Mark E Peeples
Research Inst Nationwide Children´s Hosp
Project start date: 2012-02-01
Project end date: 2017-01-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Mark E Peeples
VISUALIZING INTERFERON-BETA INDUCTION IN VIVO
Mark E Peeples, Professor
Research Inst Nationwide Children´s Hosp, Nationwide Children´s Hospital, Columbus, Oh 43205-2664
Grant 5R21AI073597-02 from National Institute Of Allergy And Infectious Diseases
Abstract: Virus infection of vertebrates triggers multiple host defense mechanisms, and type I interferons (IFNs) are among the earliest of these. Type I (or 1 and 2) IFNs are best known for their antiviral functions. IFN-1/2 synthesis is induced by virus infection of all cell types. When neighboring cells are exposed to secreted IFN-1/2, the binding of these ligands to the IFN-1/2 receptor leads to the transcriptional upregulation of the many interferon-stimulated genes which mediate the anti-viral state. This very early innate immune response to viral invasion is very important for limiting viral replication and spread. But while the type I IFN response can contain a virus infection, an antigen- specific T cell response is needed for resolution. Recent progress in the area of IFN gene regulation and function has led to the realization that, in addition to their antiviral effects, IFN-1/2 also have a role in promoting an effective and appropriate adaptive immune response to viral infection. IFN-2 and IFN-1 secreted from infected cells and plasmacytoid dendritic cells can also promote antigen presentation, lymphocyte activation, and lymphocyte survival. As type I IFNs have such an important role in anti-viral defense, it is not surprising that all viruses studied have found ways of inhibiting IFN-1/2 production and or action. These mechanisms vary among pathogens, and their effectiveness can influence viral virulence, chronicity, and ability to reinfect. Our laboratory is interested in how different respiratory viruses induce type I IFNs in vivo, and how production of these cytokines affects the development of virus specific lymphocyte responses. Our ability to study these dynamic processes would be markedly enhanced if we were able to trace the source and timing of IFN production during an active infection. This application describes our plans to derive indicator mice carrying a gene for green or red fluorescent protein that is driven by a type I IFN promoter. These reagents will allow virologists to study the ability of many different viruses to activate and/or suppress type I IFN production in vivo and in vitro. PUBLIC HEALTH RELEVANCE Virus infection triggers multiple host defense mechanisms, and the cytokines called type I interferons (IFNs) are among the earliest of these. This very early innate immune response to viral invasion is important for limiting viral replication and spread. As type I IFNs have such an important role in anti-viral defense, it is not surprising that all viruses studied have found ways of inhibiting IFN production and or action. This application describes our plan to derive indicator cell lines and transgenic mice that will fluoresce when the IFN genes are activated. These reagents will allow virologists to study the ability of many different viruses to activate and/or suppress type I IFN production in vivo and in vitro
Keywords: 0-11 years old; ATGN; Affect; Aged 65 and Over; Allergic; Animal Model; Animal Models and Related Studies; Animals; Anti-Viral Response; Antigen Presentation; Antigens; Antiviral Agents; Antiviral Drugs; Antiviral Response; Antivirals; Area; Attenuated; Blast Transformation; Blastogenesis; Blood Serum; Cell Line; Cell Lines, Strains; Cell Maturation; CellLine; Cells; Child; Child Youth; Childhood; Children (0-21); Clinical; Cytokine Signal Transduction; Cytokine Signaling; Dendritic Cells; Disease; Disorder; Effectiveness; Elderly; Elderly, over 65; Endogenous Interferon Beta; Epithelial; FP593; Family; Gene Action Regulation; Gene Expression Regulation; Gene Regulation; Gene Regulation Process; Generations; Genes; Grippe; Host Defense Mechanism; Human, Child; IFN; IFN-Beta; IFNb; Immune response; Immunity; Immunization; Immunologic Stimulation; Immunological Stimulation; Immunostimulation; In Vitro; Individual; Infant; Infection; Influenza; Interferon Beta, Natural; Interferon Type I; Interferon, Fibroblast; Interferon-beta; Interferons; Laboratories; Lead; Life; Ligand Binding; Link; Literature; Lymphoblast Transformation; Lymphocyte; Lymphocyte Activation; Lymphocyte Stimulation; Lymphocyte Transformation; Lymphocytic; Mammals, Mice; Mediating; Memory; Mice; Mice, Transgenic; Murine; Mus; Nasal; Natural human interferon beta; Nose; Nose, Nasal Passages; Paramyxoviridae; Pb