<p> HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered HDL profile exacerbated by downmodulation and impairment of ATP-Binding Cassette Transporter A1 (ABCA1) activity by the HIV-1 protein Nef. Nef has been shown to increase delivery of cholesterol to lipid rafts, sites of viral assembly and egress, by inhibition of ABCA1 cholesterol efflux functionality and reduction of ABCA1 protein levels through lysosomal degradation. Important mechanistic details of ABCA1 inactivation and degradation by Nef, and whether these two processes are intimately linked or separable are still to be defined. The studies presented here were designed to identify cellular co-factors for ABCA1-mediated cholesterol efflux that may be targeted by Nef to achieve ABCA1 inactivation. In these studies, a novel cellular factor, the ER-resident lectin chaperone calnexin, was shown to be involved in a physical interaction with ABCA1 that is disrupted by Nef. Nef was found to bind and redistribute calnexin and reduce binding and co-localization of ABCA1 with calnexin. In vitro knockdown of calnexin via RNAi reproduced several previously described biochemical effects of Nef, including redistribution of ABCA1, increased ABCA1 membrane localization, and reduced ABCA1 recycling. Importantly, knockdown of calnexin also resulted in reduced ABCA1-mediated cholesterol efflux, but without the Nef-mediated reduction in ABCA1 protein levels, suggesting that Nef utilizes a bipartite mechanism to inactivate and degrade ABCA1 and that these functions may be separable. Despite the lack of effect of calnexin knockdown on ABCA1 protein levels, interference with the ABCA1-calnexin interaction was critical for Nef-mediated functional impairment of ABCA1. This was shown with a Nef mutant defective in interaction with calnexin which was incapable of preventing ABCA1-calnexin interaction and was also defective in impairing ABCA1-mediated cholesterol efflux activity. Thus, these studies identified a novel mechanism by which HIV-1 Nef impairs functional activity of cholesterol transporter ABCA1 by blocking its interaction with calnexin. Calnexin acts as an ABCA1 functional chaperone, limiting total and cell surface ABCA1 expression while increasing ABCA1-mediated cholesterol efflux. Combined with the demonstration that Nef increases delivery of ABCA1 to lysosomes, these results suggest the Nef-mediated impairment of ABCA1 function involves reduced interaction with calnexin followed by delivery of ABCA1 to lysosomes for degradation.</p>

<p> Viruses have long been noted to be composed simply of nucleic acid and protein. This thesis describes this confluence of science of viruses at the interface of genomics and proteomics. Chapter 2 describes the discovery of klassevirus, a new picornavirus in pediatric diarrhea. Chapter 3 shows that klassevirus is likely a human pathogen given the seroconversion of klassevirus-positive individuals against a klassevirus non-structural protein that is not present in the picornavirus virion. Subsequent work failed to obtain a culturable virus from klassevirus-positive stool samples, enabling the transition to culture-independent methods of characterizing picornavirus-host protein interactions. Chapter 4 describes the use of affinity purification mass spectrometry to discovery a novel picornavirus 3A-ACBD3-PI4KB complex that promotes viral replication in the enteroviruses and kobuviruses. Chapter 5 extends upon the methodology to describe a novel host protein interactor of ACBD3 (TBC1D22A/B), whose interaction is altered specifically by the kobuvirus 3A protein. This complex also demonstrates significant interaction with the klassevirus 3A protein, suggesting that the AP-MS work may inform the biology of the uncultured virus. Finally, chapter 6 describes future directions that are opened up by this work.</p>

