Craig Eugene Cameron - US grants

DESCRIPTION (Applicant's Description): The long-term objectives of this proposal are to develop further 5-aminoimidazole-4-carboxamide ribotide transformylase/inosine monophosphate cyclohydrolase (ATIC) as a c h e m otherapeutic target for the treatment of cancer and to create methotrexate-resistant variants of ATIC suitable for use in hematopoietic stem c e ll support thereby expanding the use of methotrexate in high-dose chemotherapy protocols. The specific aims of this proposal are as follows: (1) to use pre-steady-state kinetic methods to define the minimal kinetic schemes for the two reactions catalyzed by ATIC; (2) to identify the regions of ATIC responsible for dimerization and substrate binding using approaches such as site-directed mutagenesis and protein footprinting; and (3) to develop a genetic screen in yeast to identify methotrexate-resistant variants of ATIC.

DESCRIPTION (provided by applicant): This is an application for renewal of a grant to study the structure, function and mechanism of the viral RNA-dependent RNA polymerase (RdRP). Positive-strand RNA viruses cause a variety of diseases in humans. Strategies to control disease outbreaks caused by this category of viruses are needed. The central hypothesis driving the proposed studies is that the viral RdRP is a tractable target for development of broad-spectrum antiviral agents. The previous funding period was devoted to the elucidation of the kinetic, thermodynamic and structural basis for nucleotide incorporation catalyzed by a prototypical RdRP, 3Dpol from poliovirus that would permit a quantitative, mechanistic comparison of the viral RdRP to cellular polymerases. All of the original aims were completed. During the next funding period, we will pursue the following specific aims: (1) Mechanistic studies of 3Dpol-catalyzed nucleotide incorporation. We hypothesize that a conformational change preceding phosphoryl transfer is a key determinant of 3Dpol fidelity. We propose to develop innovative methodologies to evaluate this step directly. We have identified conditions for the 3Dpol reaction in which phosphoryl transfer is the sole rate-limiting step. This advance permits us to interrogate the chemical mechanism directly. We will test the hypotheses that one of the two metal ions required for catalysis functions to orient the 3' nucleophile of the primer for catalysis and that phosphoryl transfer employs general acid-base catalysis. A conformational change exists after phosphoryl transfer. We hypothesize that this step represents another fidelity checkpoint. We will refine existing approaches and develop new approaches to study this step directly. (2) Structural basis for fidelity of 3Dpol-catalyzed nucleotide incorporation. We will employ a novel genetic selection strategy to isolate poliovirus mutants with an antimutator phenotype in order to uncover structural determinants of fidelity. This approach has already provided a link between a site remote from the catalytic center and the conformational change preceding phosphoryl transfer, an unexpected yet exciting finding. We will continue to test predictions of our structural model for the 3Dpol ternary complex in order to define all of the residues participating in the hydrogen-bonding network in the ribose-binding pocket that is required for incorporation fidelity. We will test the hypothesis that a loop of unknown function and unique to RdRPs and reverse transcriptases located in the palm subdomain of 3Dpol is required for incorporation fidelity. (3) Determinants of 3Dpol-primer/template complex stability. A crystal structure for a ternary complex of 3Dpol does not exist. If we can assemble 3Dpol-primer/template complexes that do not dissociate on the timescale required for crystallization, then we will be in a better position to fill this gap. We will pursue two approaches that may permit us to achieve this goal, and, at the same time, provide insight into the mechanisms and surfaces employed by 3Dpol to maximize stability of the 3Dpol-primer/template complex.