element; Phase; Pneumovirus; Process; Production; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); RSV Vaccines; RSV proteins, Respiratory syncytial virus; Reagent; Receptor Protein; Relative; Relative (related person); Reporter; Reporting; Resolution; Respiratory Infections; Respiratory Syncytial Virus Vaccines; Respiratory System, Nose, Nasal Passages; Respiratory Tract Infections; Respiratory syncytial virus; Respiratory syncytial virus RSV proteins; Role; Science of Virology; Sensitization, Immunologic; Sensitization, Immunological; Serum; Source; T-Cells; T-Lymphocyte; Technology; Thymus-Dependent Lymphocytes; Time; Transgenic Mice; Tropism; Up-Regulation; Up-Regulation (Physiology); Upregulation; Vaccinated; Veiled Cells; Vertebrate Animals; Vertebrates; Viral; Viral Diseases; Virology; Virulence; Virus; Virus Diseases; Virus-Cell Interaction; Virus-Cell Membrane Interaction; Viruses, General; advanced age; cell type; children; cultured cell line; cytokine; disease/disorder; drFP583; ds red protein; dsFP593; elders; flu infection; geriatric; heavy metal Pb; heavy metal lead; host response; immunogen; immunogenic; immunogenicity; immunoresponse; in vivo; influenza infection; interest; late life; later life; lymph cell; men; men`s; model organism; older adult; older person; pathogen; pediatric; proteins, Respiratory syncytial virus; public health relevance; receptor; red fluorescent protein; respiratory; respiratory infection virus; respiratory virus; response; senior citizen; social role; thymus derived lymphocyte; vertebrata; viral infection; virology; virus development; virus infection; youngster
Project start date: 2008-05-23
Project end date: 2010-04-30
Budget start date: 1-MAY-2009
Budget end date: 30-APR-2010
PFA/PA: PA-06-181
5R21AI073597-02 (2009): $216000
Translational Regulation In Sendai Virus
Mark E Peeples, Professor
Research Inst Nationwide Children s Hosp Nationwide Children s Hospital Columbus, Oh 432052664
Grant 5R01AI047430-05 from National Institute Of Allergy And Infectious Diseases IRG: VR
Abstract: The long-term goal of this proposal is to understand the regulation of protein synthesis in mammalian viruses. Our laboratory has focused on the role of mRNA structure on translation. The P/C mRNA of Sendai virus provides an excellent model to dissect translation machinery. It uses five translation start sites to express five proteins (P, C, C , Yl and Y2) in differential amounts. Expression of these proteins is not in accordance with the tenets of ribosome scanning, internal ribosome entry, or the ribosome shunting model. Our studies indicated that the C-AUG must occur in +1 orientation in relation to the P-AUG for the efficient synthesis of C protein. Importantly, all the known viral bicistronic mRNAs with overlapping reading frames have their second AUG in +1 orientation. Thus, one aim of the proposal is to test the hypothesis that the +1 orientation of the downstream reading frame in a bicistronic mRNA is crucial for its efficient initiation. This concept will be examined by constructing mRNAs with three overlapping open reading frames. Appropriate placement of AUG start sites will test the validity of this hypothesis. Second, non-AUG start sites are used efficiently in the P/C mRNA. The 5 UTR of the mRNA appears to be important for translation initiation at non-AUG codons. Third, the Y2 start site (fourth AUG) locus has a high propensity for translation initiation. Chimeric mRNAs will be constructed to define the loci that enhance translation of C and Y2 proteins. Preliminary results have indicated that long-range interactions in the mRNA play a role in the initiation of all P/C mRNA encoded proteins. To test this, secondary structure of the entire P/C mRNA will be analyzed using both secondary and higher order structure-probing reagents to establish structure-function relationships. Creating deletions within the coding regions of P and C proteins will localize internal regulatory sequences. Fourth, synthesis of C and certain cellular proteins is resistant to cycloheximide inhibition. Elements and mechanism that allow cycloheximide resistance will be defined. Finally, P/C mRNA binding proteins that are involved in translation regulation will be identified and characterized. Recent studies have shown that the internal initiation can occur on the A site of the ribosome without the initiator methionine tRNA, eIF2 or GTP hydrolysis. This initiation mechanism can explain some of our results. We will test this model for the P/C mRNA. In essence, the proposed studies will define the mechanisms that allow the regulated expression of the polycistronic P/C mRNA. Moreover, these studies will provide insights as to how mRNA structure and its interacting proteins regulate the protein synthesis machinery in mammalian cells.
