Walter J. Atwood - US grants

DESCRIPTION: (Adapted from the applicant's abstract) The initial event in the life cycle of a virus is its interaction with specific receptors present on the surface of a cell. Understanding these interactions is important to our understanding of viral tropism and tissue specific pathology associated with viral disease. The human polyomavirus, JCV, is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease, progressive mulifocal leukoencephalopathy (PML). PML occurs most frequently in immunosuppressed patients and its incidence has risen dramatically as a result of the AIDS pandemic. PML is the direct result of JCV multiplication in oligodendrocytes, the myelin producing cell in the CNS. In vivo, JCV has been detected in oligodendrocytes, astrocytes, lymphoid tissue, and peripheral blood of PML patients. In vitro, JCV infects human glial cells and, to a limited extent, human B lymphocytes. The initial step in infection of cells by JCV is at the level of attachment and entry. This critical event in the life cycle of JCV has not been investigated due to the lack of a convenient cell culture system for efficient propagation of JCV. We have overcome this problem by taking advantage of a chimeric JCV/SV40 hybrid virus that can be efficiently propagated in the human glial cell line, SVG. Our preliminary data demonstrate that highly purified JCV binds to a limited number of trypsin sensitive receptors on SVG cells. This has led us to hypothesize that infection of SVG cell by JCV is mediated by a specific proteinaceous cell surface receptor. This hypothesis will be tested by fulfilling the following three specific aims: 1) determine the biochemical nature of cell surface molecules that are important in mediating infection of SVG cells by JCV, 2) determine the relationship between virus binding to SVG cells and infectivity, and 3) identify a specific SVG cell surface receptor for JCV. The identification of a specific JCV receptor on SVG cells will yield new insights into the early events of the JCV life cycle. As this is a critical step in the establishment of infection this work may lead to the development of novel therapies to prevent or treat JCV-induced disease.

DESCRIPTION (provided by applicant): The initial event in the life cycle of a virus is its interaction with receptors present on the surface of a cell. Understanding these interactions is important to our understanding of viral tropism, spread, and pathogenesis. The human polyomavirus, JCV, is the etiological agent of the fatal central nervous system (CNS) demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV has also been associated with several human tumors, including oligoastrocytoma and medulloblastoma. Following primary infection, JCV establishes a lifelong persistent infection in kidney and lymphoid tissues. In a minority of individuals, the virus spreads to the CNS, infecting both oligodendrocytes and astrocytes. The mechanisms that restrict JCV tropism for these cells and tissues and the mechanisms that allow for the spread of JCV from the periphery to the CNS are not understood. Our laboratory has been studying early events in the life cycle of JCV. We have determined that: 1. an N-linked glycoprotein containing terminal alpha (2-6) linked sialic acid is a critical component of the JCV receptor; 2. JCV and the related polyomavirus, SV40, do not share receptor specificity; and, 3. JCV, unlike SV40, enters cells by clathrin dependent receptor mediated endocytosis. The long term goals of our research are to define the role of virus receptors in mediating infection of cells and in determining viral tropism, spread, and pathogenesis in the infected host. Our working hypothesis, which is based on our previous work, is that JCV receptor interactions are critical determinants of viral entry, tropism, and spread within the host. We will address this hypothesis by asking the following questions: 1. What is the identity of the JCV receptor? 2. What are the cell and tissue distributions of JCV receptors? and 3. How does JCV penetrate the plasma membrane and target its genome to the nucleus? The data resulting from these studies will yield novel insights into the pathogenesis of JCV induced disease and may lead to novel therapies to prevent or treat these diseases.

