<p>Many RNA viruses, including enteroviruses, remodel host ER membranes to form platforms with unique lipid components to assemble replication complexes and synthesize new viral RNA. Cholesterol is a critical component of cellular membranes regulating fluidity and being indispensable for proper assembly and function of membrane based protein-lipid complexes. Here we show that enteroviruses harness the clathrin mediated endocytosis (CME) pathway to transfer free cholesterol from the plasma membrane to the viral replication organelles (VROs). We show that cholesterol is responsible for regulating viral protein processing and facilitates viral RNA synthesis, and disrupting CME causes cellular cholesterol pools to be stored in lipid droplets obstructing the transfer to VROs and inhibiting viral replication. In contrast, we found that the presence of excess intracellular cholesterol, as in cells lacking caveolins or those from patients with Niemann-Pick disease, stimulates viral replication. We demonstrate that, the redistribution of free cellular cholesterol and the cellular recycle dynamics are affected during infection. The CME rate of uptake does not change during the initial 2 hours of infection while the rate of cellular endosomal recycling is inhibited resulting in a net decrease of free cholesterol at the plasma membrane, and facilitating the access and active transfer of cholesterol from enriched internal cellular compartments to VROs. Our findings indicate that cholesterol is critical for enteroviral replication and that CME has an important role in the enteroviral life cycle and in the host cellular cholesterol homeostasis. </p>

Thesis (Ph.D.)--Yale University, 2006. / (UMI)AAI3243691. Adviser: William C. Summers. Source: Dissertation Abstracts International, Volume: 67-12, Section: A, page: 4675.

Thesis (Ph.D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 68-01, Section: B, page: 0144. Adviser: Warner C. Greene.

The processes that regulate viral vector vaccine-elicited cellular and humoral immune responses remain poorly defined. Thus, in this thesis, the role of CD4+ T cells – master regulators of adaptive immunity – in modulating adenovirus (Ad) vector-elicited cytotoxic CD8+ T cell responses and transgene-specific antibody responses was investigated. CD4+ T cell help is critical for the induction of CD8+ T cell and antibody responses, but the mechanisms and timing of help to each of these two arms of the immune system are distinct. CD4+ T cell help is required immediately and continuously for one week to drive functional CD8+ T cell effector differentiation and prevent dysfunction. Elevated signaling via PD-1, decreased IL-2 signaling, and increased non-canonical NFAT signaling all appear important for driving this CD8+ T cell dysfunction and impairing effector functionality. Absence of CD4+ T cells at the time of Ad vector immunization prevents the development of antigen-specific antibody responses. However, if the CD4+ T cell population is allowed to recover then fully functional antigen-specific antibody responses develop without the re-administration of antigen. Thus, CD4+ T cell help is absolutely required for the development of antibody responses following Ad vector immunization, but, intriguingly, help can be provided at a time separate from initial antigen exposure. Collectively, CD4+ T cell help and the appropriate timing of this help are critical for the generation of optimal CD8+ T cell and antibody responses following Ad vector immunization. These data advance our understanding of how CD4+ T cells regulate Ad vector-elicited cellular and humoral immune responses, and may improve rational vaccine design. / Medical Sciences

In mice, the Cmv1/Ly49h locus expressed on Natural Killer (NK) cells determines innate resistance to Murine Cytomegalovirus Virus (MCMV). NK cells provide the first line of defence against infections and tumors through cytokine production or direct cytotoxicity. Acquisition of MCMV resistance in transgenic mice expressing Ly49H, hereafter FVB-Tg (Ly49h), demonstrated the critical role of Ly49H in clearance of the infection, and provided an ideal model to characterize the role of NK cells in host defence. The scoring of viral titers in the visceral organs of Ly49H transgenic (FVB-Tg (Ly49h)) mice and their MCMV-susceptible counterparts, FVB, indicated a tissue specific effect of Ly49H independent of genetic background in spleen, lung, kidney and thymus. In the liver, the presence of Ly49H was associated with increased numbers of inflammatory foci, suggesting that Ly49H may facilitate localization of NK cells to the vicinity of infected cells. To identify genes critical to the initial control of virus replication, comparative gene expression analysis of explanted spleen NK cells from FVB-Tg (Ly49h) and FVB mice was carried at 36 hours post-infection. This allows for the onset of Ly49H related mechanisms of host resistance. In contrast to whole spleen samples, RT-PCR from purified NK cells from either mouse strain did not detect MCMV gene expression, indicating that NK cells are not productively infected. Out of 16,000 genes analyzed by microarray, 35 showed greater than 2.5-fold expression difference between resistant and susceptible mice. Genes involved in NK cell proliferation, cytotoxicity and in cell-mediated immunity, such as the early T lymphocyte activation-1 gene (Eta-1 or osteopontin), showed enhanced expression in NK cells from resistant mice. On the other hand, NK cells from susceptible mice showed increased expression of pro-inflammatory cytokines such as IFN-gamma, MIP2, and TNF-associated receptors, indicating that antiviral cytokines are not sufficient to control viral replication in the absence of Ly49h, and that direct killing of virus-infected cells by NK cells expressing Ly49H is required for successful clearance of MCMV. To characterize the response of NK cells during MCMV clearance further, gene expression patterns were studied in FVB-Tg (Ly49h) during the course of infection. Out of 22690 genes analyzed, the expression of 225 genes was significantly changed at 3 days post-infection, when the effect of Cmv1/Ly49h is strongest and the viral load is highest. At later time points, the number of genes affected and the level of gene expression gradually returned to normal, in parallel with a decrease in viral titer, indicating that altered expression patterns co-varied with viral burden. More than 50% of the genes upregulated were involved in cell proliferation, metabolism, and transcription, while about 15% were involved in NK cell cytotoxic function, indicating that NK cell blastogenesis and direct killing of infected cells are crucial for MCMV-resistance. Altogether, our results indicate that the differential pattern of expression between resistant and susceptible mice depends on the presence or absence of Ly49h as well as on signals emanating from productively infected cells, such as macrophages and dendritic cells. Genes differentially expressed on MCMV-susceptible NK cells may serve as useful biomarkers for HCMV susceptibility in risk populations, graft recipients in particular.