Keywords: genetic regulation, genetic translation, parainfluenza virus type 1, virus RNA, RNA binding protein, chemical structure function, messenger RNA, open reading frame, virus genetics, genetic mapping
Project start date: 2002-08-01
Project end date: 2007-05-31
5R01AI047430-05 (2006): $278735
5R01AI047430-04 (2005): $285441
7R01AI047430-03 (2004): $294858
5R01AI047430-02 (2003): $285440
1R01AI047430-01A2 (2002): $264065
Fusion Protein In Respiratory Syncytial Virus Entry
Mark E Peeples, Professor
Research Inst Nationwide Children s Hosp Nationwide Children s Hospital Columbus, Oh 432052664
Grant 5R01AI047213-06 from National Institute Of Allergy And Infectious Diseases IRG: VR
Abstract: Respiratory syncytial virus (RSV), is the single most important infectious cause of severe respiratory disease in infants. Infection begins when the virion attachment protein binds to a cellular receptor and the virion membrane fuses with the target cell membrane. RSV has three surface glycoproteins in a position to perform the attachment and fusion functions G (glycoprotein), F (fusion), and the small hydrophobic (SH) protein. Laboratory strains of RSV attach to glycosaminoglycans (GAGs) to initiate infection, but this characteristic might be selected by passage in cultured cells. The first Specific Aim of this study will compare the viral glycoprotein gene sequences derived directly from patient samples to those from cultured virus from the same patient. The second Specific Aim will examine the ability of the RSV F protein to bind to target cells, an idea derived from the surprising fact that both the G and SH genes can be deleted from RSV without loss of infectivity. This characteristic, unique among paramyxoviruses, strongly suggests that the RSV F protein is able to attach to cells, and together with its well-established role in membrane fusion, to initiate infection. F protein attachment to target cells will be explored using radioactively labeled recombinant RSV virions that contain the F protein as their only viral glycoprotein. These viruses also express the green fluorescent protein (GFP), allowing easy assay of infectivity and determination of the efficiency of infection once the virus is bound. The third Specific Aim will identify the region(s) of the F protein involved in cell attachment and fusion, through blocking studies with Fspecific monoclonal antibodies and overlapping peptide libraries, and through targeted mutations in the F protein. These mutations will be incorporated into recombinant virions expressing GFP, and their ability to support infection tested. The overall goal of this project is to understand the entry mechanism of RSV for eventual use in designing antiviral agents.
Keywords: respiratory infection, respiratory syncytial virus, virus infection mechanism, virus protein, binding site, blocking antibody, gene mutation, nucleic acid sequence, protein sequence, infant human (0-1 year), patient oriented research, site directed mutagenesis, tissue /cell culture
Project start date: 2001-06-01
Project end date: 2007-04-30
5R01AI047213-06 (2005): $291370
5R01AI047213-04 (2004): $1
5R01AI047213-03 (2003): $290000
5R01AI047213-02 (2002): $290000
Sponsored Links Excellgen http://Excellgen.com
1R01AI047213-01A2 (2001): $298904
7R01AI047213-05 (2004): $291999
NDV M PROTEIN--VIRION ASSEMBLY AND NUCLEAR LOCATION
Mark E Peeples, Professor
Rush University Medical Center
1653 W Congress Pkwy
chicago, Il 60612
Grant 5R01AI029606-05 from National Institute Of Allergy And Infectious Diseases IRG: EVR
Abstract: Paramyxoviruses are thought to multiply exclusively in the cytoplasm of a host cell. Progeny virus particles are formed by budding from the plasma membrane, a process orchestrated by matrix (M) protein. The M protein probably interacts with the plasma membrane, the cytoplasmic domain of the viral glycoproteins and the viral nucleocapsid. However, the specific interactions and the mechanisms by which these components are gathered together to initiate budding are not understood. In addition, a recent unexpected finding may point to a new role for the M protein in its relationship with its host cell the M protein is the only viral protein produced by Newcastle disease virus (NDV) which accumulates in the nucleus, specifically the nucleolus, of an infected cell. Interestingly, NDV is also the most efficient paramyxovirus at inhibiting host protein synthesis. In this study, both the assembly and nuclear targeting of the M protein of NDV will be examined. The interaction between the M protein and the viral fusion (F) glycoprotein will be dissected 1) genetically, by pinpointing the M protein mutation in mutants defective in this interaction; and 2) physically, by defining the portion of the F cytoplasmic domain peptide which interacts with the M protein and vice versa. The nuclear and nucleolar targeting of the M protein will be dissected 1) genetically, by transient expression of deletion and site-specific mutants of the M protein gene and analysis of the effects on M protein targeting; and 2) functionally, by quantifying virus production and the inhibition of host protein synthesis in enucleated cells and in a stably transfected cell line expressing the M protein. With a better understanding of the virus assembly process and the viral inhibition of host protein synthesis, therapies might to targeted to control paramyxovirus infections. Such therapies are particularly important for diseases such as bronchiolitis or pneumonia in infants, caused by another paramyxovirus, respiratory syncytial virus. No safe, effective vaccine has been developed for this infection, despite intensive efforts. Even if such a vaccine is developed, it may not be efficacious during the period of greatest risk, the first six months of life. A better understanding of paramyxovirus pathogenesis and assembly might provide clues for alternative treatments to control virus infection and dissemination