[unreadable] DESCRIPTION (provided by applicant): The arsenal of antiviral agents that may be useful for treatment of infections by RNA viruses classified as either category A, B or C pathogens consists of one compound, ribavirin. Ribavirin is the only broad-spectrum antiviral nucleoside employed clinically. Development of compounds with antiviral activity greater than that of ribavirin has been thwarted by the absence of a clear understanding of the mechanism of action of this compound. Current dogma states that ribavirin functions by inhibiting inosine monophosphate dehydrogenase (IMPDH), a cellular enzyme required for de novo synthesis of GTP. Our preliminary studies with a model RNA virus suggest an alternative mechanism of action for ribavirin. Briefly, RTP is a substrate for viral RNA-dependent RNA polymerases (RdRP) and is incorporated opposite both cytidine and uridine. The lack of specific incorporation of ribavirin causes an increase in the mutation frequency of the virus beyond that capable of supporting viability of the virus population. These data suggest that ribavirin is a lethal mutagen of RNA virus genomes. The long-term goal of the collaborative research presented in this proposal is the development of lethal mutagenesis into a useful therapeutic regimen for the treatment of RNA virus infections. This goal will be accomplished by pursuing the following specific aims: [unreadable] 1. Identify leads for development [unreadable] (a) by evaluating the mutagenic activity of a subset of compounds from appropriate libraries provided to us by Ribapharm, Inc. (a subsidiary of ICN Pharmaceuticals, Inc.) and the Medical Research Council (United Kingdom), and [unreadable] (b) by synthesizing and characterizing ribonucleoside analogs containing pseudo bases that are known or predicted to pair ambiguously; [unreadable] 2. Evaluate the ability of ribonucleotide reductase to utilize mutagens as substrates and/or effectors in order to obtain information on potential issues of safety; [unreadable] 3. Establish dengue virus type 2 as a model system for evaluation of lethal mutagenesis by characterizing the mechanism of action of ribavirin against this virus; [unreadable] 4. Develop cell-based assays to screen for inhibitors and lethal mutagens of dengue virus. [unreadable] Validation of lethal mutagenesis as an antiviral strategy by identifying novel mutagens and demonstrating efficacy against important pathogens without heritable genetic consequences should provide the impetus for pursuit of this strategy by the pharmaceutical industry. The availability of cell-based assays to screen for lethal mutagens should greatly facilitate the discovery effort. Lethal mutagens should have broad-spectrum activity against RNA viruses, thus better equipping this nation to deal with the potential use of RNA viruses as biological weapons. [unreadable] [unreadable]

Project Abstract This is an application for renewal of a grant to study picornavirus genome replication. Picornaviruses represent an existing and emerging threat to US public health. Although protein factors and genetic elements required for picornavirus genome replication are known and appear to be conserved, a clear understanding of the mechanisms employed to produce picornaviral RNA is lacking. The long-term objective of this program is to reconstitute picornavirus genome replication in vitro from purified components. We have achieved all of the major objectives of the previous funding period. In addition, we have solved the first crystal structure for a picornaviral 3CD protein, developed the technology to study 3C-RNA interactions by using nuclear magnetic resonance spectroscopy, and discovered that the 3CD protein has both pre- and post-replication functions. During the next funding period, we will continue our studies of picornavirus genome replication as well as explore our newly discovered function for 3CD by pursuing the following specific aims: (1) Define the mechanism of assembly and structural organization of the picornavirus VPg uridylylation complex by using molecular genetic, biochemical and biophysical approaches; (2) Define the molecular basis for sequence- and structure-specific RNA recognition by 3C by using nuclear magnetic resonance spectroscopy; and (3) Elucidate the function of 3CD in formation of replication complexes.

