MECHANISM OF HIV DNA INTEGRATION
Frederic D Bushman, Professor
Salk Institute For Biological Studies La Jolla, Ca 920371099
Grant 5R01AI034786-08 from National Institute Of Allergy And Infectious Diseases IRG: ARRC
Abstract: Adapted From Applicant s ) This application proposes to study the process of HIV integration by (1) purifying preintegrative complexes to near homogeneity and determine the protein composition and stoichiometry, (2) characterizing the structure of isolated preintegrative complexes in detail using electron microscopy and assays for protein-DNA interactions, and (3) to build upon the exciting finding that HMG I(Y) is a likely component of the preintegrative complex by examining its role in the integration process in vivo and in vitro.
Keywords: DNA binding protein, human immunodeficiency virus 1, virus DNA, virus integration, protein structure /function, electron microscopy, human tissue, protein purification
Project start date: 1993-07-01
Project end date: 2001-06-30
5R01AI034786-08 (2000): $345516
Sponsored Links Excellgen http://Excellgen.com
Mechanism Of HIV DNA Integration
Frederic D Bushman, Professor
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 5R01AI034786-14 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: In order to replicate, a retrovirus such as HIV must integrate a cDNA copy of its RNA genome into a chromosome of the host. Although the cDNA integration system is a promising target for antiretroviral therapy, no clinically useful inhibitors have yet been developed. Our proposed studies are designed to provide mechanistic information useful in developing such inhibitors. Integration in vivo is carried out by a sub-viral particle containing the viral cDNA complexed with proteins. Such "preintegration complexes" (PICs) can be isolated from infected cells and when presented with a target DNA in vitro can carry out integration. We have been engaged in a long term study of the composition, organization and activities of PICs. We propose to continue this work in the renewal and investigate the implications with studies of viral replication in cell culture. We propose three specific aims 1) we will further improve methods for the large-scale production of PICs and their biochemical analysis, particularly the detection and quantitation of complex components using LC-MS and other methods. 2) We will investigate the organization of PICs using chromatin immunoprecipitation (ChIP) assays, EM, and activity based tests. 3) We will investigate the function of PIC components using cells and viruses containing appropriate mutations. Virus growth will be monitored using quantitative PCR assays, including a new method we devised for quantitating integration in vivo. Highlight of our studies include the findings of three cellular proteins associated with PICs, the Ku heterodimer and the architectural DNA binding protein HMG I (Y). The discovery of Ku protein in PICs together with other work has lead to a new model for the role of the host cell non-homologous DNA end joining pathway in retroviral growth. Long term, these studies, together with data on protein structures, will provide a high-resolution picture of the organization and function of preintegration complexes.
Keywords: DNA binding protein, human immunodeficiency virus 1, virus DNA, virus integration, complementary DNA, protein structure function, virus replication, chicken, genetically modified animal, immunoprecipitation, polymerase chain reaction, tissue /cell culture
Project start date: 1993-07-01
Project end date: 2006-05-31
5R01AI034786-14 (2005): $356625
5R01AI034786-13 (2004): $356625
5R01AI034786-11 (2003): $418050
5R01AI034786-10 (2002): $418050
5R01AI034786-03 (1995): $204689
5R01AI034786-02 (1994): $189880
5R01AI034786-07 (1999): $335451
5R01AI034786-06 (1998): $325682
5R01AI034786-04 (1996): $220859
Grants awarded to Frederic D Bushman
MASSIVELY PARALLEL ANALYSIS OF INTEGRATION IN THERAPEUTIC GENE TRANSFER
Frederic D Bushman, Professor
University Of Pennsylvania, 3451 Walnut Street, Philadelphia, Pa 19104
Grant 1R01AI082020-01 from National Institute Of Allergy And Infectious Diseases
Abstract: To treat genetic diseases by therapeutic gene transfer, it is usually necessary to integrated the therapeutic gene into a chromosome of the host cell. However, this has led to clinical adverse events in patients receiving gene therapy for SCID-X1, in which integration of retroviral vectors activated cellular proto-oncogenes, leading to transformation of gene-corrected cells. Thus the gene therapy community has become intensely focused on the question of where gene transfer vectors integrate in the human genome. The FDA has even mandated that integration sites be analyzed as a step in monitoring for possible adverse events. The Bushman laboratory has established a unique collection of technologies for analyzing integration site populations based on DNA bar coding, pyrosequencing, and custom bioinformatic tools. Using these high throughput methods, populations of integration site sequences can be generated of up to 105 bases of sequence in a single one day run. In one published study, we generated and analyzed the placement of 40,000 unique sites of HIV DNA integration in the human genome. Here we propose to apply these methods to systematic analysis of patients from the French SCID-X1 trial. We have initiated massively parallel sequencing studies of longitudinal DNA samples from some of the French SCID-X1 patients, with the goal of understanding the evolution, ecology, and ultimate fate of transduced cells. So far, we have generated ~128,000 integration site sequence reads from 80 patient samples for a total of ~33,000,000 bases of DNA sequence. For the first time, we can begin to estimate the numbers of transduced cell clones contributing to the gene-corrected cell pool. We can ask how populations of gene corrected cells change over time. In preliminary studies, we find a troubling decline in clone diversity in our two best-studied patients, suggesting "clone burn out". We can ask whether insertional activation of genes involved in growth control leads to outgrowth of clones even in the absence of clinical adverse events. For those patients who suffered adverse events and were subsequently treated by chemotherapy, we will ask how the chemotherapeutic treatment affected the size, diversity and dynamics of the gene-corrected cell pools. We will also analyze adverse events in animal models and, longer term, integration sites generated in new clinical trials. We propose to work in close collaboration with the French SCID-X1 team to complete the following Specific Aims Aim 1. Determine the total numbers of integration sites present in samples from SCID-X1 patients. Aim 2. Determine how the number and distribution of integration sites changes over time in SCID-X1 patients. Aim 3. Determine integration site locations and relationship to genotoxicity in preclinical models and new clinical trials. Success has been achieved with human gene therapy for SCID-X1, but adverse events due to insertional activation of proto-oncogenes and leukemia have caused severe setbacks. Here we propose to apply massively parallel pyrosequencing to analyzing vector integration in samples from the historic SCID-X1 trial