Project start date: 1990-04-01
Project end date: 1996-03-31
5R01AI029606-05 (1994): $155077
Respiratory Syncytial Virus Based Vectors For CFTR
Mark E Peeples, Professor
University Of North Carolina Chapel Hill
office Of Sponsored Research
chapel Hill, Nc 27599
Grant 5P01HL051818-150003 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: Numerous viral vectors have been developed with the ultimate goal of efficiently delivering replacement genes to human organs, without causing damage to the targeted cells. However, the luminal cells of the human airway epithelium are generally refractive to most vectors. Respiratory viruses like respiratory syncytial virus (RSV) are unique in that they specifically target these cells. Typical infection causes cell death after several days however it is now possible to alter RSV in such a way that it may no longer be cytotoxic. As a result, RSV could be a useful vector for delivering a transgene, such as the CFTR channel, to the respiratory epithelial cells of cystic fibrosis patients. We now know that RSV specifically infects the ciliated cells in primary cultures of human well-differentiated airway epithelial (HAE) cultures developed by the PPG cell culture core, and is cytotoxic on these cells over the course of a week. In this proposal, a number of reagents will be further developed for airway specific gene delivery. Specifically, attenuated vaccine candidate strains of RSV will be tested for lack of cytotoxicity in HAE cells. In addition to attenuated RSV strains, recent work with RSV ´replicons´ that lack the viral glycoprotein genes and therefore are unable to spread are now available for further vector development. To generate infectious vector from these replicons, the viral glycoproteins will be provided from integrated gene copies in an inducible manner similar to retroviral packaging systems. Non-replicating RSV also provide the opportunity to deliver controlled (dose dependent) amounts of the CFTR transgene which may be enough to correct a genetic defect such as cystic fibrosis. RSV vectors capable of producing the highest amounts of vector in producer cells and the lowest cytotoxicity in target HAE cells will be engineered to carry CFTR transgene. Unique to this PPG is the ability to test resulting RSV-CFTR vectors in HAE cultures derived from cystic fibrosis patients for repair of bioelectric defects, restoration of airway surface liquid depth, and the restoration of mucociliary clearance
Keywords: chloride channel, cystic fibrosis, gene delivery system, respiratory syncytial virus, transfection /expression vector, virus genetics attenuated microorganism, biotechnology, cytotoxicity, gene expression, gene therapy, genetic transduction, glycoprotein, microorganism immunology, respiratory epithelium, vaccine, virion, virus infection mechanism, virus protein, virus replication human tissue, microarray technology, tissue /cell culture
Generation Of A Single-Cycle Virus To Study The Pathogenesis Of Nipah Virus
Mark E Peeples, Professor
Research Inst Nationwide Children s Hosp Nationwide Children s Hospital Columbus, Oh 432052664
Grant 5R21AI069014-02 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: In 1999, an outbreak of respiratory disease in Maylasian pigs spread to humans causing febrile encephalitis with a high mortality rate. The cause of this outbreak was identified as Nipah virus (NiV), a relative of Hendra virus that had recently emerged in horses and humans in Australia. The reservoir of both viruses appears to be fruit bats, though the route of transmission to humans and animals is not clear. Since 1999, there have been three outbreaks of NiV in Bangladesh. NiV infection has a high mortality rate in humans, between 40% and 75%, and can cause severe disease in a number of animals of agricultural importance. Therefore, NiV is a candidate for use in bioterror. Our understanding of NiV infection has been hampered by its lethal nature and the need to use BSL-4 conditions to study it. The goal of this application is to make NiV studies easier to perform. Based on knowledge gained from studying another paramyxovirus, respiratory syncytial virus (RSV), a plasmid will be constructed containing a copy of the NiV genome in which the two glycoprotein genes will be replaced with genes for the green fluorescent protein and for an antibiotic resistance gene. The resulting NiV "replicon" should be able to replicate autonomously, as does the existing RSV replicon. Glycoproteins will be provided in trans to generate "single-cycle virus" (SCV), as has been successful for the RSV replicon. Because the Ni-SCV will be infectious for one round only, studies under lower level BSL conditions should be possible. Once the Ni-SCV has been generated, Ni-SCV infection will be compared to infection with NiV. This novel Ni-SCV system will be used to study the pathogenesis of NiV, including the ability of NiV to interfere with the innate immune response. Initial studies will focus on the P/C/V/W genes to dissect the role of each of these proteins in the induction of interferon, in the context of NiV infection. In the future, work with the Ni-SCV could be expanded to explore the induction or suppression of chemokine and cytokine expression, and other pathogenic mechanisms, as well as to provide a means to screen candidate antiviral agents.
