the ability of the HIV to generate variants able to evade their protective effects. Understanding the effects of these interventions on virus evolution could aid in the design of targeted antiviral strategies. We addressed this in a cohort of SIV infected non-human primates given short-term antiviral drug treatment (ART) with and without DNA vaccinations. We hypothesized that the most potent therapies (e.g. those that suppress virus burden to the greatest degree) would limit virus evolution. Our results supported this hypothesis. There was no apparent vaccine effect however. These results could indicate that the immune response was not strong enough to induce changes in the global virus population, evolution must be monitored at the epitope level to be revealed, or the most informative time points were unavailable due to low virus burdens. We additionally hypothesized that infection of the gut associated lymphoid tissue may render this organ as a reservoir for expression of unique viral genotypes. We demonstrated that high plasma virus loads were associated with high tissue virus loads and wide dissemination of genotypes. In contrast, the lymphoid tissues on animlals that controlled their virus burden contained genotypes not expressed in other organs. Our results have important implications on studying virus evolution in vivo by demonstrating that large populations and potentially numerous virus genes need to be analyzed.

The pXO1 and pXO2 plasmids of B. anthracis are both necessary for full virulence, and understanding the mechanisms by which these plasmids replicate would be helpful in combating anthrax and the spread or use of these plasmids in other systems. A 5-kilobase region of the pXO1 plasmid was cloned into an E. coli vector and replicates when introduced into B. anthracis. Deletion analysis indicated that a 158bp region containing a stem-loop structure contains the origin of replication. Mutational analysis showed that open reading frame 45 (repX) of pXO1 is required for the replication of the miniplasmid in B. anthracis. Interestingly, repX showed limited homology to bacterial FtsZ proteins that are involved in cell division. A mutation in the predicted GTP binding domain of RepX abolished its replication activity. RepX was purified as an MBP- as well as His- N-terminal fusion by overexpression in E. coli and had strong GTPase activity. RepX also bound to DNA weakly and non-specifically. A potential origin of replication (ori) and replication initiator gene, repS of plasmid pXO2 was cloned into an E. coli vector (pBSCm) which was shown to replicate in B. anthracis, B. cereus, and B. subtilis. The mini pXO2 replicon could not be established in B. subtilis polA mutant, suggesting that DNA Pol I is required for plasmid replication. A countertranscript encoded by the repS promoter region was identified which may control pXO2 copy number by inhibiting repS expression. RepS of pXO2 was overexpressed and purified from E. coli as an MBP fusion at the amino terminal end. DNA binding experiments using double-stranded (ds) and single-stranded (ss) substrates showed that MBP-RepS specifically binds to a 60-bp ds sequence containing the putative pXO2 origin of replication and that a central 20-bp region containing the putative start site for replication and the 5 side of the origin is important for this binding. MBP-RepS also bound to ss DNA non-specifically. A cell-free system from plasmid-negative B. anthracis cells that promotes robust replication of rolling-circle replicating (RCR) plasmids was developed and adapted to study the replication of plasmid pXO2 in vitro. This system showed that pXO2 replication requires RepS supplied in trans and directional transcription into the origin.