<p>This work investigates the functional heterogeneity of influenza virus quasispecies through quantitative analysis of cellular responses to the entry of noninfectious biologically active particles, the effect of reassortment of gene segments on the generation and function of these particle subpopulations, and the potential of these subpopulations as <i>in vitro</i> correlates of <i>in vivo</i> effectiveness of live-attenuated influenza vaccines (LAIVs). </p><p> For the first time, the clonogenic assay was used to show that populations of most influenza A viruses contained cell-killing particles in excess of infectious particles when tested in the same host cell. Thus, a new class of influenza virus particles was revealed &ndash; noninfectious cell-killing particles which required the synthesis of a specific viral polymerase subunit to kill cells and the expression of NS1 protein to temporally delay apoptosis/cell-killing. </p><p> The noninfectious cell-killing particles were clearly distinguished from the well known defective-interfering particles by differences in their numbers in standard influenza virus populations, their temporal appearance and quantity during serial high multiplicity propagation in mammalian and chicken cells, an inability of defective-interfering particles to kill cells or interfere with the cell-killing capacity of noninfectious cell-killing particles, genetic requirements (a small DI RNA &sim;350 nt and a large RNA &sim;2,300 nt for defective-interfering and noninfectious cell-killing particle activities, respectively), and the extracellular T&half; at 40.5 &deg;C (&sim;40h and &sim;85h for noninfectious cell-killing particles and defective-interfering particles, respectively). </p><p> Specific exchange of the <i>NS</i> gene segment from lethal A/HK/156/97 (H5N1) (NS1: E92, or E92D) virus for the cognate <i>NS</i> gene segment of A/PR/834 (H1N1) (NS1: D92) virus caused <i>de novo</i> generation of large defective-interfering particle subpopulations and >10-fold enhancement of interferon-inducing particle efficiency. These changes were attributed to dysfunction of the H5N1 virus <i>NS1</i> gene. </p><p> Populations of two effective LAIVs (Vac⁺) in chickens were characterized by high defective-interfering to interferon-inducing particle ratios and induction of large amounts of interferon in chicken cells. Interferon is an antiviral cytokine that acts as a potent natural adjuvant of adaptive immune responses in chickens. Populations of two ineffective LAIVs (Vac^-) in chickens had lower defective-interfering to interferon-inducing particle ratios and induced less interferon. Unexpectedly, these phenotypes were reversed in mammalian cells. Populations of Vac^- (in chickens) LAIV candidates were excellent interferon inducers with high defective-interfering to interferon-inducing particle ratios in mammalian cells. In contrast, populations of Vac⁺ (in chickens) LAIV candidates were poor interferon inducers with low defective-interfering to interferon-inducing particle ratios in mammalian cells. As predicted by the <i>in vitro</i> screen, the Vac phenotypes were reversed <i>in vivo</i> (in mice) relative to chickens. </p><p> Overall, this study shows that the majority of noninfectious particles of influenza virus are biologically active, reassortment can change the subpopulation make of influenza virus, and a high defective-interfering to interferon-inducing particle ratio is a strong <i>in vitro</i> correlate of the effectiveness of self-adjuvanting LAIVs. Taken together, these attributes of an influenza virus population represent a novel ensemble of <i>in vitro</i> parameters that may be used to distinguish between Vac⁺ and Vac^- LAIV candidates. </p>

<p> Recombinant adeno-associated virus (rAAV) is a widely used gene therapy vector. Although a wide range of rAAV serotypes can effectively enter most cell types, their transduction efficiencies (i.e., transgene expression) can vary widely depending on the target cell type. Integrins play important roles as co-receptors for rAAV infection, however, it remains unclear how integrin-dependent and -independent mechanisms of rAAV endocytosis influence the efficiency of intracellular virus processing and ultimately transgene expression. In this thesis, I examined the contribution of integrin-mediated endocytosis to transduction of fibroblasts by rAAV2. I found that promoting AAV2/integrin binding with Mn⁺⁺ greatly enhanced (~17-fold) rAAV2 transduction independently of cell binding and endocytosis. Subcellular localization studies of rAAV2 demonstrated that integrin activation by Mn⁺⁺ promoted AAV2 aggregation on &alpha;5 and &beta;1 integrins and recruitment of the cytosolic integrin effector protein vinculin. Focal adhesion kinase (FAK), a down stream effector of integrin signals, was essential for AAV/integrin complex endocytosis and transduction, but not AAV2 recruitment to integrins. Recruitment of FAK to AAV2/integrin complexes was increased by transiently trapping the endocytic event at the plasma membrane by pharmacologic inhibition of dynasore. This also increased the size of AAV2 clusters found beneath the cell at FAK/integrin complexes resembling immature filopodia and caused a large, FAK-dependent (75-fold) increase in AAV2 transduction. These findings support a model whereby integrin activation at the cell surface can redirect rAAV2 toward a FAK-dependent entry pathway that is more productive for cellular transduction. This pathway appears to be conserved for other rAAV serotypes that contain a capsid integrin-binding domain (AAV1 and 6).</p>

<p> Herpes Simplex Virus Type 1 is a member of the <i>alphaherpesvirinae </i> subfamily within the family <i>Herpesviridae</i>. This virus has both a lytic and latent cycle. Primary infection occurs when the virus enters epithelial cells around the mucosal lining of the nose and mouth. Within the epithelial cells, the virus undergoes an active lytic infection, causing an ulcerated blister, more famously known as a 'cold sore' or 'fever blister'. Once HSV enters the nearby sensory neurons the genome is transported to the neuronal cell body where its latency associated transcripts are activated and the virus remains in a dormant latent cycle until reactivation, when the virus is transported back down the axon to the epithelial cells at or near the site of initial infection. The Virion Host Shutoff protein is a tegument protein from HSV1 and acts as a ribonuclease, degrading both cellular and viral mRNAs, making the course of viral infection more efficient. A study by Saffran, Read and Smiley uncovered an unexpected new function of Vhs: stimulation of translation from some IRESs. An IRES is a section of mRNA with a high level of secondary structure, capable of inducing cap-independent translation. In similar experiments utilizing a bicistronic reporter transcript, I sought to discover whether or not these two functions of the Vhs protein could be de-coupled. Experiments involved dually transfecting HeLa cells with different Vhs mutants across a range of Vhs plasmid concentrations and the bicistronic reporter construct. Levels of reporter activity were measured from cell lysates 36 hours after transfections and provided a measurement of the control at the level of translation. As the cellular Bip IRES element was present between the cistrons, the 3' cistron provided a measure of IRES stimulation. The Results revealed examples of Vhs mutants in which the two activities had been separated. It is unknown what role IRES stimulation could play during Herpesvirus infection, although it is interesting to note that some HSV1 genes have IRES like elements within the 5' UTR. Future experiments can be done to investigate whether or not Vhs is actively recruiting transcription initiation factors to these IRES elements.</p>