Keywords: AIDS Virus; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Adverse Experience; Adverse event; Affect; Animal Model; Animal Models and Related Studies; Bar Codes; Base Sequence; Bio-Informatics; Bioinformatics; Blood (Leukemia); Cells; Cellular Oncogene; Chromosomes; Classification; Clinical; Clinical Trials; Clinical Trials, Unspecified; Codes, Bar; Collaborations; Collection; Communities; Custom; DNA; DNA Integration; DNA Sequence; Deoxyribonucleic Acid; Ecology; Environmental Science; Evolution; Gene Transfer; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Generalized Growth; Genes; Genetic Condition; Genetic Diseases; Genetic Intervention; Genome, Human; Goals; Growth; HIV; HTLV-III; Hereditary Disease; Human; Human Genome; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human, General; Insertional Activations; Intervention, Genetic; LAV-HTLV-III; Laboratories; Leukemias, General; Location; Longitudinal Studies; Lymphadenopathy-Associated Virus; Man (Taxonomy); Man, Modern; Methods; Molecular Biology, Gene Therapy; Molecular Disease; Monitor; Nucleotide Sequence; Patients; Population; Pre-Clinical Model; Preclinical Models; Proto-Oncogenes; Publishing; Reading; Retroviral Vector; Retrovirus Vector; Running; Sampling; Site; Systematics; Technology; Therapy, DNA; Time; Tissue Growth; Virus-HIV; Work; base; burnout; c-ONC; cell transduction; cellular transduction; chemotherapy; clinical investigation; gene correction; gene therapy; gene transfer vector; gene-corrected; genetic disorder; genetic therapy; genotoxicity; hereditary disorder; leukemia; long-term study; model organism; nucleic acid sequence; ontogeny; population based; protooncogene; public health relevance; success; therapeutic gene; tool; transduced cells; transfer of a gene; vector
Relevance: Success has been achieved with human gene therapy for SCID-X1, but adverse events due to insertional activation of proto-oncogenes and leukemia have caused severe setbacks. Here we propose to apply massively parallel pyrosequencing to analyzing vector integration in samples from the historic SCID-X1 trial
Project start date: 2009-06-15
Project end date: 2013-05-31
Budget start date: 15-JUN-2009
Budget end date: 31-MAY-2010
PFA/PA: PA-07-070
1R01AI082020-01 (2009): $384890
THE MACAQUE GUT MICROBIOME IN HEALTH, LENTIVIRAL INFECTION AND IBD
Frederic D Bushman, Professor
Tulane University Of Louisiana, 6823 St Charles Ave, New Orleans, La 70118
Grant 5P51RR000164-48_8856 from National Center For Research Resources
Abstract: This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The vertebrate gut harbors a vast community of bacterial mutualists, the composition of which is modulated by the host immune system. Many gastrointestinal (GI) diseases are expected to be associated with disruptions of host-bacterial interactions, but relatively few comprehensive studies have been reported. We have used the rhesus macaque model to investigate forces shaping GI bacterial communities. We used DNA bar coding and pyrosequencing to characterize 141,000 sequences of 16S rRNA genes obtained from 100 uncultured GI bacterial samples, allowing quantitative analysis of community composition in health and disease. Microbial communities of macaques were distinct from those of mice and humans in both abundance and types of taxa present. The macaque communities differed among samples from intestinal mucosa, colonic contents, and stool, paralleling studies of humans. Communities also differed among animals, over time within individual animals, and between males and females. To investigate changes associated with disease (AIDS), samples of colonic contents taken at necropsy were compared between healthy animals and animals with colitis and undergoing antibiotic therapy. Communities from diseased and healthy animals also differed significantly in composition. This work provides comprehensive data and improved methods for studying the role of commensal microbiota in macaque models of GI diseases and provides a model for the large-scale screening of the human gut microbiome
Keywords: Animals; Antibiotic Therapy; Antibiotic Treatment; Autopsy; Bar Codes; CRISP; Codes, Bar; Colitis; Communities; Computer Retrieval of Information on Scientific Projects Database; DNA; Data; Deoxyribonucleic Acid; Disease; Disorder; Feces; Female; Funding; Gastrointestinal Diseases; Gastrointestinal Diseases and Manifestations; Gastrointestinal Tract, Feces; Genes, rRNA; Grant; Health; Human; Human, General; Immune system; Individual; Infection; Institution; Intestinal Mucosa; Investigators; Macaca; Macaca mulatta; Macaque; Mammals, Mice; Mammals, Primates; Man (Taxonomy); Man, Modern; Methods; Mice; Modeling; Murine; Mus; NIH; National Institutes of Health; National Institutes of Health (U.S.); Primates; Reporting; Research; Research Personnel; Research Resources; Researchers; Resources; Rhesus; Rhesus Macaque; Rhesus Monkey; Ribosomal RNA Genes; Role; Sampling; Screening procedure; Shapes; Source; Taxon; Time; United States National Institutes of Health; Work; body system, allergic/immunologic; disease/disorder; gastrointestinal; gastrointestinal disorder; improved; male; microbial community; microbiome; necropsy; organ system, allergic/immunologic; postmortem; rRNA Genes; screening; screenings; social role; stool; treatment of bacterial diseases; treatment of bacterial infectious disease
Project start date: 2009-05-01
Project end date: 2010-04-30
Budget start date: 1-MAY-2009
Budget end date: 30-APR-2010
5P51RR000164-48_8856 (2009): $62691
SEQUENCING AND VIRAL EVOLUTION CORE
Frederic D Bushman, Professor
University Of Pennsylvania, 3451 Walnut Street, Philadelphia, Pa 19104
Grant 1U19AI082628-01_5499 from National Institute Of Allergy And Infectious Diseases
Abstract: The new deep sequencing methods offer tremendously improved means of characterizing genetic diversity. The Sequencing and Viral Evolution Core, under the direction of Dr. F. Bushman, will perform deep sequencing to support.the IPCP team. The core will analyze viral populations in the presence and absence of therapy to monitor the effects of treatment, characterize the effects of zinc-finger nucleases on genomic DMA, and quantify lentiviral vector integration sites to monitor for possible genotoxicity. The Bushman laboratory has published extensively on applications of deep sequencing, and will adapt these methods to support the proposed projects. The effects of the gag and pol epitope targeting will be assessed by quantifying longitudinal changes in viral quasispecies that accumulate selectively in the presence of pressure from therapy. For HIV viruses replicating in cells with the CXCR4 and CCR5 knockouts, deep sequencing will be used to characterize viral envelope sequences present prior to therapy and their evolution under therapy. Deep sequencing will be used to characterize the genomic structure at the site of gene deletion mediated by zinc finger nucleases and scan for possible associated chromosomal abnormalities. Deep sequencing will also be used to follow integration site selection by lentiviral vectors, and monitor for possible genotoxicity due to vector integration. Our specific aims are as follows Specific Aim 1 DNA barcoding and pyrosequencing for analysis of viral mutations in response to gag and pol epitope targeting (Projects 1 and 2), and zinc finger mediated CXCR4 and CCR5 knockout in CD4 T cells (Projects 2 and 3). Specific Aim 2 Longitudinal analysis of lentiviral vector integration sites using pyrosequencing (Projects 1 and 2)
