MOLECULAR GENETICS OF THE RECEPTOR FOR MVH

Kathryn V Holmes, Professor
Microbiologyuniversity Of Colorado Denver
grants And Contracts, Mail Stop F428
aurora, Co 800450508

Grant 5R01AI025231-20 from National Institute Of Allergy And Infectious Diseases, IRG: EVR

Keywords: glycoprotein, receptor, virus Coronaviridae, RNA, X ray crystallography, acid, angiogenesis, base, cat, cell, cell line, dendritic cell, emotion, family, gene, gene mutation, genetics, genome, human, immune response, infection, lead, learning, macrophage, membrane fusion, metastasis, microorganism culture, model, mutant, myelinopathy, neoplasm /cancer blood supply, play, protein, protein S, protein structure, protein structure function, receptor binding, receptor expression, role, swine, temperature, virion, virus disease, virus infection mechanism, virus receptor

Project start date: 1988-02-01

Project end date: 2008-12-31

5R01AI025231-20 (2007): $525523

Sponsored Links Lab Supply Mall http://www.labsupplymall.com

MOLECULAR GENETICS OF THE RECEPTOR FOR MHV

Kathryn V Holmes, Professor
University Of Colorado Denver Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 5R01AI025231-15 from National Institute Of Allergy And Infectious Diseases, IRG: VR

Abstract: Emerging viral diseases sometimes arise when viruses mutate to acquire the ability to infect cells of a new species. Coronaviruses generally cause disease in only one animal species. The viruses are readily transmitted to other members of the same species, but they are transmitte poorly or not at all to other species. This species specificity of coronavirus infection and disease is determined in part by species-specific differences among the cell membrane glycoproteins that coronaviruses use as receptors to enter susceptible host cells. Receptors for mouse, human, pig, dog and cat coronaviruses have been identified and their cDNAs cloned. When the recombinant receptor proteins are expressed in virus-resistant cell lines, they become susceptible to infection with the appropriate coronavirus. Rarely, host range mutants of viruses occur that have gained the ability to infect cells from another animal species. Because not only the major receptor but a large number of alternative receptors for murine coronaviruse MHV have been identified and host range mutants of the virus have been selected, this is an excellent model system for the study of how mutations that affect virus species specificity may be selected. The proposed experiments will examine how murine coronaviruses acquire the ability to infect cells from many other species, and determine whether such host range mutants can infect the new species in vivo and cause disease. The hypothesis that host range mutants of viruses are selected in cells or tissues that express low levels of the natural virus receptor in addition to alternative receptors of lower efficiency will be tested. Using a combination of genetic, biochemical, and molecular techniques with reagents that have been developed to study MHV receptors, mutations will be identified in the virus attachment glycoprotein that affect receptor specificity and identify what receptors these mutant viruses use to infect cells of a different species. The molecular mechanism by which expression of viru receptors is down-regulated in persistently infected cells will also be studied.

Keywords: molecular genetics, murine hepatitis virus, virus infection mechanism, virus receptor, carcinoembryonal antigen, emerging infectious disease, gene mutation, mutant, receptor binding, receptor expression, virus genetics, virus protein, flow cytometry, hamster, laboratory mouse, laboratory rat, molecular cloning, nucleic acid sequence, site directed mutagenesis, tissue /cell culture

Project start date: 1988-02-01

Project end date: 2003-06-30

5R01AI025231-15 (2002): $350040

5R01AI025231-14 (2001): $339823

5R01AI025231-13 (2000): $331643

5R01AI025231-12 (1999): $325858

5R01AI025231-10 (1997): $225504

5R01AI025231-07 (1994): $194120

MOLECULAR GENETICS OF THE RECEPTOR FOR MVH

Kathryn V Holmes, Professor
University Of Colorado Denver Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 5R01AI025231-19 from National Institute Of Allergy And Infectious Diseases, IRG: EVR

Project start date: 1988-02-01

Project end date: 2007-12-31

5R01AI025231-19 (2006): $527884

5R01AI025231-18 (2005): $535833

5R01AI025231-17 (2004): $527465

MOLECULAR GENETICS OF THE RECEPTOR FOR MHV

Kathryn V Holmes, Professor
University Of Colorado Denver Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 5R01AI025231-09 from National Institute Of Allergy And Infectious Diseases, IRG: VR

Project start date: 1988-02-01

Project end date: 1998-06-30

5R01AI025231-09 (1996): $216926

5R01AI025231-05 (1992): $171062

CORONAVIRUS/RECEPTOR INTERACTIONS

Kathryn V Holmes, Professor
Henry M. Jackson Fdn For The Adv Mil/med Advancement Of Military Medicine, Inc. Rockville, Md 20852