It is difficult to induce protective immunity against most RNA viruses. However, there is strong evidence that humoral and especially cellular immune responses play crucial roles in the control of established RNA viral infections. Thus, an ideal vaccine should be able to induce strong specific antibody titer as well as a robust T-helper and T-cell cytotoxic response. Here, correlates of protective immunity against HIV-1, HCV and SARS-CoV were assessed. Monocistronic and polycistronic DNA constructs containing structural HIV-l, and SARS genes were designed. The structural proteins (HIV- gpl20, gag, pol, HCV-core, E1/E2 and SARS-NC) were also expressed, purified and characterized in mammalian and bacterial cell lines. HLA-A2.1 and B6 mice were immunized with different combinations of DNA constructs, recombinant proteins and novel adjuvants. Humoral responses were measured by titrating of specific antibodies and cell-mediated immune responses were identified by Th1/Th2 cytokine expression, lymphocyte proliferation, intracellular cytokine staining, HLA-peptide dimer assay, and ELISPOT. The first study in HIV-1 showed that a combination of DNA single constructs, protein and adjuvant induce a higher immune response compared to the DNA or/and protein alone. In the second HIV-1 approach, a synergistic effect between HIV/HCV antigens was detected that may lead to induction of multi-specific immune responses against both HIV and HCV. In the third study (SARS project), a high level of specific SARS-CD8+ T-cell response was demonstrated in mice that received DNA encoding the SARS-nucleocapsid, protein and XIAP (X-link inhibitor of apoptosis) as an adjuvant.

Lassa virus (LASV), Junin virus (JUNV) and several other members of the Arenaviridae induce severe, often fatal hemorrhagic fevers, and are classified as Biosafety Level 4 and NIAID Biodefense Category A agents. In addition to high case fatality rates, arenaviruses have many features that enhance their potential as bioweapons. Arenaviruses have relatively stable virions, do not require passage via insect vectors, are transmitted easily by human-to-human contact and can be spread by simple means of dispersal. The ease of travel to and from endemic areas also permits easy access to LASV and other arenaviruses for use as bioweapons. A cluster of hemorrhagic fever cases in the United States caused by any arenavirus would be a major public health incident. The potential use of arenaviruses as biological weapons directed against civilian or military targets necessitates development of effective commercial diagnostics. The ultimate goal of our project is to develop and validate multiagent diagnostic immunoassays for arenaviruses using recombinant antigens. These assays can be used to determine the attack agent following a deliberate release, and allow the virus used to be distinguished from other hemorrhagic fever viruses, such as dengue virus or Ebola virus, that may have similar case presentations. Development of rapid immunodiagnostic assays will also improve treatment of arenaviral diseases, facilitate studies to understand their prevalence and natural history, and ultimately lead to vaccines for preventing these major causes of morbidity and mortality / acase@tulane.edu

Approximately 170 million are infected with the hepatitis C virus (HCV) world wide and in the United States an estimated 2.7 million are HCV RNA positive. Of those infected with HCV, 54-86% fail to clear the virus and develop a chronic infection. The chronic phase can last many decades and can ultimately lead to end stage liver disease. The efficiency of the current therapy for a HCV genotype 1 infection, the dominate genotype in the United States, is a 30% sustained viral response (SVR) rate To develop more efficient drugs and therapies, a better understanding of the HCV life cycle is required. For this project we focused our efforts on the initial phase of the HCV life cycle, the process of viral entry. First, we attempted to utilize biophysical assays to identify peptide inhibitors of HCV entry, a method previously used successfully in our laboratory for the SARS coronavirus. Second, we set about measuring the kinetics of a successful HCV cell culture strain (HCVcc) infection of naive Huh7 cells and how it relates to viral entry and decay. Finally, we examined the role viral particle density has on early infection kinetics of HCVcc We were able to identify four peptide inhibitors of HCV pseudo particle (HCVpp) infectivity where two of the peptides showed activity in our biophysical assays indicating that in the case of HCV, utilization of biophysical assays is not an efficient method to identify peptide inhibitors of HCV. Our examination of HCVcc infection kinetics, in vitro, revealed that HCVcc early infection kinetics is a slow process requiring 24 hours to reach completion with particle decay being a significant factor in deciding final infectious titers. When we studied the relationship between particle density and HCVcc early infection kinetics, we did not find a difference in early infection kinetics between different density fractions of HCVcc. We did, however, observe that during the process of infection the density profile of the viral inoculum shifts dramatically toward lighter density. We observed the same effect during HCVcc decay process as the density profile shifted toward lower densities as a function of time spent at 37&deg;C / acase@tulane.edu