Keywords: AIDS Virus; Aberrant Chromosome; Abnormalities, Chromosomal; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Adverse Experience; Adverse event; Antigenic Determinants; Appearance; Binding Determinants; Bio-Informatics; Bioinformatics; C-C CKR-5 Gene; C-C Chemokine Receptor Type 5 Gene; CC-CKR-5 Gene; CCCKR5 Gene; CCR-5 Gene; CCR5; CCR5 gene; CD195 Antigen Gene; CD4 Positive T Lymphocytes; CD4 T cells; CD4 lymphocyte; CD4+ T cell; CD4+ T-Lymphocyte; CD4-Positive Lymphocytes; CD8; CD8B; CD8B1; CD8B1 gene; CHEMR13 Gene; CKR-5 Gene; CKR5 Gene; CMKBR5 Gene; CXC-R4; CXCR-4; CXCR4; CXCR4 gene; Cells; Cells, CD4; Cellular Oncogene; Chemokine (C-C) Receptor 5 Gene; Chromosomal Aberrations; Chromosomal Alterations; Chromosome Aberrations; Chromosome Alterations; Chromosome Anomalies; Chromosome abnormality; Cytogenetic Aberrations; Cytogenetic Abnormalities; D2S201E; DNA; DNA Sequence; Data; Deoxyribonucleic Acid; Effectiveness; Epitopes; Evolution; FB22; Gagging; Gene Deletion; Gene variant; Gene-Modified; Generalized Growth; Genes; Genetic Alteration; Genetic Change; Genetic Diversity; Genetic Variation; Genetic defect; Genomics; Growth; HIV; HIV-1 Fusion Co-Receptor Gene; HM89; HSY3RR; HTLV-III; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; ITX; Immune response; Immunologically Directed Therapy; Immunotherapy; Insertional Activations; Intervention; Intervention Strategies; Knock-out; Knockout; LAP3; LAV-HTLV-III; LCR1; LESTR; LYT3; Laboratories; Lentiviral Vector; Lentivirus Vector; Lymphadenopathy-Associated Virus; Mediating; Methods; Monitor; Mother Cells; Mutation; NPY3R; NPYR; NPYRL; NPYY3R; Patients; Population; Position; Positioning Attribute; Pressure; Pressure- physical agent; Progenitor Cells; Programs (PT); Programs [Publication Type]; Proto-Oncogenes; Publishing; Reading; Reflex, Pharyngeal; Reporting; Running; Sampling; Scanning; Site; Stem cells; Structure; T-Cells; T-Lymphocyte; T4 Cells; T4 Lymphocytes; Technology; Therapeutic; Thymus-Dependent Lymphocytes; Tissue Growth; Variant; Variation; Variation (Genetics); Viral; Viral Genome; Virus; Virus-HIV; Viruses, General; Zinc Finger Domain; Zinc Finger Motifs; Zinc Fingers; allelic variant; base; c-ONC; cost; gene deletion mutation; genome mutation; genotoxicity; helper T cell; host response; immune therapy; immunoresponse; improved; interest; interventional strategy; longitudinal analysis; nuclease; ontogeny; pressure; programs; protooncogene; response; thymus derived lymphocyte; tool; treatment effect; vector
Relevance: The new 454/Roche pyrosequencing method allows up to 100,000,000 bases of DNA sequence to be determined in a single one day run. The core will use this method to evaluate the effectiveness of therapy and monitor for possible adverse events
Project start date: 2009-04-01
Project end date: 2014-03-31
Budget end date: 31-MAR-2010
PFA/PA: RFA-AI-08-018
1U19AI082628-01_5499 (2009): $272743
MODIFYING HIV TO INTEGRATE AT PRESELECTED DNA SITES
Frederic D Bushman, Professor
Salk Institute For Biological Studies La Jolla, Ca 920371099
Grant 5R01AI037489-06 from National Institute Of Allergy And Infectious Diseases IRG: ARRC
Abstract: The investigators propose to develop retroviral derivatives capable of integrating into preselected DNA sites. They have previously reported the construction and characterization of a fusion protein composed of HIV integrase linked to the site specific DNA-binding domain of l repressor. This hybrid protein directed integration selectively to target DNAs containing sites recognized by the repressor domain. More recently they have found that i) they can create biologically active HIV proviruses encoding integrases fused to DNA-binding domains, and ii) preintegration complexes containing such fusions direct integration preferentially into target DNA containing appropriate sites.
Keywords: human immunodeficiency virus, nucleic acid sequence, recombinant virus, transfection /expression vector, vaccine development, vector vaccine, virus integration, cAMP response element binding protein, chimeric protein, gene mutation, integrase, polymerase chain reaction, site directed mutagenesis, tissue /cell culture, transfection, vaccinia virus
Project start date: 1996-12-01
Project end date: 2001-11-30
5R01AI037489-06 (2000): $239889
5R01AI037489-05 (1999): $232901
5R01AI037489-04 (1998): $226118
2R01AI037489-03 (1997): $224093
BROAD SPECTRUM THERAPEUTICS TARGETING RESOLVASE ENZYMES
Frederic D Bushman, Professor
University Of Pennsylvania, 3451 Walnut Street, Philadelphia, Pa 19104
Grant 1U01AI082015-01 from National Institute Of Allergy And Infectious Diseases
Abstract: text here that is the new information for your application. This section must be no Project Summary (Change of Scope) Modified Project Summary (Change of Scope) U01 A1082015-O1 "Broad Spectrum Therapeutics Targeting UO1 AI082015-01 "Broad Spectrum Therapeutics Targeting Resolvase Enzymes" For applications in biodefense, it is desirable for small molecule inhibitors to biodefense, target multiple category A-C agents because it is difficult and expensive to develop even one small molecule inhibitor. We propose to develop inhibitors of molecule inhibitor. We propose Holliday junction resolvase enzymes, which are found in several category A-C Initial studies focused on resolvase enzymes agents. Initial studies focused on resolvase enzymes found in poxviruses. These enzymes catalyze a required replication step in which concatemers of the viral genomic DNA are cleaved into unit length genomes for packaging. The cleaved into unit length genomes for packaging. enzyme is also important in pathogenic fungi such as Coccidioldes, the causative Coccidioides, Fever, where agent of Valley Fever, where it is involved in mitochondrial DNA replication. In involved in mitochondrial DNA replication. previous work we developed a high throughput assay and screened >133,000 Our best compound so far has an IC50 small molecules for inhibitory activity. Our best compound so far has an 1C50 against purified resolvase of -100 nM, 1C50 against virus of 3 uM. We have -100 nM, IC50 against virus of 3 uM. revised our Aims in accordance with the reviewers comments, and to maximize our progress over the projected two years (instead of five years) of funding. We projected two years (instead of five years) of funding. will emphasize new compound identification, iterative compound synthesis, and increasingly stringent assays to develop inhibitors of poxvirus resolvases that are active in animal models. Inhibitors active against poxviruses in vivo will also be models. Inhibitors active against poxviruses tested in pathogenic fungi in an effort to develop "dual use" pharmacotherapy. pathogelllic Funding of the project will generate jobs for more than five people (5.45 FTEs summed over all the participating groups). and two pieces of equipment will be purchased (a fluorescence plate reader and a -80¿C freezer) from American sources. advancing the goals of the ARRA. sources, thereby advancing the goals of the ARRA. -(} -¿O D)¿ ... 