Grant 5U01AI026075-08 from National Institute Of Allergy And Infectious Diseases, IRG: SRC

Abstract: We have isolated and characterized a 100-120 kDa glycoprotein which serves as the receptor for mouse hepatitis virus, a murine coronavirus. Immunoaffinity purification of the receptor, cloning and expression of the receptor gene revealed that the receptor is a member of the immunoglobulin superfamily of glycoproteins, serologically related to proteins in the carcinoembryonic antigen family. A monoclonal antibody which binds to the receptor on mouse cells blocks MHV infection in vitro and partially protects mice from infection in vivo. We will characterize the domains of the receptor glycoprotein and the viral attachment glycoprotein which interact using recombinant DNA technology and protein chemistry as well as ultrastructural and X-ray crystallography. The information obtained will be used to develop drugs which interfere with the virus-receptor interaction, and these will be tested for protection from MHV infection in vitro and in a mouse model system. Analogous studies will be done to characterize the receptor for human coronavirus HCV229E which causes colds. Development of anti-receptor drugs may provide a useful approach to the prevention and treatment of coronavirus infections of man and animals.

Keywords: Coronaviridae, antiviral agent, host organism interaction, murine hepatitis virus, protein structure function, virus infection mechanism, virus receptor, antiidiotype antibody, antireceptor antibody, carcinoembryonal antigen, common cold, messenger RNA, molecular site, protein engineering, receptor binding, receptor expression, synthetic peptide, virion, virus genetics, virus protein, X ray crystallography, computer simulation, electron microscopy, human tissue, in situ hybridization, laboratory mouse, molecular cloning, protein sequence, recombinant DNA, site directed mutagenesis, western blotting

Project start date: 1988-04-01

Project end date: 1995-06-30

5U01AI026075-08 (1994): $263692

CORONAVIRUS-RECEPTOR INTERACTIONS

Kathryn V Holmes, Professor
Henry M. Jackson Fdn For The Adv Mil/med Advancement Of Military Medicine, Inc. Rockville, Md 20852

Grant 5U01AI026075-07 from National Institute Of Allergy And Infectious Diseases, IRG: SRC

Abstract: We have isolated and characterized a 100-120 kDa glycoprotein which serves as the receptor for mouse hepatitis virus, a murine coronavirus. Immunoaffinity purification of the receptor, cloning and expression of the receptor gene revealed that the receptor is a member of the immunoglobulin superfamily of glycoproteins, serologically related to proteins in the carcinoembryonic antigen family. A monoclonal antibody which binds to the receptor on mouse cells blocks MHV infection in vitro and partially protects mice from infection in vivo. We will characterize the domains of the receptor glycoprotein and the viral attachment glycoprotein which interact using recombinant DNA technology and protein chemistry as well as ultrastructural and X-ray crystallography. The information obtained will be used to develop drugs which interfere with the virus-receptor interaction, and these will be tested for protection from MHV infection in vitro and in a mouse model system. Analogous studies will be done to characterize the receptor for human coronavirus HCV229E which causes colds. Development of anti-receptor drugs may provide a useful approach to the prevention and treatment of coronavirus infections of man and animals.

Keywords: Coronaviridae, antiviral agent, host organism interaction, mouse hepatitis virus, protein structure function, virus infection mechanism, virus receptor, antiidiotype antibody, antireceptor antibody, carcinoembryonal antigen, common cold, messenger RNA, molecular site, protein engineering, receptor binding, receptor expression, synthetic peptide, virion, virus genetics, virus protein, X ray crystallography, computer simulation, electron microscopy, human tissue, in situ hybridization, laboratory mouse, molecular cloning, protein sequence, recombinant DNA, site directed mutagenesis, western blotting

Project start date: 1988-04-01

Project end date: 1995-03-31

5U01AI026075-07 (1993): $214275

5U01AI026075-06 (1992): $204040

Molecular Pathogenesis Of Infectious Diseases

Kathryn V Holmes, Professor
Microbiologyuniversity Of Colorado Denver

Grant 5T32AI052066-07 from National Institute Of Allergy And Infectious Diseases, IRG: MID