Keywords: Nipah virus, infection, replicon, virus, Australia, Bangladesh, Chiroptera, Paramyxovirus, antibiotic, antiviral agent, base, cell, cell line, chemokine, conditioning, copying, cytokine, cytoplasm, drug resistance, encephalitis, environment, fruit, gene, genome, glycoprotein, green fluorescent protein, horse, human, hyperthermia, immune response, interferon, lead, molecular weight, plasmid, prevention, protein, protein C, respiratory syncytial virus, role, suppression, swine, virion, virulence
Project start date: 2006-04-01
Project end date: 2009-03-31
5R21AI069014-02 (2007): $206823
1R21AI069014-01 (2006): $177500
Sponsored Links Excellgen http://Excellgen.com
IDENTIFICATION OF A CELL RECEPTOR FOR HEPATITIS B VIRUS
Mark E Peeples, Professor
Rush University Medical Center 1653 W Congress Pkwy Chicago, Il 60612
Grant 5R01AI025586-08 from National Institute Of Allergy And Infectious Diseases IRG: VR
Abstract: Hepatitis B virus (HBV) infection is a major, worldwide health problem. It is estimated that 300,000 individuals in the U.S., alone, are infected each year. Approximately 10% of infected individuals are unable to clear the virus infection, often resulting in residual chronic infection and its devastating consequences, cirrhosis and primary liver cancer. Worldwide, over 300 million people are chronically infected with HBV. Interferon therapy has resulted in the clearance of infection in 10-30% of chronic HBV patients, but the majority remain refractory to treatment. Although effective vaccines are available, they have only recently begun to be distributed widely in the U.S., mostly to children. HBV, like other viruses, is most vulnerable to intervention before it enters its major target cell, the hepatocyte. This project is aimed at identifying and understanding these early interactions of HBV, especially its attachment to hepatocytes. No cell lines are capable of supporting HBV replication due to an early block, probably the absence of the HBV receptor. If the HBV receptor could be identified, agents that block virus attachment to this receptor might be designed for therapeutic use. Furthermore, identification of the HBV receptor might lead to cell culture and transgenic mouse systems in which to study the early stages of HBV infection to develop new antiviral drugs. A number of HBV receptor "candidates" have been described, but none has been demonstrated to be physiologically relevant. In this proposal, the early events in HBV infection will be studied in a primary human hepatocyte system. These are the only cultured cells that can be infected with HBV. Preliminary experiments have indicated that a prolonged inoculation time is necessary for maximal infection. The first two specific aims of this proposal are to determine the cause of this slow rate of attachment, to test the candidate HBV receptors proposed by this laboratory and by others, and to use a monoclonal antibody blocking approach to identify the HBV receptor. The third specific aim is to identify the HBV receptor by genetic means. A cells expressing a cDNA library prepared from human liver tissue will be inoculate5U01AG012505-04
Keywords: 1997
Project start date: 1988-07-01
Project end date: 1999-08-31
5R01AI025586-08 (1997): $248011
5R01AI025586-05 (1992): $97578
Mark E Peeples
Research Inst Nationwide Children´s Hosp
Project start date: 2011-12-01
Project end date: 2016-11-30
IDENTIFICATION OF A CELL RECEPTOR FOR HEPATITIS B VIRUS
Mark E Peeples, Professor
Rush University Medical Center 1653 W Congress Pkwy Chicago, Il 60612
Grant 5R01AI025586-07 from National Institute Of Allergy And Infectious Diseases IRG: VR
Project start date: 1988-07-01
Project end date: 1998-08-31
5R01AI025586-07 (1996): $194551