10 1120AM (GMT-0400) 11 20AM (GMT-014OO) 06/19/2009 too r=´ Ofd mN 0)_ d-0 =ii =d0 a-Ec w-w goo 06/19/2009 1031 FAX 06/19/2009 215 9557 215 898 9557 UPENN MICROBIOLOGY t012/025 IiZl 012/025
Keywords: 35 kDa major secretory protein, Vaccinia virus; AIDS Virus; ATGN; Abscission; Absorption; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Address; Affect; Aflatoxins; Agreement; Air; American; Americas; Anatomic; Anatomical Sciences; Anatomy; Animal Model; Animal Models and Related Studies; Animals; Animals, Laboratory; Antibiotic Agents; Antibiotic Drugs; Antibiotics; Antigens; Applications Grants; Arg-Tyr-Leu-Pro-Thr; Aspergillus; Assay; Authorization; Authorization documentation; Award; Back; Bar Codes; Bioassay; Biocontrols; Biologic Assays; Biological Assay; Biological Terrorism; Bioterrorism; Blast Cell; Blasts; Boxing; Budgets; CD2 gene; CD8 Cell; CD8 lymphocyte; CD8+ T-Lymphocyte; CD8-Positive Lymphocytes; CD8-Positive T-Lymphocytes; COAD; COPD; Calendar; Calibration; California; Care, Health; Cataloging; Catalogs; Categories; Cell Culture Techniques; Cell Therapy; Cells; Certification; Chemicals; Chest; Chronic Obstructive Airway Disease; Chronic Obstructive Lung Disease; Cleaved cell; Clinical; Coccidioides; Coccidioidomycosis; Code; Codes, Bar; Coding System; Collaborations; Coloring Agents; Comment; Comment (PT); Comment [Publication Type]; Commentary; Commentary (PT); Communicable Diseases, Emerging; Communities; Complement; Complement Proteins; Complementary DNA; Confidential Information; Consultations; Contracting Opportunities; Contracts; Cooperative Agreements; Cooperative Agreements, U-Series; Crohn`s disease; Crohn`s disorder; Cruciform Cutting Endonuclease; Cruciform DNA; Cruciform DNA Resolving Endonuclease; Custom; DNA; DNA Integration; DNA Replication; DNA Synthesis; DNA Vaccines; DNA biosynthesis; DNA, Complementary; DNA, Cruciform; DNA, Mitochondrial; Data Banks; Data Bases; Databank, Electronic; Databanks; Database, Electronic; Databases; Deoxyribonucleic Acid; Development; Diagnostic; Diagnostic tests; Diet; Dimensions; Direct Costs; Disclosure; Disease; Disorder; Dorsum; Drug Formulations; Drug Therapy; Drug resistance; Drugs; Dyes; Economic Income; Economical Income; Editorial Comment; Editorial Comment (PT); Emerging Communicable Diseases; Engineering; Engineerings; Enteritis, Granulomatous; Enzyme Tests; Enzymes; Equipment; Event; Excision; Excretory function; Extirpation; F3 protein; FLR; Face; Failure (biologic function); Fax; Ferrata cell; Fever; Fever Therapy; Fluorescence; Formulation; Formulations, Drug; Foundations; Funding; Gene Deletion; Gene Expression; Gene Transfer; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Modified; Gene-Tx; Generalized Growth; Genes; Genetic; Genetic Intervention; Genetics-Mutagenesis; Genome; Genome, Human; Genomics; Genotype; Goals; Government; Grant; Grant Proposals; Grants, Applications; Growth; Gut Factor; HIV; HIV Infections; HTLV-III; HTLV-III Infections; HTLV-III-LAV Infections; Health; Healthcare; Hematohistioblast; Hemocytoblast; Hemohistioblast; High Throughput Assay; Histologic; Histologically; Holliday Junction DNA; Holliday Junction Resolvases; Holliday Junctions; Home; Home environment; Human; Human Genome; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human, General; Hyperthermia; Hyperthermia, Therapeutic; IACUC; IC50; ITX; Image; Immune; Immune response; Immunologically Directed Therapy; Immunotherapy; In Vitro; Income; Individual; Induced Hyperthermia; Infection; Infectious Diseases, Emerging; Information Disclosure; Information, Confidential; Inhibitory Concentration 50; Institution; Institutional Animal Care and Use Committee; Instruction; Intermediary Metabolism; Intervention, Genetic; Investigators; Jobs; LAV-HTLV-III; Laboratories; Laboratory Animals; Laboratory Research; Lead; Legal; Length; Libraries; Liquid substance; Lung; Lung diseases; Lymphadenopathy-Associated Virus; METBL; Magnaporthe; Mails; Mali; Mammals, Mice; Man (Taxonomy); Man, Modern; Manuals; Mediation; Medical; Medication; Metabolic Processes; Metabolism; Methods; Mice; Microbiology; Minor; Miscellaneous Antibiotic; Mitochondrial DNA; Modeling; Molecular Biology, Gene Therapy; Molecular Biology, Mutagenesis; Monitor; Mother Cells; Murine; Mus; Mutagenesis; NIH; Naked DNA Vaccines; Names; Naphthyridines; National Institutes of Health; National Institutes of Health (U.S.); Negotiating; Negotiation; Occupations; Paint; Paper; Partner in relationship; Pathogenesis; Pattern; Pb element; Pennsylvania; Performance; Permission; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phase; Phenotype; Phone; Pisum sativum; Pneumonia; Pneumonitis; Policies; Powder dose form; Powders; Poxviridae; Poxvirus officinale; Poxviruses; Pressure; Pressure- physical agent; Price; Principal Investigator; Privacy; Process; Process of absorption; Professional Postions; Progenitor Cells; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Proteins; Protocol; Protocols documentation; Public Health; Published Comment; Publishing; Pulmonary Body System; Pulmonary Disease, Chronic Obstructive; Pulmonary Diseases; Pulmonary Disorder; Pulmonary Inflammation; Pulmonary Organ System; Pyrexia; Reader; Refrigeration; Removal; Reporting; Research; Research Personnel; Research Resources; Research, Laboratory; Researchers; Resistance; Resolution; Resolvase; Resources; Respiratory Disease; Respiratory Disorder; Respiratory System; Respiratory System Disease; Respiratory System Disorder; Respiratory System, Lung; Respiratory system (all sites); Respiratory tract structure; Rice; Rights; Risk; Rodent Model; Role; Running; SRBC; Safety; Salaries; Sampling; Science; Science of Anatomy; Science of Microbiology; Screening procedure; Secure; Security; Services; Shipping; Ships; Site; Smallpox; Smallpox Vaccine; Source; Staging; Stainless Steel; Steel; Stem cells; Sum; Surgical Removal; System; System, LOINC Axis 4; T-Lymphotropic Virus Type III Infections, Human; T11; T8 Cells; T8 Lymphocytes; TGase II; TXT; Telefacsimile; Telefax; Telephone; Temperature; Testing; Text; Therapeutic; Therapeutic