Abstract: This application is for the first competing renewal of our pre-doctoral training program on The Molecular Pathogenesis of Infectious Diseases at the University of Colorado School of Medicine. This emerging area of research encompasses molecular biological and biochemical studies on microbial virulence factors, host resistance mechanisms, regulation of gene expression during infection, structure-function analysis of microbial and host cell molecules that play key roles in infections, analysis of interactions of bacteria and viruses with differentiated host cells in vitro, and studies on bacterial and viral infections in animal models using either wild type or genetically modified strains of microbes and host animals. The highly interactive members of our training faculty have proven productivity for innovative research in these areas, successful research funding from NIH and other granting agencies, and extensive experience in training pre-doctoral students in the multidisciplinary approaches needed for cutting edge research on the pathogenesis of infectious diseases. In addition to performing their mentored research projects, our trainees will participate in a comprehensive program of didactic courses, seminars, journal clubs, and research-in-progress meetings which will train them to identify important new research questions, design well-controlled experiments using a wide variety of molecular, cellular and in vivo techniques, critically evaluate and present their results, and understand and apply principles of ethics to their research. Students are selected for appointment to this training program only after they have completed all of their required courses, passed their preliminary and comprehensive examinations and begun their dissertation research. The breadth of our integrated, interdepartmental training program will prepare students for innovative careers in academia or biotechnology. This dynamic new training program has been extremely successful. It has fostered new research collaborations and grants, improved the curriculum, and fostered interdisciplinary interactions among the pre-doctoral trainees. In the 4 years since our training program on the Molecular Pathogenesis of Infectious Diseases began, our training faculty increased from 19 to 21, and the steady state number of predoctoral trainees in faculty labs grew from 21 to 31. Currently this training grant provides only one year of support for 3 trainees each year. We request 4 positions in years 06-07 to provide a 2-year commitment for 1 of the 3 trainees appointed each year and 5 positions in years 08-10 to fund one additional trainee whose research will focus on molecular mechanisms of infection related to biodefense or emerging infectious disease agents

Project start date: 2002-08-01

Project end date: 2012-08-31

2T32AI052066-06 (2007): $117510

SARS Coronavirus: Inhibition Of Entry

Kathryn V Holmes, Professor
Microbiologyuniversity Of Colorado Denver

Grant 5P01AI059576-05 from National Institute Of Allergy And Infectious Diseases, IRG: ZAI1

Abstract: The Sudden Acute Respiratory Syndrome (SARS) epidemic has spread to 30 countries and killed more than 800 people in the 7-month period from November 2002, to June 2003. The epidemic is a major public health emergency that has closed hospitals and schools and caused quarantines, travel advisories, and severe economic losses. No drugs or vaccines with proven efficacy are available to treat or prevent SARS. Little is known about the new, emerging coronavirus (SARS-CoV) that apparently jumped from wild animals into humans, and is now transmitted from person to person. The 6 principal investigators on this Program Project Grant have planned a coordinated analysis of the spike (S) glycoprotein on the envelope of the SARS-coronavirus to learn what species the virus can potentially infect, study how the S protein works as a molecular key to allow the virus to enter host cells, and develop new anti-viral drugs and vaccines to combat SARS-CoV. The first goals are to identify the cellular receptor for the virus, learn how it initiates virus infection, and develop antibodies and candidate anti-viral drugs that block virus infection of human cells. Dr. Holmes, who has studied receptors for other human and animal coronaviruses intensively, is the principal investigator of this Program Project Grant and Project 1. Dr. DeMartini (Project 2) has studied how viruses cause diseases of the lung in animals. He will identify animals susceptible to SARS-CoV infection, and study SARS lung pathology. Dr. Hodges (Project 3) and Dr. Wang (Project 4) will study the viral spike protein using biophysical, biochemical and functional assays with Dr. Holmes (Project 1), and by X-ray crystallography to learn what parts of the protein bind to the receptor, where antibodies target the spike, and how the spike causes membrane fusion. Dr. Wentworth (Project 5) and Dr. Mason (Project 6) will develop transgenic mice that express the human receptor for SARS-CoV and use them to learn how SARS-CoV causes disease. Mr. Shallow, (Administrative Core A) will coordinate the administration of the Projects that will be done in 5 institutions, and Dr. Holmes will coordinate the scientific interactions between the Projects. Dr. Pearson (Virus Core B) will work with the infectious SARS-CoV in the Biosafety Level 3 lab in collaboration with investigators from all of the other projects. Dr Gill (Protein Core C) will direct the synthesis and purification of viral spike proteins, receptor proteins and antibodies that are essential for each of these projects. This research will help to explain the origin of SARS-CoV, explain the pathology of SARS, develop candidate vaccines and drugs against SARS, and test them in new animal models for SARS

Keywords: SARS virus, antiviral agent, communicable disease control, host organism interaction, protein protein interaction, severe acute respiratory syndrome, virus infection mechanism, virus protein, virus receptor

Project start date: 2004-06-15

Project end date: 2009-05-31

5P01AI059576-04 (2007): $1761568

5P01AI059576-03 (2006): $1764074

5P01AI059576-02 (2005): $1715730

1P01AI059576-01 (2004): $1838728

Molecular Pathogenesis Of Infectious Diseases

Kathryn V Holmes, Professor
University Of Colorado Denver/hsc Aurora Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 5T32AI052066-05 from National Institute Of Allergy And Infectious Diseases, IRG: AITC