The initial event of the life cycle of a virus is its interaction with receptors present on the surface of a cell. Understanding these interactions is important to our understanding of viral tropism, spread, and pathogenesis. The human papillomavirus, JCV, is the etiological agent of the fatal central nervous system (CNS) demyelinating disease. progressive multi-focal leukoencephalopathy (PML). JCV has also been associated with several human tumors, including oligoastrocytoma and medullablastoma. Following primary infection, JCV establishes a lifelong persistent infection in kidney and lymphoid tissues. In a minority of individuals, the virus spreads to the CNS, infecting both oligodendrocytes and astrocytes. The mechanisms that restrict JCV tropism for these cells and tissues and the mechanisms that allow for t6he spread of JCV from the periphery to the CNS are not understood. Immunosuppressive illness, including AIDS, is a major risk factor for the development of PML and PML remains an important and life threatening disease in these patients. Our laboratory has been studying early events in the life cycle of JCV. We have determined that an N- linked glycoprotein containing terminal alpha (2-6) linked sialic acids is a critical component of the JCV receptor; that this component of the receptor is expressed in a tissue specific manner; that JCV binds selectively to cells in vivo that are associated with infection. that infection of some non-permissive cells is blocked at a step preceding early viral gene expression; and, that JCV, unlike SV40 enters cells by clathrin dependent receptor mediated endocytosis. The long term goals of our research are to define the role of virus receptors in mediating infection of cells and in determining viral tropism, spread, and pathogenesis in the infected host. Our working hypothesis, which is based on our previous work, is that JCV receptor interactions are critical determinants of viral entry, tropism, and spread within the host. We will address this hypothesis by asking the following questions: 1. What is the identity of the JCV receptor? 2. What are the cell and tissue distributions of JCV receptors? and 3. How does JCV penetrate the plasma membrane and target its genome to the nucleus? The data resulting from these studies will yield novel insights into the pathogenesis and target its genome to the nucleus? The data resulting from these studies will yield noel insights into the pathogenesis of JCV induced disease and may lead to novel therapies to prevent or treat these diseases.

DESCRIPTION (provided by applicant): This application seeks support for the 2nd, 3rd, and 4th International Symposia on Polyomaviruses and Human Diseases. The 2nd International meeting will be held in Sapporo, Japan from June 11-13, 2004. The third International meeting will be held in Philadelphia in June of 2005, and the fourth International meeting will be held in Barcelona, Spain in June of 2006. These meetings have become the primary international conference for scientists and clinicians studying diseases associated with human polyomaviruses. These symposia are unique in that they bring together clinicians and basic scientists interested in developing therapeutic strategies to treat polyomavirus induced human disease. Specifically, we are requesting funds to defray the costs of 1. plenary session speakers, 2. travel grants for graduate students and post-doctoral fellows, 3. preparation of on-site registration materials, and 4. poster session costs. The Specific Aims of the 2nd International Symposium on Polyomaviruses and Human Diseases are to provide a forum for the dissemination of new information related to 1. the impact of human polyomaviruses in disease, 2. the epidemiology of polyomavirus associated disease, 3. the basic biology underlying polyomavirus associated disease, and 4. the development of treatment strategies to prevent or treat disease associated with polyomavirus infections. This meeting will provide a forum to foster collaborations between basic researchers and clinicians that should lead to novel methodologies to treat patients suffering from human polyomavirus associated disease. The attendance is estimated at 80 people based on the success of the 1st International Symposium held last year in Italy.