Effect; Therapeutic Intervention; Therapy, Cell; Therapy, DNA; Thermotherapy; Thick; Thickness; Thorace; Thoracic; Thorax; Time; Tissue Growth; Toxicology; Tracts, Respiratory; Trademark; U-Series Cooperative Agreements; United States; United States National Institutes of Health; Universities; V (voltage); Vaccine Antigen; Vaccines; Vaccines, DNA; Vaccines, Recombinant DNA; Vaccinia; Vaccinia virus; Vaccinia virus complement-control protein; Validation; Variola; Viewpoint; Viewpoint (PT); Viral; Viral Diseases; Virulence; Virus; Virus Diseases; Virus-HIV; Viruses, General; Wages; Weight; Work; Yeasts; absorption; ing; anatomy; arginyl-tyrosyl-leucyl-prolyl-threonyl; base; biodefense; biosignature; cDNA; cell-based therapy; cleaved; clinical data repository; clinical data warehouse; complement-control protein, Vaccinia virus; contactin; contactin 1; cost; data repository; density; design; designing; digital; disease/disorder; drug resistant; drug/agent; efficacy testing; eleocolitis; ethenylbenzene, homopolymer; excretion; facial; failure; febrile; febris; fluid; fungus; gene deletion mutation; gene product; gene therapy; genetic therapy; granulomatous enterocolitis; heavy metal Pb; heavy metal lead; high throughput screening; histidine kinase; host response; imaging; immune therapy; immunogen; immunoresponse; improved; in vivo; inhibitor; inhibitor/antagonist; interest; intervention therapy; liquid; lung disorder; mate; meetings; microbiome; model organism; mouse model; mtDNA; mutant; neopolyoxin C; neuronal cell surface protein F3; nikkomycin; nikkomycin Z; ontogeny; pathogen; pea; peer; polystyrene latex; pox virus; pressure; prevent; preventing; pricing; proctolin; programs; protein-histidine kinase; public health medicine (field); public health relevance; pulmonary; recombinant vaccinia virus; regional enteritis; relational database; resection; resistance to Drug; resistant; resistant strain; resistant to Drug; respiratory; respiratory tract; response; scaffold; scaffolding; screening; screenings; seal; small molecule; small pox; small pox vaccine; social role; solid state; styrene copolymer; styrofoam; therapeutic gene; therapeutic target; tissue-type transglutaminase; tool; transfer of a gene; transglutaminase 2; transglutaminase C; transglutaminase II; transglutaminase TGM2; variola major; viral infection; virus infection; voltage
Relevance: Modified Enter the text here that is the new public health relevance information for your application. Using Using no more than two or three sentences, describe the relevance of this research to public health. relevance of this research to public health. no than Public Health Relevance Modified Public Health Relevance The United States of America is at risk of terrorist attacks using poxviruses or using pathogenic fungi. Both are also current medical problems. We propose to fungi. Both are medical problems. develop improved small molecule therapy targeting the Holliday junction resolving enzymes encoded by these pathogens as new treatments for infection. resolving Z-0 13 s-0¿ Have 06/19/2009 11 :20AM (GMT-O´4:OO) 11:20AM (GMT-04:00) 06/19/2009 10:32 FAX 06/19/2009 215 9557 215 898 9557 UPENN !IICROBIOLOGY MICROBIOLOGY L l!IJ 015/025 Program OlrectorlPrlnclpallnvestigator:J:!ushman, Frederic Dixon Frederic Dixon Program Director/Principal investigator:ushman, FREDERIC BUSHMAN, FREDERIC 10.08 cm ttl effort ACTIVE U19 AI078675 (Weiner, P1) U19A1078675 (Weiner, PI) 1/1/08-12131/13 1/1/08-12/31/13 0.96 calendar months NIH/IPCAVD NIH/ IPCAVD $152,853 Improving DNA Vaccines The goal of this project is to use pyrosequencing and DNA bar coding to study immune pressure, in project s coding order to guide the design of improved DNA vaccines. vaccines. R01 A1052845 (Bushman, P1) ROl AI052845 (Bushman, PI) 4/1/08-3/31/13 2.4 calendar months NIH $225,000 Favored Sites for HIV cDNA Integration in the Human Genome is to improve our understanding of HIV DNA integration mechanisms. The goal of this project s to improve our understanding of HIV DNA integration mechanisms. MARCE RFP (Bushman, P1) (Bushman, PI) 3/1/09-2128/14 3/1109-2/28/14 MARCE $175,000 Inhibition of DNA Modifying Enzymes of Category A-C Agents Inhibition Agents The goal of this project is to develop therapeutics against Category A-C Agents Cam ... .´.. P´) 0 o¿´ RFAAI08018 (June, P1) 3/1109-2128/14 RFAAIO8OI8 (June, PI) 3/1/09-2/28/14 0.96 calendar months NIH $173,248 Combination immunotherapy with gene modified CD4 and CD8 cells and stem cells modified The goal of this project is to use deep sequencing to analyze samples from cell therapy trials to treat HIV. O´^> ´co a0. ::2 coy E en, P´) 0.96 calendar months ado i´4 R01 A1082020 (Bushman, P1) 6/15/09-5/31/13 2.4 calendar months ROl AI082020 (Bushman, PI) NIH $250,000 Massively Parallel Analysis of Integration in Therapeutic Gene Transfer SCID-X1 The goal of this project is to use deep sequencing to analyze integration in SCID-X1 gene therapy. fl= o´´ E E PENDING´ Effort will be adjusted on pending grants if funded. PENDING * funded. LewiS, Co-Pis) 711109-6/30/13 UH2HGOO4994 UH2HG004994 (Wu, Bushman, Lewis, Co-PIs) 7/1/09-6/30/13 NIH $700,000 Diet, Genetic Factors, and the Gut Microbiome in Crohn´s Disease The goal of this project is to analyze the role of gut flora in Crohn´s disease (/f 61.y _¿oo U01 Al (Bushman PI) 611/09-5/31/14 2.4 calendar months U01 AI 082015 (Bushman P1) 6/1/09-5/31/14 this grant NIH/NIAID Broad Therapeutics Targeting Resolvase Enzymes Broad Spectrum Therapeutics Targeting Resolvase Enzymes The goal of this project its to carry out basic and pharmacological studies of resolvases. 0x¿ RFA HL-09-006 (Coliman and Bushman Co-PIs) (Collman Co-Pis) 9/30/09-8/29/14 calendar months 2.4 calendar months NIH/NHLBI $525,000 Human Respiratory Tract Microbiome in Health, HIV Infection and HIV Lung Disease of project is use sequencing respiratory microbiome The goal of this project is to use high density deep sequencing to examine the respiratory microbiome in HIV infected and uninfected individuals with COPD. individuals with COPD. -´I =-O PA-06-181 (Bushman, P1) (Bushman, PI) 12/1/09-11130/11 1.2 calendar months 12/1/09-11/30/11 NIH $125,000 Deep-sequencing for HIV genotyping in resource-limited settings resource-limited of project is method use in resourceThe goal of this project is to develop a method for genotypic drug resistance testing for use in resourcelimited settings. 3 ¿2_ 3¿¿ OVERLAP: . 