Abstract: The Molecular Pathogenesis of Infectious Diseases is an area of great interest to an interdepartmental group of 19 faculty members at the University of Colorado Health Sciences Center. This is a newly emerging area for research that encompasses molecular biological and biochemical studies on microbial virulence factors, host resistance mechanisms, regulation of gene expression during infection, structure-function analysis of microbial and host cell molecules that play key roles in infections, analysis of interactions of bacteria and viruses with differentiated host cells in vitro, and studies on infections with genetically modified bacteria and viruses in genetically modified animal models. Our faculty group has a proven record of innovative research in this area. The faculty are training 21 predoctoral students in the multidisciplinary approaches needed to understand and eventually control infectious diseases. Predoctoral students to be supported by the Molecular Pathogenesis of Infectious Diseases Graduate Student Training Grant will learn to identify and solve important research problems in medical microbiology, molecular structure, cellular interactions with bacteria and viruses, and in vivo analysis of bacterial and viral diseases. Highly qualified predoctoral students at the University of Colorado School of Medicine who are doing research on the molecular pathogenesis of bacterial or viral diseases with NIH-funded mentors on the training grant faculty will be selected for appointment to the training grant after they have completed all of their required courses, passed their preliminary and comprehensive examinations, and begun their dissertation research. We request 4 predoctoral student positions for the first year of the grant, 5 for the second year, and 6 for each successive year. Each selected student will be supported by the training grant for a minimum of 2 years during the period of dissertation research. A comprehensive program of seminars, journal clubs, and research-in-progress meetings will train the students to identify important research questions, design well-controlled experiments using a wide variety of molecular, cellular and in vivo techniques, and critically evaluate and present the results. Training in scientific communication skills and ethics in research are important components of the program. The breadth of this integrated, interdepartmental training program will prepare the students well for careers in innovative research and teaching in academia or biotechnology in the important area of the pathogenesis of infectious diseases.

Project start date: 2002-08-01

Project end date: 2007-07-31

5T32AI052066-05 (2006): $86183

5T32AI052066-04 (2005): $86057

5T32AI052066-03 (2004): $85934

5T32AI052066-02 (2003): $83209

1T32AI052066-01 (2002): $77221

GORDON CONFERENCE ON ANIMAL CELLS AND VIRUSES

Kathryn V Holmes, Professor
Gordon Research Conferences
west Kingston, Ri 02892

Grant 1R13AI034315-01 from National Institute Of Allergy And Infectious Diseases, IRG: MID

Abstract: Support is requested for the meeting of the Gordon Research on Animal Cells and Viruses to be held at Tilton Academy in New Hampshire June 21-25, 1993. This meeting will bring together experts in diverse fields of virology and cell biology in an environment which will stimulate discussion and interaction. In addition to partial support for speeches, support for 10 young investigators is requested. Topics for the meeting will include 1. Viral Gene Expression 2. Nucleic Acid Translation and Transport 3. Nucleic Acid Replication 4. Virus Assembly 5. Viral Penetration and Disassembly 6. Host-dependent Replication of Viruses 7. Viruses, Oncogenes and Cell Cycle Regulation 8. Interactions of Viruses with the Immune System 9. Viral Pathogenesis From research stimulated by such a meeting we anticipate important new contributions to viral pathogenesis and therapy as well as basic molecular biology of eukaryotic cells

Keywords: meeting /conference /symposium, virus cell cycle, gene expression, host organism interaction, travel, virus cytopathogenic effect, virus multiplication

Project start date: 1993-06-01

Project end date: 1994-05-31

1R13AI034315-01 (1993): $3000

MOLECULAR GENETICS OF THE RECEPTOR FOR MVH

Kathryn V Holmes, Professor
University Of Colorado Denver/hsc Aurora Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 2R01AI025231-16 from National Institute Of Allergy And Infectious Diseases, IRG: EVR