Clostridium perfringens, a Gram-positive and spore-forming anaerobe, is a significant pathogen of both humans and domestic animals. Among the many toxins produced by this bacterium, the C. perfringens enterotoxin (CPE) is one of the principal contributors to C. perfringens human disease via its role in both foodborne and non-foodborne gastrointestinal illness. Produced in massive quantities during sporulation in the intestine, CPE begins its action by binding to host cells and forming an SDS-sensitive small complex. At physiologic conditions, CPE then associates with additional proteins to form large SDS-resistant complexes in the plasma membrane, the formation of which coincide with membrane permeability alterations of the cell. The two species of large complex have been reported to have molecular masses of ~155 and ~200 kDa, and recent compositional analysis of the complexes has shown that the former complex contains both CPE and claudin, while the latter contains CPE, claudin, and occludin. Prior structure-function analysis of CPE has defined regions at the N- and C-termini involved with cytotoxic and binding activities of the toxin, respectively. Despite the important findings contributed from previous studies of CPE, several significant questions remain regarding the molecular aspects of CPEs mechanism of action. In this thesis dissertation, research is presented aimed to answer three specific structure-function and mechanistic questions about CPE action. Site-directed mutagenesis enabled the identification of two residues in the N-terminal cytotoxicity region of CPE that were crucial for the formation of the CPE large complexes, and likely function in oligomerization of the toxin. In addition, a novel pre-pore step was defined by deletion mutagenesis of a 25 amino acid region of CPE proposed to be involved in membrane insertion. Lastly, CPE was determined to have hexameric stoichiometry in both of the large complexes, prompting a reevaluation of their molecular masses. Several new insights into CPE activity have been gained by the work presented here within, and models for the molecular mechanism of action and structure-function relationships of CPE are updated.

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes two important diseases, chickenpox and herpes zoster, separated by a prolonged period of latent infection in which no disease occurs. VZV remains a serious public health concern despite the availability of licensed vaccines for the prevention of chickenpox during childhood and zoster in the elderly. The VZV ORF66 protein kinase is a well-established mediator of VZV tropism and pathogenesis in vivo, but information regarding the molecular mechanisms of ORF66 function is limited. ORF66 is orthologous to the alphaherpesvirus US3 kinase family, which is involved in regulating diverse aspects of the host environment. ORF66 has been implicated in nuclear exclusion of the VZV principle transcriptional regulator and tegument protein, IE62, during late-stage VZV infection; and in downmodulation of class I major histocompatibility complex surface expression, which is critical for the identification and elimination of infected cells by the adaptive immune system. However, the mechanisms underlying these processes are not well-resolved. In this work, I have significantly extended our understanding of known ORF66 functions during VZV infection, and I have established novel functions for both ORF66 and the IE62 protein. Purification of ORF66 allowed the development of an in vitro kinase assay, and I used this to prove that ORF66 directly phosphorylates IE62 adjacent to its nuclear localization signal to mediate nuclear exclusion. Additional investigations of IE62 nucleocytoplasmic trafficking revealed a novel nuclear export activity, which was multi-dimensionally regulated by intrinsic IE62 nuclear retention and phosphorylation by kinase(s) other than ORF66. With regard to the ORF66 role in the regulation of MHC-I surface presentation, I developed assays with recombinant VZV expressing enhanced green fluorescent protein-tagged ORF66 to establish that ORF66 affects the cellular environment such that early MHC-I biogenesis is delayed, resulting in a reduction in total surface MHC-I. MHC-I studies indicated highly novel ORF66 nuclear distribution patterns, and a further examination of these revealed that ORF66 may regulate nucleocapsid morphogenesis or trafficking in VZV-infected nuclei. In sum, this work represents a major contribution to VZV biology and provides insights into a multi-functional mediator of VZV persistence and pathogenesis.