0 (DO ..0 PHS 39812590 (Rev. 11/07) 398/2590 ´20 o6¿ p¿QU 1.8 calendar months 1.8 464 cr.. 0´o 0.2 22_ a)>_ >.S2 O-- Page_ Page 14 other Support Fonnat Page Other Support 06/19/2009 11:20AM (GMT-04:00) 11 :20AM (GMT-014:00) 06/19/2009 10:32 FAX 06/19/2009 215 898 9557 215 9557 UPENN MICROBIOLOGY E IiZl 016/025 Program Direetor/l´rincipallnvestlgator: Bushman. Frederic Dixon Program Director/Principal Investigator: Bushman, Frederic Dixon ISAACS, S.N. ACTIVE 6.6 cm 3.60 calendar U01 AI066333 (lsaacs, P1) 7/5/2005 - 6/30/2010 U01 A1066333 (Isaacs, PI) NIH/NIAID $225,000 Smallpox vaccine and vaccinia complement control protein Th is project examines the deletion of the gene encoding the vaccinia virus complement control protein as a of protein as a This more strategy to generate a safer, but more efficacious smallpox vaccine. U01 A1077913 (Isaacs, P1) 5/1/2008 - 4/30/2013 5/1/2008-4/30/2013 U01 A1077913(lsaacs, PI) ~$425,000 -$425,000 NIH/NIAID NI H/N lAl D Optimizing the formulation of a protein based smallpox vaccine ¿0O The goal of this project is to develop a safe and effective protein-based smallpox vaccine using variola proteins. .O. 0<0 0020 1.2 calendar E This project will investigate viral and cellular proteins involved in poxvirus entry into cells. PENDING OYERLAP OVERLAP m z %20 PHS 398/2590 (Rev. 11/07) 11107) PHS em. U54 AI057168 (Levine, overall PI for MARCE) 3/1/2009 - 2/28/2014 1.8 calendar U54 A1057168 (Levine, overall P1 for MARCE) NIH/NIAID $235,000 Middle Atlantic Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Program 2: Emerging Virus Entry into Host Cells: Strategies for Inhibition Cells: 2: Inhibition Project 2.4: Mechanism of Poxvirus Entry into Cells (Gary Cohen. P1) of Entry into Cells (Gary Cohen, PI) 2.4: ACV page~ Page 15 Other Support Format Page Fonmat 06/19/2009 11 :20AM (GMT-0´4:00) 11:20AM (GMT-04:00) 06/19/2009 10:33 FAX 06/19/2009 215 9557 215 898 9557 UPENN MICROBIOLOGY IiZl 017/025 Program DirectorlPrincipallnvestigator: Program DiiactorlPrincipal Investigator: Bushman. Frederic Dixon Bushman, MADRID, PETER B. 4.2 cm ACTIVE DTRAlTMTI DTRA(TMTI Contract No.: HDTRA1-07-C-0083 No.: HDTRAI-07-C-0083 of Performance: 07118/07-07/17109 Period of Performance: 07118/07-07/17/09 Title: An Accelerated Path to Safe and Effective Therapeutics (APSET) for Bioterrorism Agents (APSEl) Title: Award Amount: $9,100,000 Time: 1.20 months Time: Mate. c13 NIHINIDDKD NII-I/NIDDKD (Subcontract to Numerate Inc.) Contract No.: 1R41DK081244-01A1 Contract No,: 1R41DK081244-O1A1 Period of Perfomance: 02101109 -- 01/31/2010 Period Perfomance; 02/01/09 0113112010 Title: Drug Engineering of Transglutaminase-2 Inhibitors of Award Amount: $54,000 Award Amount: Time: 3.00 months Time: PENDING NIH/NIAIO NIH/NIAID Period of Performance: 07/01/09 - 06/30/11 07101/09 06130111 Period of gyrase b histidine kinases broad-spectrum Title: Dual action inhibitors of gyrase b and histidine kinases as broad-spectrum antibiotics. Award Amount: $3,200,000 Time: 6.00 months OVERLAP . "Q. s.N C0= o¿A) t11 E PHS 398/2590 (Rev. 11/07) oar3E 01= coo ¿c. Page_ Page 2¿ Other Support Format Page Fonnat Page 06/19/2009 11 :20AM (GMT-0´4:00) 11:20AM (GMT-04:00) 06/19/2009 10:33 FAX 06/19/2009 215 898 9557 215 UPENN MICROBIOLOGY IiZl 018/025 t018/025 PrinClpallnvestlgatorlProgram Director: Bushman, Frederic D. Pflncipal Investigator/Program Director Bushman Frederic D. PHS 39812590 OTHER SUPPORT 398/2590 ORBACH, MJ 5.5 ttl cm ACTIVE 1 P01 Al061 31 0-01 Galgiani (P1) P01 A1061310-01 Galgiani (PI) 06/15/04 - 05/31/10 3.0 calendar NIHINIAID No cost extension NIH/NIAID Induction of Fungal Dormancy as a Potential Disease Therapy, Host Control in Coccidioldomycosis Therapy, Coccidioidomycosis The major goal of this project is to explore the genetic basis of quiescence in Coccidioides spp. using global CoccidiOides gene expression analysis and mutagenesis of differentially regulated genes. This grant is in a no-cost extension year. Role: Project Leader Role: OVERLAP: No overlap. -15 o´3 .N.´ fan 0627159 Soderlund, C. (P1) Soderlund, C. (PI) 12/01/06 -11/30/09 1.0 calendar 12/01/06 - 11/30/09 Foundation No cost extension National Science Foundation Community Database for studying rice-rice blast interactions. a platform This will The goal of this project is to set up a community annotation platform for the Magnaporthe genome. This will include addition of mutant information generated by a previous NSF project along with encouraging addition participation by the research community. This grant is in a no-cost extension year. community. This grant is in a no-cost extension year. Role: Role: Co-PI OVELAP: No overlap. -15yct (¿d MJ (PI) 58-6435-8-315 Orbach, MJ (P1) 8/1/08-07/31/13 0.5 calendar USDA Cooperative Agreement $28,622 Influence of Agrichemicals on Aspergillus Communities and Aflatoxin Management. Aflatoxin Management. The goals of this project are to determine whether the application of agrichemicals aimed at weed control affects reSistance of fungi in the fields to those chemicals. If so, resistant strains of Aspergillus species used as resistance chemicals. so, reSistant biocontrol agents will be selected for and tested for efficacy as biocontrol agents compared to non-resistant biocontrol strains. OVERLAP: No overlap. =ENO 3 PENDING U01 Al08201 5-01 (Orbach - subcontract) UO1 A1082015-01 1.0 calendar NIH this grant $73,554 Broad Spectrum Therapeutics Targeting Resolvase Enzymes Broad Coccidioides on The goal of this research is to determine the effect of eliminating the resolvase gene from Coeckiloides on for virulence of the fungus for mammalian hosts and to develop small molecules targeting this gene for therapy. for the current proposal. OVERLAP: This is for the current proposal. .-. o 17 06/19/2009 11:20AM (GMT-04:00) 11 :20AM (GMT-0L:00) -10 .2´y E"w 460 035 o4) ¿¿¿f7¿mW C ELL 4¿"5 +-´ ID.. 06/19/2009 10:33 FAX 06/19/2009 215 9557 215 898 9557 UPENN MICROBIOLOGY IiZl 019/025 Program Di_orlP,incipallnvestigator: Director/Principal Invesugator: Bushman. Frederic Dixon Bushman, Frederic Dixon SHUBITZ. LISA F. F. SHUBITZ, ACTIVE 11.6cm AI06131 0-030001 (Galgiani) 06/15/04 -05/31/09 0.5 calendar 1 P01 A1061 310-030001 (Galgiani) 06/15/04 -05/31/09 no cost extension NIH/NIAID Histological Marker of Resistance to Coccidioidomycosis, Host Control in Coccidioidomycosis The develop histopathological responses infection in the The major goals of this project are to develop histopathological profiles of host responses to infection in the lungs of genetically resistant mice and compare this profile to the responses of genetically susceptible mice, responses of genetically susceptible mice, and to identify vaccine formulations and protocols that result in histologic patterns closest to those found formulations in histologic patterns those found and with resistance. 150 7Z´ 00- 1 P01 A1061 310-030004 (Galgiani) P01 AI06131 0-030004 0.5 calendar 06/15/04 -. 