Abstract: Our long term goal is to understand the molecular mechanisms by which viruses occasionally jump from their normal host species to a new species, a process that may lead to emerging viral diseases in the new host species. We will focus on the interactions of the spike glycoprotein of murine coronavirus MHV with host cell receptors in the CEA family of glycoproteins. Several labs have shown that persistent infection of murine cell lines with MHV leads to markedly reduced expression of the CEACAM1a receptor glycoprotein. In the persistently infected cultures, viruses with mutations in their spike glycoproteins and some other genes rapidly replace the wild type virus. The mutations in the spike genes are associated with acquisition of the ability of the virus to replicate in cell lines from cats, pigs, rats, monkeys and humans. We will identify amino acid residues in both the viral spike glycoprotein and the CEACAM1a receptor that determine the specificity of binding virus. We will study the effects of mutations in S and in the receptor upon the specificity of virus-receptor interactions. We will continue our work on determining the crystal structures of CEACAM1 proteins to learn how the functional CEACAM1a receptor differs from CEACAM1b proteins from MHV-resistant mice and human CEACAM1. We will engineer spike and receptor proteins that will form co-crystals in order to determine the crystal structure of the complex. We will analyze the conformational changes in the viral spike protein induced by soluble receptor or by pH 8 at 37 degrees C that are associated with membrane fusion and virus entry. To develop strains of inbred mice that are resistant to MHV infection, we will manipulate the Ceacam1gene in mice to reduce or eliminate expression of CEACAM1 proteins. We will also substitute chimeric Ceacam1a/b genes for the normal Ceacam1 gene in mice. The animals will be tested for MHV-susceptibility and immune responses. These studies will provide important information about the mechanism of changing receptor specificity in a model virus that causes disease in its natural host.

Keywords: molecular genetics, murine hepatitis virus, virus infection mechanism, virus receptor, carcinoembryonal antigen, conformation, emerging infectious disease, gene mutation, host organism interaction, mutant, receptor binding, receptor expression, virus genetics, virus protein, X ray crystallography, flow cytometry, laboratory mouse, molecular cloning, nucleic acid sequence, site directed mutagenesis, tissue /cell culture, transgenic animal

Project start date: 1988-02-01

Project end date: 2007-12-31

2R01AI025231-16 (2003): $331772

CORONAVIRUS RECEPTOR INTERACTIONS

Kathryn V Holmes, Professor
University Of Colorado Denver Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 5U01AI026075-13 from National Institute Of Allergy And Infectious Diseases, IRG: SRC

Abstract: Human coronaviruses cause 15 to 30% of common colds which are associated with economic losses worldwide. We recently discovered that the receptor for human coronavirus HCV-229E is a membrane glycoprotein called human aminopeptidase N (hAPN) or CD13. Several monoclonal antibodies directed against hAPN prevent virus from binding to hAPN and prevent infection. Our goal is to develop peptides or peptidomimetics that inhibit virus- receptor interactions and are sufficiently non-toxic that they can be used in the respiratory tract to prevent HCV-229E infection or reduce the shedding of infectious virus. To do this, we will use mutational analysis to identify the residues required for binding of the receptor with the virus glycoprotein and/or anti-hAPN MAbs. Peptides or other drugs targeted to these binding sites will be tested for their ability to block virus-receptor interactions and prevent infection of cultured cells. Because no animals are susceptible to HCV-229E infection, we will make transgenic mice expressing hAPN in the respiratory epithelial cells, and challenge them with HCV-229E virus intranasally. If they are susceptible to infection as predicted, then potential anti-human coronavirus drugs that are effective and non-toxic in vitro can be tested in vivo in these animals. We will also investigate the nature of the receptor for human coronavirus HCV-OC43 in order to develop receptor-targeted anti-viral drugs that could be used in combination with drugs that block interactions of HCV-229E and human rhinoviruses with their specific receptors. A combination drug like this would be suitable for more than 90% of common colds in man. The mouse coronavirus MHV, which is closely related to HCV- OC43, is an important model for coronavirus infection in its natural host. Molecular interactions of MHV spike glycoprotein with isoforms of the biliary glycoprotein that serve as the MHV receptor (MHVR) will be studied with biosensor, genetic and structural techniques. The resulting three dimensional structural data will be used to generate candidate drugs to prevent or treat MHV infection of the respiratory or enteric tracts. These will be tested for efficacy and toxicity in vitro and then in vivo. These detailed studies of coronavirus-receptor interactions are also expected to elucidate the effects of receptors upon the virus attachment glycoproteins and provide new insight into the early steps of coronavirus attachment and penetration into cells.

Keywords: Coronaviridae, antiviral agent, host organism interaction, murine hepatitis virus, protein structure /function, virus infection mechanism, virus receptor, common cold, disease model, drug design /synthesis /production, drug screening /evaluation, messenger RNA, nonhuman therapy evaluation, receptor binding, receptor expression, virion, virus genetics, virus protein, X ray crystallography, electron microscopy, enzyme linked immunosorbent assay, histopathology, laboratory mouse, recombinant DNA, transgenic animal

Project start date: 1988-04-01

Project end date: 1999-08-31

5U01AI026075-13 (1998): $308705

5U01AI026075-12 (1997): $296961

CORONAVIRUS-RECEPTOR INTERACTIONS

Kathryn V Holmes, Professor
University Of Colorado Denver Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 5R01AI026075-18 from National Institute Of Allergy And Infectious Diseases, IRG: VR