Hepatitis C virus (HCV) has become a worldwide problem. Roughly 3% of world population are estimated to be infected with the virus, producing high rates of progressive liver disease, leading to cirrhosis and hepatocellular carcinoma. The present therapy, a combined administration of pegylated interferon-alpha (IFN) and ribavirin is costly and only successful in 50% of patients infected with HCV. It is also associated with serious side effects. Thus, there is an urgent need for better tolerated and more effective treatment modalities. A therapeutic vaccine may be the solution. / Recent efforts to produce efficient vaccines require not only the identification of potential viral antigens but also vaccine adjuvants or enhancers of immunity. Dendritic cells (DC) are being considered one such adjuvant for the activation of CD4+ and CD8+ T-cells. As potent antigen presenting cells, they are capable of capturing antigens, processing them into peptides, and presenting them on products of the MHC to T cells. For such reasons, peptide loading of antigens onto DCs to enhance T cell responses is becoming of increasing interest. Using cell penetrating peptides, or motifs capable of transporting cargo freely across cell membranes, we have developed a peptide based delivery system suitable for the transport of all HCV proteins into immature DCs. In our studies we demonstrated that 3.1% of immature DCs internalized the reporter cargo, eGFP. This system was then optimized to 53.81 % in target HeLa cells. / Another area of recent focus is the regulation of HCV translation and replication. Positive stranded viruses such as HCV use the genomic RNA as a common template for translation as well as for RNA replication, both proceeding in inverse directions. Thus, specific regulatory mechanisms must be in place in order to coordinate these two antagonistic processes. In this study, we investigated the role of HCV Core protein as a translational inhibitor and enhancer of replication. Using several transient and stable in vivo reporter assays, we showed that Core expression inhibited HCV IRES-mediated translation in trans, in a dose-dependent manner. Furthermore, HCV Core protein is able to dramatically inhibit HCV translation in the Huh7 replicon system, more so than the bicistronic reporter systems tested and subsequently increase total levels of replicon RNA by 1.5 log fold and thus, affect replication. We believe that Core may indeed be the sought regulator of translation and replication.

<p> Hepatitis B virus gives rise to chronic infection in 350 million people world wide, and infection can result in the development of liver cirrhosis, liver failure or hepatocellular carcinoma. Current therapies merely treat the infection and prevent progression, because there is no cure. There have been promising studies in the clearance of HBV DNA in murine models with the use of iNKT stimulating lipid &alpha;GalCer. However, iNKT stimulation of &alpha;GalCer has not been an effective measure in anti-viral or anti-cancer clinical trials. It is speculated that these differences between human and mouse reactivity to &alpha;GalCer is based on the differences of CD1d/NKT lipid presentation systems between the two species. Previously, our lab has generated an hCD1d KI model to support a human like NKT cell environment. In order to investigate the role of invariant NKT cells in HBV and to allow us to test potential therapeutic lipids for HBV in a more human like environment, we generated a novel HBVtg hCD1d-KI model. Thus in this study we seek to characterize the population of iNKT cells in this new HBVtg hCD1d-KI model. As reported in human chronic HBV patients, we found that the iNKT cells are significantly lower in this new HBV-transgenic model. Among the decreased levels of iNKT cells, there was a significant decrease in the population of CD4+ iNKT cells accompanied by a significant increase of CD4- iNKT cells. Similarly, there was an overall significant decrease of total NKT Cells. There was an overall increase in CD8+ conventional T Cells, and a significant increase of total conventional T cells. Our study also found that conventional T cells had and significant upregulation of PD-1, which was restricted to the CD8+ population. In our study, the levels of PD-1 among the iNKT cell populations were not affected; suggesting the decrease of iNKT cells was not related to PD-1 expression. Altogether, our results support that our new HBVtg can a useful new model for study of HBV pathogenesis as well as exploration and validation of novel anti-HBV therapeutic approaches.</p>