DESCRIPTION (provided by applicant): This is an application to obtain funds to replace our existing Typhoon 8600 variable mode imager with a Typhoon 9410, a related instrument with enhanced capabilities. The existing instrument is only five years old but has been in high demand, ~7000 scans during the past fiscal year. Over the past few years, the number of service calls and the severity of the corresponding problems have increased dramatically. The manufacturer no longer sells the 8600, so support for this instrument may be limited. Thirteen investigators, supported by 19 grants from NIH, rely on this instrument on a daily basis to perform their research. Several other investigators, funded by other agencies, rely on this instrument as well. The use of the typhoon has almost completely eliminated the use of expensive X- ray film for detection of radionuclides. However, X-ray film is still used for chemiluminescence applications, especially Western blotting. The 8600 lacks the blue laser required for use of the most sensitive chemifluorescence reagents available for Western-blotting applications;therefore, we have been forced to use chemiluminescence almost exclusively. The 9410 has the required blue laser. Acquisition of a Typhoon 9410 will enhance chemifluorescence capabilities by increasing sensitivity, ensure uninterrupted progress on the 19 NIH-supported research projects, and eliminate the cost associated with the use of X-ray film for chemiluminescence applications. PUBLIC HEALTH RELEVANCE: The NIH-funded projects that will benefit from the purchase of the Typhoon 9410 focus on topics ranging from virus replication and pathogenesis to cancer with the ultimate goal of preventing, diagnosing and/or treating diseases afflicting citizens of the United States and around the world.

DESCRIPTION (provided by applicant): This is an application for renewal of a grant to study to the mechanism and fidelity of nucleotide incorporation by the replicative polymerase of RNA viruses, the RNA-dependent RNA polymerase (RdRp). Over the past ten years, the world has witnessed the emergence of SARS, the spread of West Nile encephalitis, and the fear of a global flu pandemic. These diseases are caused by RNA viruses. In addition, the threat of intentional release of RNA viruses as weapons or agents of terror has increased substantially. The long-term goal of this research program is to develop strategies to treat and/or prevent RNA virus infection by targeting the RdRp. Our program has employed a prototypical RNA virus, poliovirus (PV), and its RdRp (3Dpol) as our model system. The previous funding period was devoted to the interrogation of the chemical mechanism for 3Dpol- catalyzed nucleotidyl transfer, elucidation of the structural basis for 3Dpol incorporation fidelity, and development of the tools to solve a crystal structure for 3Dpol in complex with primed template and nucleotide. We have made outstanding progress towards completion of all our aims. We have obtained new insight into the chemical mechanism for nucleotidyl transfer. We discovered a link between RdRp incorporation fidelity and pathogenesis. We discovered a connection between RdRp dynamics and incorporation fidelity. Together, our studies lead to the very provocative hypothesis that RdRp incorporation fidelity is a target for antiviral and vaccine development that will be elaborated during the next funding period. We will pursue the following specific aims: (1) Elucidate additional roles for the general acid in polymerase function;(2) Identify novel determinants and mechanisms of polymerase fidelity;and (3) Establish dynamics-function relationships for the RdRp. PUBLIC HEALTH RELEVANCE: RNA viruses represent an existing and emerging threat to US public health. Achievement of the goals of the application will provide novel targets and mechanisms for development of vaccines and inhibitors to prevent and to treat infections by RNA viruses.