Abstract: Human coronaviruses cause 15 to 30 percent of common colds, yet there has been little investigation of these human pathogens. Our long term goal is to develop anti-viral drugs to prevent and/or treat human coronavirus infections of the upper respiratory tract. We anticipate that these drugs would be used in combination with drugs to prevent or treat colds due to rhinoviruses which are now being developed in other laboratories. By covering the viruses that cause most common colds, such combination drugs would make it unnecessary to identify the type of common cold virus causing early symptoms before initiating therapy. Our strategy for developing anti-coronavirus drugs is to use information about viral spike protein and receptor structure and function to identify small molecules that may block interactions of the viral spike glycoprotein with its cellular receptor glycoprotein. Our lab has identified receptors for human, murine and feline coronaviruses. We have expressed and purified soluble recombinant receptor glycoproteins, and are testing them for receptor activities. We will use mutagenesis to identify the amino acids and domains of the viral spike proteins and receptor glycoproteins that interact, and test the mutant proteins for binding activity and the ability to induce or undergo conformational changes that lead to membrane fusion and virus entry. We will analyze the structures of the spike proteins and receptor glycoproteins using cryo-electron microscopy and X-ray crystallography. The resulting structural models will be used to develop small molecules that block virus binding and/or receptor induced conformational changes in the spike protein. These molecules will be tested in cell culture model systems for toxicity and for the ability to block virus infection. Non-toxic drugs with anti-viral activity in cultures will be tested in animal model systems for the ability to block coronavirus infections of the respiratory tract. Our experimental models are the interactions of two coronaviruses, mouse hepatitis virus (MHV) and human coronavirus 229E (HCoV-229E), with their receptors, MHVR and hAPN, respectively. The murine model is more advanced than the human model, and will provide information and strategies that will facilitate our studies on human coronavirus-receptor interactions. The MHV model will also provide fundamental information on how an immunoglobulin-related receptor initiates infection with an enveloped virus. The 229E model will elucidate how the enzyme APN acts as a virus receptor. Inhibition of HCoV-229E respiratory infection by candidate drugs will be studied in transgenic mice that express the HCoV-229E receptor, hAPN, in the respiratory epithelium.

Keywords: Coronaviridae, antiviral agent, common cold, drug design /synthesis /production, murine hepatitis virus, protein structure function, receptor binding, virus protein, virus receptor, disease /disorder model, drug screening /evaluation, model design /development, receptor expression, structural model, virus infection mechanism, X ray crystallography, cryoelectron microscopy, laboratory mouse, site directed mutagenesis, tissue /cell culture, transgenic animal

Project start date: 1988-04-01

Project end date: 2005-06-30

5R01AI026075-18 (2003): $348198

5R01AI026075-17 (2002): $339439

5R01AI026075-16 (2001): $330936

5R01AI026075-15 (2000): $322681

INTERNATIONAL SYMPOSIUM ON CORONAVIRUSES And ARTERIVIRUSES

Kathryn V Holmes, Professor
University Of Colorado Denver Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 1R13AI040964-01 from National Institute Of Allergy And Infectious Diseases, IRG: MID

Abstract: Funds are requested to provide travel expenses for 8 junior scientists to attend the VIIth International Symposium on Corona viruses and Arteriviruses to be held in Segovia, Spain from May 10-15, 1997. Corona viruses and arteriviruses are large enveloped, plus-strand RNA viruses that cause widespread and economically important diseases of man and domestic animals. In addition to their importance in human and veterinary medicine, these viruses have a unique pattern of RNA replication and transcription with a very high frequency of recombination. A central focus of the meeting will be on the processing and function of the proteins encoded by the large, unique 5  open reading frame of these viruses that is processed by viral and possibly cellular proteases to form the viral RNA polymerase. The meeting will also address the roles of receptors and viral proteins in determining viral species specificity, pathogenesis, relevant immune responses, coronavirus evolution, and control of coronavirus and arterivirus diseases.