EXCEED THE SPACE PROVIDED. It is proposed to establish a COBRE comprised of 5 research projects headed by junior and early career faculty and 3 research core facilities. The scientific theme of the Center will be cancer signaling networks, namely, the molecular mechanisms by which signaling networks relevant to carcinogenesis are regulated: transcription, protein phosphorylation and protein degradation. Some of the focal points of the proposed research will be DNA repair, Wnt and IGF-1 signaling, proteome-wide analysis of protein phosphorylation, ubiquitin-mediated proteolysis, global RNA polymerase II machinery, transgenic mouse models of carcinogenesis, and bioinformatic inference of network systems. Cancers under investigation will include hepatocellular carcinoma and ovarian follicular cancer. Two of the investigators will continue from the current COBRE (Drs. Singer, Zhitkovich), and 3 are newly hired junior faculty. The proposed research core are Mouse Transgenics, Genomics, and Bioinformatics. The first two carry over from the current Center, while the latter will be established as a new core facility. Each project leader and core director has an assigned senior faculty mentor. The mentors comprise the IAC which is chaired by the PI. The EAC evaluates all aspects of the Center's operation and advises the PI and the IAC. The PI is also advised by the Steering Committee of the Brown Center for Genomics and Proteomics, and reports to the Dean of the Medical School and the senior administration. The of the program is to establish both a thematic focus and the caliber of science required for a transition to a PPG funded by the NCI. The following Specific Aims are proposed to achieve these goals: Aim 1: To investigate the role of the gonad-specific transcriptional coactivator TAF4b in the development of ovarian granulosa cell cancer; Aim 2: To examine the function of Wnt signaling networks in the development of hepatocellular carcinoma and to address whether targets in this pathway may be useful for cancer therapy; Aim 3: To describe and functionally test, on a proteome- wide basis, cascades of tyrosine protein phosphorylationtriggered by receptor crosslinking in mast cells and by IGF-1stimulation in hepatocellular carcinoma cells; Aim 4: To elucidate the mechanisms by which Cullin 3 complexes recognize regulatory proteins causally connected to cell cycle aberrations found in cancer and target them for ubiquitin-mediatedproteolysis; Aim 5: To discover novel mechanisms by which normal levels of oxidative stress found in all cells participate in generating DNA damage that ultimately leads to the development of cancer. '

DESCRIPTION (provided by applicant): Progressive Multifocal Leukoencephalopathy (PML) is a major life threatening complication in patients with underlying immunosuppressive disorders (e.g. AIDS) and in patients undergoing immunotherapy for autoimmune diseases such as multiple sclerosis, Crohn's disease, severe plaque psoriasis, systemic lupus erythematosis, hematologic malignancies, and rheumatoid arthritis. The disease is paradoxically caused by a common human polyomavirus following activation from a latent to a lytic phase of growth. There are several critical gaps in our understanding of the basic biology of PML. First, the anatomical site of virus latency is not known but kidney, tonsil, bone marrow, and brain have all been postulated to be involved. Second, the mechanisms that govern latency versus lytic growth of the virus are not well understood. Third, the mechanisms of viral spread to the CNS and within the CNS are not known. We hypothesize that virus induced signals reprogram the cellular environment to promote replication and spread and that epigenetics plays a role in governing the balance between latency and activation. The experiments proposed here will define these signals and the molecular pathways involved in viral pathogenesis. These mechanisms and pathways may be amenable to pharmacological intervention.

The administrative core will serve to facilitate communication between the three projects and the cores. This will be accomplished by arranging for teleconference calls as needed. The projects also propose to hold live videoconferences as often as needed to facilitate data sharing and analysis and it will be the responsibility of the administrative core to arrange these. An FTP site has been established at Brown so members of each team can upload large data files for analysis. The administrative core will track and analyze expenditures submitted to Brown from the Pis of projects #1 and #3 and from core B. Ms Tammy Glass has experience in these activities as she currently manages a multi-component COBRE account for Dr. Atwood, Invoices will be reviewed monthly by Dr. Atwood and by Ms Glass and when approved will be sent to accounts payable for immediate payment. The administrative core will assist in the preparation of annual progress reports. This will be done yearly well in advance of the deadlines for submitting progress reports. Ms. Glass will be responsible for collating these reports electronically and forwarding them to the appropriate awarding institute.