Developing an effective lentiviral vaccine has been an elusive goal, largely due to immune evasion mechanisms of lentiviruses. Lentiviral envelope (Env) proteins pose a major obstacle to vaccine development due to extreme antigenic variation, but research with equine infectious anemia virus (EIAV) has indicated Env to be a primary determinant of vaccine efficacy. We have developed an attenuated EIAV vaccine capable of protecting horses from disease after homologous challenge. However, when variant challenge strains with divergent Env proteins are utilized, vaccine protection is incrementally decreased with increasing divergence from the homologous Env. I hypothesize there may be Env-specific immune responses associated with vaccine protection and believe antigenic variation may have profound effects on immune recognition. Utilizing thymidine incorporation and chromium release assays on PBMC from vaccinated horses challenged with the homologous strain, I identified broadly reactive regions of Env recognized by T-helper and CTL cells. With PBMC from vaccinated ponies challenged with divergent EIAV strains I identified Env-specific immune responses associated with vaccine protection from disease. Although I was unable to correlate antibody neutralization with protection, I found one T-helper and eight CTL peptide responses associated with vaccine protection. Three of the CTL peptides were located in variable domains of Env. To understand the effects of variation on immune recognition, I utilized sera and PBMC from vaccinated and variant infected ponies to analyze the cross-reactivity of humoral and cellular immune responses. Due to limited proliferative activity, I was unable to fully analyze the effects of Env variation on lymphoproliferation; however, CTL analysis indicated Env variation had profound effects on immune recognition in EIAV vaccinated and infected ponies. The most remarkable effects of Env variation were observed in in vitro neutralization assays, where there was no detectable cross-reactivity of neutralizing antibodies. Collectively, these results indicate that eliciting key immune responses to more conserved regions of the EIAV Env may be crucial in developing an effective EIAV vaccine. The peptide-specific responses identified in this dissertation could serve as important targets for future EIAV vaccines and this analysis may be a valuable complement to ongoing work in SIV and HIV.

Muscular dystrophy is a devastating disease in which no treatment or cure exists. A promising therapy for muscular dystrophy is the transplantation of cells that are able to contribute to existing muscle fibers or generate new muscle fibers. While several cell populations have been shown to demonstrate this phenomenon in mouse models, the mechanism by which these cells are able to differentiate into myogenic cells is largely unknown. The goal of this research was to (i) create HSV vectors that are useful for expressing muscle development genes in developing embryos in culture (ii) design strategies to produce and characterize large and diverse HSV vector libraries of expressed genes from different cells or tissues and (iii) design methods by which this expression vector library can be screened for expressed gene functions that participate in skeletal muscle lineage determination. The outcome of these studies were intended to provide a method for exploiting the high level infectivity and growth of HSV in embryonic stem (ES) cells and germ layer derivatives, the virus ability to accommodate large DNA inserts, and the ability to create vector libraries potentially useful in selection of lineage determining genes based on complementation of virus growth. The engineering and characterization of a replication defective HSV vector that is useful for the delivery of genes to embryonic stem cells and their derivatives was described. The deletion of ICP4, ICP22 and the conversion of the immediate early genes ICP0 and ICP27 to the early gene class by promoter exchange generated a vector, JDββHE. JDββHE was found to be non-toxic to ES cells and vector infection of ES cells did not interfere with germ layers formation. This vector was engineered to be dependent on a single IE gene ICP4 for replication. Gene expression from the JDββHE HSV vector, including the expression of eGFP and the early myogenic gene Pax3, was demonstrated in infected ES cells and cells of the embryoid body in a robust and transient manner. In order to discover genes which contribute to muscle differentiation, a cDNA library was constructed within HSV viral vectors in order to be used in the identification of novel gene functions which play a role in the activation early myogenic promoters. For this purpose, the HSV genome was modified to contain BAC elements as well as the Gateway recombination system, which facilitated efficient incorporation of cDNAs into the vector genome. HSV cDNA vectors expressed gene products at the transcriptional and protein level. Functional analysis of the cDNA inserts demonstrated that the library consisted of at least 15,000 unique genes. Some of these genes have been shown to participate in biological functions related to muscle differentiation, such as Id1 and Cand2. Finally, a conditional replication strategy was developed by which the recently produced HSV cDNA library could be used to identify genes that activate early myogenic promoters. This study exploited the characteristic of the JDββHE vector in which viral replication can be restored upon introduction of the essential viral gene ICP4. By placing this essential viral gene under the control of the early myogenic promoter Pax3, viral replication was found to be dependent on activation of this cellular promoter in a Pax3-expressing rhabdomyosarcoma cell line. In a similar manner, HSV cDNA library vectors that express a Pax3 activator will be able to replicate in the presence of the conditional ICP4 construct, thereby capturing the relevant gene product by the release of viral progeny into the supernatant. The future use of this strategy to identify gene products that specifically activate an early myogenic promoter would lead to the testing of these gene products for their ability to induce downstream myogenic gene expression and differentiation. In this manner, a better understanding of early muscle differentiation might be achieved and applied to the use of stem cells in transplantation studies using muscular dystrophy models.