Keywords: Coronaviridae, Togaviridae, meeting /conference /symposium, DNA directed RNA polymerase, virus protein, travel

Project start date: 1997-04-01

Project end date: 1998-03-31

1R13AI040964-01 (1997): $4000

MOLECULAR GENETICS OF THE RECEPTOR FOR MHV

Kathryn V Holmes, Professor
University Of Colorado Denver Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 2R01AI025231-11 from National Institute Of Allergy And Infectious Diseases, IRG: VR

Abstract: Emerging viral diseases sometimes arise when viruses mutate to acquire the ability to infect cells of a new species. Coronaviruses generally cause disease in only one animal species. The viruses are readily transmitted to other members of the same species, but they are transmitte poorly or not at all to other species. This species specificity of coronavirus infection and disease is determined in part by species-specific differences among the cell membrane glycoproteins that coronaviruses use as receptors to enter susceptible host cells. Receptors for mouse, human, pig, dog and cat coronaviruses have been identified and their cDNAs cloned. When the recombinant receptor proteins are expressed in virus-resistant cell lines, they become susceptible to infection with the appropriate coronavirus. Rarely, host range mutants of viruses occur that have gained the ability to infect cells from another animal species. Because not only the major receptor but a large number of alternative receptors for murine coronaviruse MHV have been identified and host range mutants of the virus have been selected, this is an excellent model system for the study of how mutations that affect virus species specificity may be selected. The proposed experiments will examine how murine coronaviruses acquire the ability to infect cells from many other species, and determine whether such host range mutants can infect the new species in vivo and cause disease. The hypothesis that host range mutants of viruses are selected in cells or tissues that express low levels of the natural virus receptor in addition to alternative receptors of lower efficiency will be tested. Using a combination of genetic, biochemical, and molecular techniques with reagents that have been developed to study MHV receptors, mutations will be identified in the virus attachment glycoprotein that affect receptor specificity and identify what receptors these mutant viruses use to infect cells of a different species. The molecular mechanism by which expression of viru receptors is down-regulated in persistently infected cells will also be studied.

Keywords: molecular genetics, murine hepatitis virus, virus infection mechanism, virus receptor, carcinoembryonal antigen, gene mutation, mutant, receptor binding, receptor expression, virus genetics, virus protein, flow cytometry, hamster, laboratory mouse, laboratory rat, molecular cloning, nucleic acid sequence, site directed mutagenesis, tissue /cell culture

Project start date: 1988-02-01

Project end date: 2003-06-30

2R01AI025231-11 (1998): $317910

HUMAN CORONAVIRUS 229E SPIKE AND RECEPTOR INTERACTIONS

Kathryn V Holmes, Professor
Microbiologyuniversity Of Colorado Denver
grants And Contracts, Mail Stop F428
aurora, Co 800450508

Grant 3R21AI044900-01A1S1 from National Institute Of Allergy And Infectious Diseases, IRG: VR

Abstract: Although cellular receptors for many human viruses have been identified in the past 10 years, we do not yet fully understand how binding of a virus to its receptor leads to penetration of the viral genome into the cell to initiate replication. We identified human aminopeptidase N (hAPN), a membrane metalloprotease, as the receptor for human coronavirus 229E. APN is the only protease shown to have virus receptor activity. The 229E viral envelope glycoprotein that binds to hAPN is the 200 kDa spike protein, S. Our goal is to analyze the molecular interactions between S and hAPN that lead to virus infection. The study of this is a novel virus-receptor system will provide new insight into how enveloped viruses bind to and fuse with cellular membranes. Human coronaviruses cause 15 to 30 percent of upper respiratory tract and sinus infections in humans of all ages, and lower respiratory tract infections and exacerbations of asthma in children. No vaccines or drugs are available to treat or prevent diseases caused by human coronaviruses. We will do structural and functional analyses of the 229E S glycoprotein and hAPN. We will introduce mutations into the S gene and the hAPN gene, express the mutant proteins, and explore the effects of the mutations on virus-receptor interactions. We will expressed anchorless, soluble S and hAPN glycoproteins in baculovirus vectors and purify the proteins to homogeneity. The structures of these glycoproteins, or peptides derived from them, will be analyzed by X-ray crystallography. We will determine whether binding of the purified receptor at neutral or acid pH, or acid pH alone can lead to conformational changes in the S protein on virions that may be associated with membrane fusion. We will select and/or engineer 229E viruses and VSV pseudotypes containing mutant S proteins and characterize the functional and antigenic changes that result from the mutations. The S gene will be sequenced by RT-PCR from human clinical specimens, and S proteins that differ significantly in amino acid sequence will be cloned, expressed in eukaryotic cells and their interactions with the hAPN receptor will compared with the wild type 229E S protein. In addition to providing a novel model system for studying virus-receptor interactions, our research on 229E S glycoprotein and hAPN may lead to development of new anti-viral drugs that block the initial stages of human coronavirus infection

Keywords: Coronaviridae, receptor binding, virus infection mechanism, virus protein, virus receptor acid base balance, gene mutation, glycoprotein structure, neutralizing antibody, recombinant virus, temperature Vesiculovirus, X ray crystallography, human tissue, laboratory mouse, molecular cloning, nucleic acid sequence, polymerase chain reaction