Hemorrhagic fever viruses cause diseases ranging from nonspecific febrile illnesses to severe vascular permeability, edema, coagulation defects, and shock. However, the mechanism that these viruses use to cause this spectrum of diseases is not well characterized. Endothelial cells are a key structural component of blood vessels and they also act as a barrier between blood and tissues. These cells serve an immunologic function by recognizing pathogens, expressing soluble mediators, and interacting with other immune system cells for their extravasation across the endothelium. The aim of this study was to isolate specific virus/endothelial cells interactions to determine factors that lead to an increase in endothelium permeability. It was determined that microvascular endothelial cells infected with Dengue virus, a member of the Flaviviridae family, induced an increase in permeability that was attributed to expression of the cytokine TNF-alpha and subsequent apoptosis of the cell. With the addition of neutralizing TNF-alpha antibodies or a general caspase inhibitor, Dengue virus-induced permeability was reduced as well as the number of apoptotic cells. Endothelial cells infected with Pichinde virus, a member of the Arenaviridae family, also exhibited an increase in permeability. Through the use of gene expression arrays and inhibitors to specific physiological functions and pathways, it was determined that the production of nitric oxide led to Pichinde virus-induced permeability. This permeability could be abrogated with the incorporation of a nitric oxide synthase inhibitor. This series of in vitro experiments has demonstrated that hemorrhagic fever viruses utilize a variety of different methods in order to cause vascular permeability in human endothelial cells. This study also provides the basework for future in vivo experiments utilizing small animal models of viral hemorrhagic fevers (VHF) / acase@tulane.edu

Primary Effusion Lymphoma (PEL) is a lymphoproliferative disease of B cell origin associated with HHV-8 infection and characterized by migration of tumor cells to serous body cavities. PEL cells originate from post-germinal center B cells yet harbor a non-B, non-T phenotype, a characteristic that has not been fully explained. In the present study we demonstrate that PEL cells have an impaired expression of B cell-specific transcription factors and this results in a decreased activity of promoters regulating essential B cell genes. PEL cells lack PU.1 expression, although its transcription partner IRF-4 is highly upregulated, leading to decreased activity of the immunoglobulin lambda and kappa light chain ETS-IRF enhancers. Expression of the B cell specific transcription factor Oct-2 and the B cell specific co-activator of octamer factors (Bob-1), which are known to regulate PU.1 expression, was also impaired. Ectopic expression of Oct-2 was able to fully restore PU.1 promoter activity in the PEL cell line BCBL-1, while PU.1 expression also reconstituted the activation of the lambdaB Ets-IRF site. In addition, protein levels of BSAP/Pax-5 and IRF-8/ICSBP were undetectable in PEL cells. The pattern of transcription factor ablation observed in PEL was found to be comparable to that observed in classical Hodgkin's disease-derived cell lines, which also lack B cell specific surface markers. Comparative analysis of gene expression by cDNA microarray of BCBL-1 cells (PEL), L-428 (cHD) and BJAB cells revealed a subset of genes that were differentially expressed in PEL cells. Among these, four genes involved in cell migration and chemotaxis were strongly upregulated in PEL cells: LTA4H, IL-16, TSP-1, and selectin-P ligand. Upregulation of LTA4H was investigated at the transcriptional level. The LTA4H promoter exhibited 50% higher activity in BCBL-1 cells than in BJAB or L-428 cells. Deletion analysis of the LTA4H promoter revealed a positive cis regulatory element active only in BCBL-1 cells in the promoter proximal region located between -76 to -40 bp. Formation of a specific DNA-protein complex in this region was confirmed by Electromobility Shift Assay (EMSA). Co-culture of BCBL-1 cells with ionophore-stimulated primary neutrophils lead to an increased production of LTB4 by transcellular biosynthesis compared to L-428 cells, demonstrating the functional significance of LTA4H upregulation. BCBL-1 cells also demonstrated increased migration even in the absence of chemotactic stimulus compared to L-428 cells. These observations indicate that (1) disruption of the B-cell specific transcriptional program is likely to contribute to the incomplete B cell phenotype characteristic of PEL cells and (2) upregulation of factors involved in cell migration and chemotaxis constitute a unique characteristic of PEL cells that may contribute to the localization of this lymphoma to serous body cavities.