<p> In most human immunodeficiency virus type 1 (HIV&minus;1) infected individuals, highly active antiretroviral therapy (HAART) suppresses viremia to levels below standard clinical detection limits and delays the progression to AIDS. More sensitive assays have demonstrated that low&minus;level residual viremia is present during HAART. Furthermore, viremia rebounds upon cessation of therapy and thus, life&minus;long treatment is required to prevent the progression to AIDS. This is presumably due to viral persistence within anatomical and cellular viral reservoirs that are not eliminated during long&minus;term HAART. During HAART, viral persistence has been principally attributed to a latent viral reservoir; however, instances of complete viral replication cycles, termed residual replication, may occur. Human studies have provided evidence for and against the hypothesis that residual replication occurs in tissues and contributes to residual viremia. </p><p> To address questions regarding viral persistence, this dissertation research utilized a rhesus macaque model of HIV&minus;1 HAART. This model uses RT&minus;SHIV, a chimeric simian immunodeficiency virus (SIV) with the HIV&minus;1 reverse transcriptase (RT) replacing the native SIV RT gene. This modification renders RT&minus;SHIV susceptible to non&minus;nucleoside RT inhibitors, which are an important component of frontline therapies. Chapter 2 describes a longitudinal phylogenetic analysis of residual viremia during 42 weeks of therapy in five macaques that initiated HAART eight weeks after infection. This is the first non&minus;human primate phylogenetic study to characterize residual viremia for a period greater than five weeks. The results did not provide substantial evidence that residual replication contributed to residual viremia. Additionally, one of the five macaques maintained a predominant plasma clone (PPC) population, which is an enigmatic feature of human residual viremia. Chapter 3 describes viral DNA diversification in lymphoid and gastrointestinal tissues relative to pre&minus;HAART viremia. Similar to many human studies, there was no discernible evidence of residual replication or selection of resistance mutations. The origins of PPC populations were also not identified. Importantly, this dissertation advances the relevancy of HAART in RT&minus;SHIV infected macaques as a model of human HAART. Thus, this model may be ideal in studies designed to improve human health, investigate the nature of viral persistence, and potentially develop a cure for HIV&minus;1.</p>

<p> Propionic acidemia is an organic acidemia that results from mutations in the PCCA or PCCB genes responsible for the two protein subunits of the propionyl-CoA carboxylase enzyme. Patients with PA have several metabolic abnormalities including elevated levels of glycine, propionylcarnitine, and methyl citrate. They also experience growth delay, developmental delay, and pathologies involving the brain, heart, pancreas, eyes, and muscles. The only viable treatment options for PA are protein restriction via a formula diet or liver transplantation, but neither of these treatments result in cures. To study the possible benefit of gene therapy for the treatment of PA we generated a mouse model of PA by introducing a hypomorphic human transgene with an A138T mutation onto a <i>Pcca</i> null mouse background. The resulting <i>Pcca-/-</i>(A138T) mice recapitulated many characteristics of PA in humans, and showed similar growth delay and biochemical perturbations. These mice were then used to study the utility of adeno-associated virus (AAV) serotype 8 and adenovirus serotype 5 expressing human PCCA. Both vectors mediated significant reductions in PA metabolite levels. Efficacy lasted for approximately 2 months in adenoviral treated mice but persisted for 1.5 years in male mice treated with AAV vector with expression remaining in the liver, heart, and skeletal muscle. Further studies examined the effect of tissue-specific treatments using alternate AAV serotypes and transcriptional regulation. When PCCA expression was restricted to the liver or muscle of treated mice metabolite levels were significantly lower in both indicating that there was likely a significant amount of these metabolites being produced within the muscle. Together these data provide evidence that PA disease is amenable to treatment with gene therapy and AAV vectors are able to mediate a significant degree of correction over long periods of time in mice. Additionally, the optimal treatment for individuals with PA will include correction of PCC activity in liver and muscle at a minimum to decrease the amount of PA metabolites such as methyl citrate being produced in these tissues. These studies also provide previously unknown insight into the molecular basis of the disease.</p>

Despite great advances in our understanding of the human immunodeficiency virus (HIV) life cycle, the mechanisms that underlie the progression of HIV from cellular entry of the viral core to stable integration of the provirus are poorly understood. Sites of integration of the HIV provirus are distributed along the full length of actively transcribed genes and appear to be determined through protein-protein interactions between the viral integrase and cellular proteins. / Two cellular proteins have been proposed to perform integration targeting roles, the chromatin-remodeling factor integrase interactor 1 (INI1/hSNF5/BAF47) and the lens epithelium-derived growth factor/transcriptional co-activator (LEDGF). Here, we report the initiation of two novel integration assays to study the contribution of INI1 and LEDGF in target site selection. Elucidating these molecular determinants and their functional implications is also of particular interest to anti-HIV therapy and could have major impact on the safety of gene therapy protocols.