DESCRIPTION (provided by applicant): The human polyomaviruses, JCV and BKV, establish lifelong persistent infection in kidney but are generally not associated with disease in healthy individuals. In immunosuppressed patients reactivation and spread of JCV to the central nervous system causes a fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). Reactivation of BKV in immunosuppressed renal transplant patients causes polyomavirus associated nephropathy (PVN) that leads to graft dysfunction and loss. Viral DMA and viral gene products from both JCV and BKV have been detected in human cancers but a causal link has not been established. Our long-term goals are to define the role of virus receptors in cellular invasion, spread, and pathogenesis. During the last funding cycle we discovered that the JCV receptor is a complex consisting of alpha(2,6) linked sialic acid and the 5HT2a receptor. Both components are critical for infection as cells missing either component are not susceptible to infection by JCV. Our efforts are now focused on identifying the critical determinants that allow JCV to productively interact with this receptor complex. As the 5HT2a receptor is a G protein coupled receptor and JCV binding to cells induces a signal that is essential for infection we will determine whether JCV signals directly through this receptor. During the last funding cycle we discovered that the receptor for BKV is an N-linked glycoprotein containing alpha(2,3)-linked sialic acid, that signaling is important for BKV infection, and that BKV does not share receptor specificity with JCV. A detailed molecular genetics approach is proposed to identify the critical residues in the BKV capsid protein VP1 that interacts with sialic acid containing receptors. We will also compare JCV and BKV induced signaling and entry pathways in the cell. Our specific aims are to 1. define the interactions between human polyomavirus capsid proteins and host cell receptors, 2. define the molecular pathways that link virus- induced signaling at the plasma membrane to transcriptional responses in the nucleus, and 3. map the infectious entry pathway leading to the nucleus and identify the viral and cellular proteins involved at each step. Data resulting from these studies will yield novel insights into the pathogenesis of human polyomavirus induced disease and may lead to novel therapies to prevent or treat these diseases.

The human polyomavirus, JCV, establishes a lifelong persistent infection and is generally not associated with disease in healthy individuals. In immunosuppressed patients reactivation and spread of JCV to the central nervous system causes a fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). Our immediate goals are to define the structural features of the JCV surface that allow for cell binding and infection and to identify molecules that antagonize these interactions. In this project we will use structural information provided by project #1 to guide mutagenesis and testing of amino acid residues in the virus capsid that are hypothesized to interact directly with cell surfaces allowing for productive infection. We will work closely with project #3 to identify and screen compounds for their ability to antagonize these interactions. In preliminary experiments we've established that two small molecular weight human alpha defensins potently inhibit infection by JCV, BKV, and SV40 most likely by binding to a shared domain on the virion surface. Specific aim #3 of this project will focus on determining the mechanism of action of alpha defensin inhibition of JCV infection. Working together with the Pis of projects 1 and 3 we will determine the structural basis for inhibition by these molecules

DESCRIPTION (provided by applicant): This program project brings together an interdisciplinary team of three scientists with unique expertise to functionally target and inhibit human polyomavirus infections. The polyomaviruses in general, and the human polyomaviruses in particular, have been shown to utilize distinct host cell carbohydrates and proteins to infect target cells and tissues. The human polyomavirus JCV is the causative agent of a fatal central nervous system demyelinating disease known as progressive multifocal leukoencephalopathy (PML). The majority of PML cases occur in patients with AIDS but recently PML has also been shown to occur in multiple sclerosis patients being treated with potent immunomodulatory drugs that inhibit immunosurveillance of the CNS. There are currently no drugs in the pipeline that target the virus directly and a major goal of this program is to identify compounds capable of directly inhibiting virus infection. This goal will be accomplished by close collaborative interactions between a team consisting of a polyomavirologist, a structural biologist, and a molecular geneticist/virologist. Project # 1 led by Professor Thilo Stehle will focus on structuraly characterizing and identifying sites on the virus that are critical for interacting with host cell surfaces. Project # 2 led by Professor Walter Atwood will introduce site-specific mutations in the virus based on these structures and functionally characterize the mutants. Project #2 will also further develop a drug lead identified in years 1-4 of this program project. Project # 3 led by Dr. Dan DiMaio will conduct genetic screens for factors that are critical for JCV invasion of host cells. The projects will be supported by a virus and pseudovirus production core at Brown University headed by Dr. Gretchen Gee. An administrative core will be housed at Brown University. The overall goal of this program is to use structural information and high throughput genetic screens to identify factors and targets that can be exploited to inhibit polyomavirus infection. The three major investigators on the team have built a strong working collaboration that is evidenced by the solid preliminary data and numerous joint publications that support this application.