05/31/09 NIH/NIAID no cost extension BSL3 Animal Laboratory, Host Control in Coccidioidomycosis The major goals of this project are to provide a murine model of coccidioidomycosis to study the hostpathogen interactions in the respiratory tract in order to understand the early immunological events that lead pathogen to either resolution of infection or progression with development of clinical disease. of infection or progression with development of 0 6-0 .m. 1.0 calendar (Galgiani) 01/01/00- 12131109 01/01/00 - 12/31/09 California Health Care Foundation $198,052 California Health Care Foundation $198,052 Project, Phase II Valley Fever Vaccine Project, Phase II is identify antigens may be The goal of this project is to identify antigens that may be useful in a practical vaccine to prevent coccidioidomycosis in humans. (p´ 03/01/09 - 2/28/11 3.0 calendar 2128/11 1 R42A1074157¿01A1 (Galgiani) R42A10741 57-01 Al (Galgiani) NIH/NIAID $303,525 Experimental Pharmacology and Chemical Studies of Nikkomycin Z - Phase II Phase II is perform pharmacological and new therapy for The goal of this project is to perform pharmacological and chemical studies on a promising new therapy for Fever. Valley Fever, U54A1065359 (Galgiani) 5/1/09-4/30/10 6.0 calendar US4AI 065359 Pacific Southwest Regional Center for Excellence $189.000 Pacific $189,000 Coccidioides Proteins as Vaccine Antigens and Diagnostic Biosignatures more Fever The major goals of this research are to create a more sensitive diagnostic test to distinguish Valley Fever from course of disease, and identify vaccine antigens that will early in other causes of pneumonia eatly in the course of disease, and to identify additional vaccine antigens that will for FDA approval. be easier to formulate for FDA approval. CO/ m.. OVERLAP: OVERLAP: PENDING U01 A1082015-01 (Orbach 0.6 calendar UO1 Al08201 5-01 (Orbach - subcontract) NIH this grant $73,554 (Initial funding period) Broad Spectrum Therapeutics Targeting Resolvase Enzymes .Coccidioides on The goal of this research is to determine the effect of eliminating the resolvase gene from Cciccidioides on virulence of the fungus for mammalian hosts and to develop small molecules targeting this gene for therapy. s-. (´p 5." -"3 -(9 PHS 398/2590 (Rev. 11/07) 11107) PHS 8 page-2.. Page Other Support Format Page support Fonnat Page 06/19/2009 11 :20AM (GMT-014:OO) 11:20AM (GMT-04:00) ffi WO.O mom= O_´ >+_ v=a arc _a0 dry 044 E moc pea U´0 fir} am´ 06/19/2009 10:34 FAX 06/19/2009 215 898 9557 215 9557 UPENN !IICROBIOLOGY MICROBIOLOGY L l!IJ 020/025 IACUC Verification IACUC Verification use and IACUC approval: Animal use and IACUC apDroval: see revised IACUC protocol We have an IACUC approval protocol number 708172, "Therapeutic Interventions After Vaccinia Virus Infection", approval date 8/812006, that we are Infection", 8/8/2006, now connecting administratively to this proposal. Animal work will not start this proposal. immediately, allowing time for administrative adjustment. .
Project start date: 2009-09-01
Project end date: 2011-08-31
Budget start date: 1-SEP-2009
Budget end date: 31-AUG-2010
PFA/PA: RFA-AI-08-001
1U01AI082015-01 (2009): $1012363
Favored Sites For HIV Integration In The Human Genome
Frederic D Bushman, Professor
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 5R01AI052845-06 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Integration of retroviral cDNA into a host cell chromosome is a central feature of viral replication, but the determinants of integration target site selection remain uncertain. We have taken advantage of the draft human genome sequence to map sites of HIV cDNA integration and assess the relationship between integration acceptor sites and chromosomal features. Mapping of 524 independent sites of HIV cDNA integration in SupT1 cells revealed that 69% of integration sites were in transcription units, indicating that active genes are favored as targets. Transcriptional profiling of the target cells revealed that transcriptional activity correlated with targeting of genes for integration. Integration hotspots were also detected, including a 2 kb chromosomal region containing 5 independent integrated proviruses, 1% of the integration events analyzed. These data reveal that HIV integration is highly biased in the human genome, providing new perspective on HIV integration mechanism. These data are also important for assessing the safety of retroviral vectors in gene therapy, and the use of retroviruses as insertional mutagenesis. To extend this study, the following specific aims will be addressed To characterize integration site selection in primary human cells; To characterize integration site selection by MLV; To investigate the mechanisms directing integration targeting; and to characterize integration sites in patient samples.
Keywords: genome, human immunodeficiency virus, molecular site, virus infection mechanism, cell cycle, chromosome, genetic transcription, host organism interaction, lymphocyte, monocyte, murine leukemia virus, suppressor T lymphocyte, virus replication, clinical research, human tissue, microarray technology
Project start date: 2002-06-01
Project end date: 2007-05-31
5R01AI052845-06 (2006): $271843
5R01AI052845-05 (2005): $286481
Sponsored Links Excellgen http://Excellgen.com
5R01AI052845-03 (2004): $367918
5R01AI052845-02 (2003): $378862
1R01AI052845-01 (2002): $379400
Mechanism Of HIV DNA Integration
Frederic D Bushman, Professor
Salk Institute For Biological Studies La Jolla, Ca 920371099
Grant 2R01AI034786-09 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: In order to replicate, a retrovirus such as HIV must integrate a cDNA copy of its RNA genome into a chromosome of the host. Although the cDNA integration system is a promising target for antiretroviral therapy, no clinically useful inhibitors have yet been developed. Our proposed studies are designed to provide mechanistic information useful in developing such inhibitors. Integration in vivo is carried out by a sub-viral particle containing the viral cDNA complexed with proteins. Such "preintegration complexes" (PICs) can be isolated from infected cells and when presented with a target DNA in vitro can carry out integration. We have been engaged in a long term study of the composition, organization and activities of PICs. We propose to continue this work in the renewal and investigate the implications with studies of viral replication in cell culture. We propose three specific aims 1) we will further improve methods for the large-scale production of PICs and their biochemical analysis, particularly the detection and quantitation of complex components using LC-MS and other methods. 2) We will investigate the organization of PICs using chromatin immunoprecipitation (ChIP) assays, EM, and activity based tests. 3) We will investigate the function of PIC components using cells and viruses containing appropriate mutations. Virus growth will be monitored using quantitative PCR assays, including a new method we devised for quantitating integration in vivo. Highlight of our studies include the findings of three cellular proteins associated with PICs, the Ku heterodimer and the architectural DNA binding protein HMG I (Y). The discovery of Ku protein in PICs together with other work has lead to a new model for the role of the host cell non-homologous DNA end joining pathway in retroviral growth. Long term, these studies, together with data on protein structures, will provide a high-resolution picture of the organization and function of preintegration complexes.