DESCRIPTION (provided by applicant): This is an application for an exploratory research grant to identify new forms of the hepatitis C virus (HCV) non- structural proteins produced in infected human liver tissue and to develop models for their formation and function. HCV continues to be a significant public-health concern of global proportion. In spite of years of effot, the inability to study the numerous genotypes of HCV in cell culture remains an obstacle to our understanding of the mechanisms employed by this virus to establish persistence, which can ultimately lead to the development of hepatocellular carcinoma (HCC). HCV non-structural protein 5A (NS5A) is unique among non-structural proteins of most positive-strand RNA viruses. Two of its three domains are intrinsically disordered, and it contains numerous sites of phosphorylation for myriad serine/threonine kinases, including cAMP-activated protein kinase (PKA), casein kinase 1 family members and casein kinase 2. We have been intrigued by the possibility that specific patterns of NS5A phosphorylation produce unique conformations and functions for this protein, thus explaining the ability of NS5A to interact with so many cellular proteins and pathways. Our experimental tests of this possibility have led to the identification of the site of NS5A phosphorylation by PKA and to the development of immunological reagents to demonstrate the existence of the PKA-phosphorylated form of NS5A in cells replicating HCV RNA of both genotypes 1b and 2a. In addition, examination of biopsies from pediatric cases of hepatitis C and tissue from an end-stage case of hepatitis C, led to the discovery that forms of NS5A observed in infected liver tissue are different than those observed in human hepatoma cell lines replicating HCV RNA. The additional forms of NS5A detected in liver tissue are remarkably similar to those observed in cell lines when caspases are activated and the abundance of the PKA- phosphorylated species correlates well with the amount of liver injury. It is our hypothesis that caspase cleavage of NS5A could provide a mechanism for HCV to monitor the antiviral state of the hepatocyte; phosphorylation of NS5A could signal to HCV that an anti-apoptotic state suitable for replication exists in the hepatocyte. These cleaved and phosphorylated forms may expand the NS5A proteome, creating forms of the protein essential for virus multiplication. If this is the case, then the inability of most HCV sequences to replicat in hepatoma cell lines may be related, in part, to the inability to produce forms of NS5A, and perhaps other non-structural proteins, that are readily produced in hepatocytes and that are required for optimal virus multiplication. Our working model will be explored by pursuing the following specific aims: (1) Identification and characterization of forms of HCV non-structural proteins produced during infection in vivo; (2) Evaluation of pro-apoptotic and pro-survival (anti-apoptotic) responses during HCV infection in vivo; and (3) Elucidation of clinical correlations. These studies have the ability to discover forms of HCV non-structural proteins unique to the infected hepatocyte and to reveal correlations between the level of these new forms and clinical outcomes. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a major cause of liver disease worldwide; only two HCV sequences and one cell line are available to study the HCV lifecycle in vitro. We have discovered that forms of some HCV non-structural proteins observed in HCV-infected liver are not produced in cell lines, and we propose that the absence of these forms in cell lines limits HCV multiplication in vitro. Therefore, completion of this study may reveal new approaches to study more HCV genotypes and facilitate the development of new antiviral therapies.

? DESCRIPTION (provided by applicant): This is a new application for an R01 grant to establish the intellectual and technical framework to permit the study of viral infection on the single-cell level to be as tractable as the study of viral infection on the population level using the plaque assay. The world is ill equipped to deal with the (re)emergence of diseases caused by RNA viruses. The viral RNA genome is replicated by the virus-encoded RNA-dependent RNA polymerase (RdRp), an enzyme that, in most cases, lacks proofreading activity and a cellular repair mechanism to usurp. As a result, each progeny genome differs from another in the population by one or more nucleotide changes. The Cameron laboratory and colleagues have discovered that the genetic diversity created by replication errors made by the RdRp permits the virus to clear bottlenecks that would otherwise lead to viral extinction. Therefore, it is becoming increasingly clear that attenuated viruses for use as vaccine strains can be created by altering the nucleotide incorporation fidelity of the RdRp. Unexpectedly, the Cameron laboratory has observed that using conventional approaches to study this class of vaccine candidate in cell culture masks the attenuated phenotype. The attenuated phenotype is only observed by evaluating the infection at the level of the single cell instead of the population. This observatio motivated the development of techniques to study viral infection on the single-cell level. Our foray into this area was funded by the PSU Huck Institutes of the Life Sciences. In this proposal, we present a set of experimental objectives that will move single-cell virology from a descriptive art to a quantitative science that can be implemented broadly by the virology community not only to understand viral population dynamics but to reveal between-individual differences that may underlie susceptibility to viral infection. Importantly, this technology is essential to advancing RdRp fidelity as a target and mechanism for viral attenuation and vaccine development, thereby addressing an urgent public-health need. We will, therefore, pursue the following specific aims: (1) Elucidate parameters governing diverse kinetics of viral genome replication at the single-cell level; (2) Establish a data and statistical analysis pipeline for th single-cell virology experiment and develop mechanistic models of infection; and (3) Enhance capabilities of the microfluidic platform for characterization of viral infections at the single-cel level.