PROJECT SUMMARY - CORE A The administrative core will serve to facilitate communication between the three projects and the cores. This will be accomplished by arranging live video conferences on a regular basis to facilitate data sharing and analysis. The administrative core will schedule these conferences. Large data files are shared between all the project and core leaders and these are managed by the administrative core. The administrative core will track and analyze expenditures submitted to Brown from the PIs of projects #1 and #3 and from core B. Ms Patricia Werner has experience in these activities as she currently manages a multi-component COBRE account for Dr. Atwood. Invoices will be reviewed monthly by Dr. Atwood and by Ms Werner and when approved will be sent to accounts payable for immediate payment. The administrative core will assist in the preparation of annual progress reports. This will be done yearly well in advance of the deadlines for submitting progress reports. Ms. Werner will be responsible for collating these reports electronically and forwarding them to the appropriate awarding institute.

PROJECT SUMMARY - PROJECT 2 The initial interaction of JCPyV with its host involves recognition of specific receptor complexes on cells. Recognition of these receptors leads to virus penetration of the host cell membrane, trafficking of the virion to the endoplasmic reticulum, and the eventual delivery of the dsDNA genome to the cell nucleus. Our team, supported by this program project, elucidated the critical components of the JCPyV receptor complex and determined that the major capsid protein VP1 was responsible for directing the virus to the ER. We developed novel tools to study not only lab adapted strains of JCPyV but also mutant forms of the virus that have been reported to arise in the brains of patients with PML. These mutant forms of JCPyV have lost the ability to recognize the sialic acid receptor and as a consequence are no longer infectious in most cell types examined. We hypothesize that these mutants either recognize alternative receptors which are yet to be identified or that they have gained the capacity to spread directly from cell to cell bypassing the requirement for cell surface receptors. Our approach in project 2 is to use pseudoviruses developed in core B to further explore potential receptor usage by these mutants. We will also explore the possibility that these mutants, are capable of direct cell to cell spread by engineering the mutations into an infectious JCPyV clone and following their growth after transfection of the genomes into different cell types grown as confluent monolayers. Regardless of the mechanism of infection (receptor mediated OR direct cell to cell spread) these viruses must all traffic to the ER to begin the uncoating process for eventual delivery of their genomes to the nucleus. In the last funding cycle we discovered that the dihydroquinozolinone compound Retro-2cycl potently prevents JCPyV trafficking to the ER and substantially reduces initial infection and infectious spread. Our goal now is to define its mechanism of action, to identify its cellular targets, and to optimize the existing compound so that a therapeutic window of inhibition can be determined.

Program Summary Progressive Multifocal Leukoencephalopathy (PML) is a major life threatening complication in patients with AIDS and in patients undergoing immunotherapy for autoimmune diseases such as multiple sclerosis, Crohn's disease, severe plaque psoriasis, systemic lupus erythematosis, hematologic malignancies, and rheumatoid arthritis. The disease is paradoxically caused by a common human polyomavirus following the loss of normal immune surveillance of the central nervous system (CNS). There are several major gaps in our understanding of the basic biology that underpins the development of PML. First, the anatomical site of virus persistence is not known but kidney, bone marrow, and brain have all been postulated to be involved. Second, the mechanisms that govern viral persistence are not well defined but changes in the viral promoter (archetype to PML-type) and regulation of a viral microRNA are thought to be critical for transition to the lytic phase. Third, the mechanisms of viral spread to the CNS and within the CNS are not known. Based on our recent work we hypothesize that free virus as well as virus enclosed in extracellular vesicles spreads from the kidney to the choroid plexus and meningeal compartments where replication in CPE cells provides the means for EV mediated invasion of brain parenchyma. Once in the brain extracellular vesicle mediated spread is likely to play the major role in spreading the infection because macroglia lack the principle cellular receptors to support infection by free virus. Our vision for the R35 application is to further our understanding of these basic mechanisms of infectious spread of virus and to use the information to identify biomarkers that can predict PML early and in easily accessible compartments well before the virus has a chance to spread to the CNS. The work should also lead to the development of novel strategies to treat and prevent PML.