Recurrent HSV-1 ocular disease results from reactivation of latent virus in trigeminal ganglia (TG), often following immunosuppression or exposure to a variety of psychological or physical stressors. HSV-specific CD8+ T cells can block HSV-1 reactivation from latency in ex vivo TG cultures in part through production of IFN-&#947. Here we establish that either CD8+ T cell depletion or exposure to restraint stress permits HSV-1 to transiently escape from latency in vivo. Restraint stress caused a glucocorticoid-associated reduction of TG-resident HSV-specific CD8+ T cells, and a functional compromise of those cells that survive, at least partly mediated by catecholamines. Together, these effects of stress resulted in an approximate 65% reduction of cells capable of producing IFN-&#947, and impairment in the ability of those cells to release lytic granules, in response to reactivating virus. We also establish that restraint stress during the primary infection results in a 54% reduction of virus-specific IL-7R&#945+ memory precursor cells in the TG at the peak of expansion. When mice stressed early were stressed again during latency, their T cell response may be further compromised. Our findings demonstrate persistent in vivo regulation of latent HSV-1 by CD8+ T cells, and strongly support the concept that stress induces HSV-1 reactivation from latency at least in part by compromising CD8+ T cell surveillance of latently infected neurons.

Most new human immunodeficiency virus (HIV) infections are acquired through vaginal or rectal mucosa, and gut mucosal tissue is a primary target of HIV infection. To generate mucosal immunity against HIV or its simian counterpart simian immunodeficiency virus (SIV), the Gram positive bacterium Clostridium perfringens was used to develop a vaccine that delivers SIV p27 to the gut and induces local T cell immunity. Under in vitro conditions, Clostridium perfringens expressing SIV p27 (Cp-p27) was found to induce dendrite cell (DC) maturation and stimulate p27-specific T cell responses. To improve intracellular delivery of p27 to DCs and thereby enhance immune priming, Cp-p27 variants expressing p27 conjugated with protein transduction domains (PTDs) at the 5 end were constructed. While internalization of p27 by DCs and gut epithelial cells was improved following exposure to the PTD-Cp-p27 variants, cellular p27-specific immune stimulation was not significantly improved compared with wild-type Cp-p27. The Cp-p27 vaccine was then tested in vivo in mice for its ability to prime gut mucosal T cell responses. First, an adjuvant optimization study with three mucosal adjuvants, cholera toxin (CT), mutant E. coli heat-labile enterotoxin (LT(R192G)), and unmethylated cytosine-phosphate-guanine oligodinucleotides (CpG ODNs) was performed to determine the best T cell immune response in the gut. While the combination of CpG ODNs and (LT(R192G)) induced the highest T cell immune response, (LT(R192G)) alone provided the best multifunctional CD8+ T cell response in the gut. Oral Cp-p27 vaccination was then tested for induction of T cell immunity in vivo in a prime-boost model by combining Cp-p27 with systemic immunization with an adenovirus expressing p27 (Ad-p27). Cp-p27 vaccination primed a strong multifunctional T cell immune response in gut lamina propria, although it could not stimulate a systemic immune response. In contrast, Ad-p27 vaccination stimulated strong systemic immunity but limited gut mucosal immunity. By sequentially delivering Cp-p27 and Ad-p27, immunity in both the gut and systemic tissues was achieved. Altogether, this study demonstrates that Cp-p27 can deliver p27 to gut T cells through dendritic cells to prime a strong, multifunctional immune response in the gut effector tissue.