Project start date: 2000-03-01

Project end date: 2002-03-30

3R21AI044900-01A1S1 (2001): $20000

1R21AI044900-01A1 (2000): $229

FASEB CONFERENCE--MICROBIAL PATHOGENESIS

Kathryn V Holmes, Professor
Federation Of Amer Soc For Exper Biology
9650 Rockville Pike
bethesda, Md 208143998

Grant 1R13AI043592-01 from National Institute Of Allergy And Infectious Diseases, IRG: MID

Abstract: adapted from application ) The FASEB Summer Research Conference entitled

Keywords: Microbial Pathogenesis: Mechanisms of Infectious Diseases

Project start date: 1998-07-04

Project end date: 1999-07-03

1R13AI043592-01 (1998): $2000

MOLECULAR GENETICS OF THE RECEPTOR FOR MHV

Kathryn V Holmes, Professor
Henry M. Jackson Fdn For The Adv Mil/med Advancement Of Military Medicine, Inc. Rockville, Md 20852

Grant 2R01AI025231-06A1 from National Institute Of Allergy And Infectious Diseases, IRG: VR

Abstract: Adapted from  s ) Coronaviruses of mice and rats are a common cause of enzootic infections in colonies of laboratory rodents, frequently compromising research projects that depend upon these animals. Many different strains of mouse coronavirus (MHV) and rat coronavirus (RCV) infect specific organs or tissues in the animals, causing a variety of diseases such as respiratory or enteric infection, neurological disease, hepatitis, adenitis, or immune disorders. has identified a murine glycoprotein (MHVR) related to carcinoembryonic antigen (CEA) as a receptor for MHV-A59. CEA-related glycoproteins of mice and rats were cloned and expressed separately in cells of other species. Surprisingly, found that several different CEA-related glycoproteins can serve as alternative receptors for MHV-A59. In murine tissues, these receptors are expressed singly or together in a developmentally regulated way. will test the hypothesis that the tissue tropism and species specificity of various rodent coronavirus strains are determined in part by the relative effectiveness of the interactions between the virus attachment proteins and alternative receptors. Biosensor technology will be utilized to quantitatively compare binding of virus attachment proteins to alternative receptors, analyze changes in the viral protein and membrane that follow receptor binding, and determine the effects of virus mutations upon interactions with receptors. The synthesis and processing of receptor glycoproteins will be studied, and the genetics of receptor expression in tissues from mice of different strains and ages will be analyzed. in vitro models for receptor selection by different coronavirus strains will be developed, and the diseases causes by virus variants selected in vitro for altered receptor specificity will be studied. The rodent coronaviruses are a unique model for the role of receptor specificity in virus pathogenesis.

Keywords: molecular genetics, mouse hepatitis virus, virus infection mechanism, virus receptor, carcinoembryonal antigen, cell fusion, chimeric protein, glycoprotein biosynthesis, glycoprotein structure, membrane fusion, posttranslational modification, protein isoform, receptor binding, receptor expression, virulence, virus genetics, virus protein, biosensor, laboratory mouse, laboratory rat, molecular cloning, site directed mutagenesis, tissue /cell culture

Project start date: 1988-02-01

Project end date: 1998-06-30

2R01AI025231-06A1 (1993): $203798

SARS Receptor Characterization/Virus Binding Blockagage

Kathryn V Holmes, Professor
University Of Colorado Denver/hsc Aurora Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 5P01AI059576-040001 from National Institute Of Allergy And Infectious Diseases, IRG: ZAI1

Keywords: SARS virus, antiviral agent, drug discovery /isolation, glycoprotein, host organism interaction, inhibitor /antagonist, protein structure function, receptor binding, virus protein, virus receptor, antiviral antibody, communicable disease control, gene mutation, intermolecular interaction, monoclonal antibody, severe acute respiratory syndrome, virus genetics, virus infection mechanism, animal tissue, cell line, human tissue, hybridoma, immunologic substance development /preparation, laboratory mouse, molecular cloning, postmortem

CORONAVIRUS RECEPTOR INTERACTIONS

Kathryn V Holmes, Professor
University Of Colorado Denver Grants And Contracts, Mail Stop F428 Aurora, Co 800450508

Grant 5U01AI026075-11 from National Institute Of Allergy And Infectious Diseases, IRG: SRC

Project start date: 1988-04-01

Project end date: 1999-06-30

5U01AI026075-11 (1996): $285668

CORONAVIRUS-RECEPTOR INTERACTIONS

Kathryn V Holmes, Professor
Henry M. Jackson Fdn For The Adv Mil/med Advancement Of Military Medicine, Inc. Rockville, Md 20852

Grant 5U01AI026075-04 from National Institute Of Allergy And Infectious Diseases, IRG: SRC

Project start date: 1988-04-01

Project end date: 1991-06-30