Keywords: DNA binding protein, human immunodeficiency virus 1, virus DNA, virus integration, complementary DNA, protein structure function, virus replication, chicken, immunoprecipitation, polymerase chain reaction, tissue /cell culture, transgenic animal
Project start date: 1993-07-01
Project end date: 2006-05-31
2R01AI034786-09 (2001): $397148
1R01AI034786-01 (1993): $187980
Host Cell Response To The Early Steps Of Hiv Infection
Frederic D Bushman, Professor
Salk Institute For Biological Studies La Jolla, Ca 920371099
Grant 5R21AI053820-02 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Human cells sense the earliest steps of infection by HIV and respond almost immediately. We propose to investigate the cellular sensing apparatus, the intermediate signaling pathways, and the effector systems mobilized in response to the early steps of HIV infection. The cellular response has evolved in part to help contain infection, for example by induction of the interferon system. Pretreatment of cells with interferon causes a change in transcriptional program, resulting in induction of host systems that drastically reduce accumulation of HIV eDNA. Another host response to infection seen in some cells involves recognition of the HIV eDNA as DNA damage, with induction of a cellular DNA damage response and consequent apoptosis of a portion of the infected cells. We will investigate the mechanism by which cells sense early viral infection and respond by inhibiting viral replication. We will use a series of recently developed methods for this study, including RNAi, fluorescence-monitored PCR, and transcriptional profiling. One highlight of our preliminary data is the first demonstration that HIV replication can be blocked specifically and efficiently by RNAi. In the parent grant, "Mechanism of HIV DNA integration," we are studying the structure and function of HIV replication intermediates ("preintegration complexes," PICs) formed during the early steps of infection. Here we propose to expand the study to investigate the host responses to the presence of these replication complexes in cells. This project is exploratory in nature, since we do not know in advance the pathways we will find, and the main techniques involved are newly developed. We expect that the study will involve considerable methods development, particularly in bioinformatics and large-scale applications of RNAi. We propose to carry out the following Specific Aims 1. Investigate RNAi in mammalian cells as 1) a tool to probe the host cell response to infection, and 2) an antiviral system in its own right. 2. Investigate the mechanism by which interferon treatment of target cells abrogates the early steps of HIV infection. 3. Use microarray technology to investigate the host cell response to early steps of infection.
Keywords: HIV infection, cellular immunity, host organism interaction, virus infection mechanism, DNA damage, disease /disorder onset, gene expression, interferon, messenger RNA, protein structure function, regulatory gene, virus genetics, virus replication, microarray technology, polymerase chain reaction
Project start date: 2002-09-30
Project end date: 2003-11-30
5R21AI053820-02 (2003): $281700
1R21AI053820-01 (2002): $281700
Chemistry Of HIV-1 Integrase Function
Frederic D Bushman, Professor
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 5R01GM068408-04 from National Institute Of General Medical Sciences IRG: ADDT
Abstract: HIV replication involves reverse transcription of the viral RNA to make a cDNA copy, and integration of that cDNA into a chromosome of the host cell. Reverse transcriptase (RT) has been exploited as a target for antiviral agents, as has viral protease (PR), which is required for maturation of the viral proteins. The third HIV-encoded enzyme, integrase (IN), has not yet been exploited as an inhibitor target. We propose to carry out a thorough study of the chemistry of HIV-1 IN function, with the dual goal of elucidating the reaction mechanism and providing data to help guide the design of inhibitors. In our first series of studies, we propose to probe the IN active site using metal rescue experiments, mutagenic probing of catalytic requirements, and tethering of new small molecules in or near the active site. The second series of studies will focus on elucidating the structure of the integrase-DNA complex. There is now considerable evidence that IN protein correctly assembled with its DNA substrates responds differently to small molecule inhibitors than does free IN protein--thus the key issue in designing IN inhibitors is understanding the full IN-DNA complex. A major complication in studying IN-DNA complexes is the non-specific DNA binding by IN that predominates after mixing IN and DNA in solution. We have devised methods for assembling homogenous and monodisperse IN-DNA complexes based on use of 1) disulfide-mediated cross-linking of IN to DNA, and 2) assembly of IN with branched DNAs resembling integration intermediates. We propose to exploit these complexes and other experimental paradigms to characterize the chemistry of HIV-1 IN function using X-ray crystallography, NMR, cryo-electron microscopy, FRET and other methods. The Specific Aims are Aim 1. Functional studies of IN-DNA complexes Aim 2. Structural analysis of IN-DNA complexes.
Keywords: human immunodeficiency virus 1, integrase, protein structure function, virus infection mechanism, virus replication, active site, antiAIDS agent, crosslink, enzyme inhibitor, enzyme mechanism, enzyme structure, small molecule, X ray crystallography, cryoelectron microscopy, fluorescence resonance energy transfer, nuclear magnetic resonance spectroscopy, site directed mutagenesis
Project start date: 2004-01-15
Project end date: 2007-12-31
5R01GM068408-04 (2007): $252481
5R01GM068408-03 (2006): $260022
Sponsored Links Excellgen http://Excellgen.com
5R01GM068408-02 (2005): $266280
1R01GM068408-01A1 (2004): $266280
MECHANISM OF HIV DNA INTEGRATION
Frederic D Bushman, Professor
Salk Institute For Biological Studies La Jolla, Ca 920371099
Grant 2R01AI034786-05 from National Institute Of Allergy And Infectious Diseases IRG: ARRC
Abstract: Adapted From Applicant s ) This application proposes to study the process of HIV integration by (1) purifying preintegrative complexes to near homogeneity and determine the protein composition and stoichiometry, (2) characterizing the structure of isolated preintegrative complexes in detail using electron microscopy and assays for protein-DNA interactions, and (3) to build upon the exciting finding that HMG I(Y) is a likely component of the preintegrative complex by examining its role in the integration process in vivo and in vitro.
Keywords: DNA binding protein, human immunodeficiency virus 1, virus DNA, virus integration, protein structure /function, electron microscopy, human tissue, protein purification
Project start date: 1993-07-01
Project end date: 2001-06-30
2R01AI034786-05 (1997): $335098
MOLLUSCUM CONTAGIOSUM VIRUS TOPOISOMERASE
Frederic D Bushman, Professor
Salk Institute For Biological Studies
la Jolla, Ca 920371099
Grant 5R01AI046222-04 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Adapted from Applicant´s ) The poxvirus molluscum contagiosum (MCV) causes benign papules in healthy people but disfiguring skin lesions in immunocompromised patients. No effective treatment for MCV infection is presently available. MCV appears to be a good candidate for chemotherapy, because antiviral agents might be applied topically rather than systemically, thereby reducing possible toxic side effects. Bushman and colleagues proposes to carry out mechanistic and pharmacological studies of the topoisomerase enzyme encoded by MCV. Studies of poxviruses have revealed that the virus-encoded topoisomerase is required for replication, indicating that inhibitors of this enzyme are likely to obstruct viral growth. A DNA encoding MCV topoisomerase has been cloned and overexpressed, and assays have been established for activity in vitro. Mechanistic studies have centered on understanding DNA-protein contacts important for function and the domain structure of the enzyme. Pharmacological studies have yielded an initial panel of enzyme inhibitors. The PI proposes to carry out extensive studies of the enzyme function, structure, and pharmacology. They will investigate the mechanism of topoisomerase function in vitro, the domain organization of the enzyme, and the structure of domains and the full-length enzyme. They will investigate the function of the enzyme in vivo, and use the assays developed to characterize inhibitors. An extensive compound screening program will be carried out to identify inhibitory small molecules. It is postulated that the combination of screening and thorough mechanistic and structural studies offers the most promising long-term route to inhibitor development. In addition, poxviruses have been implicated as possible biological warfare agents; thus poxvirus topoisomerase inhibitors might be use for treating such infections
Keywords: DNA topoisomerase, Poxviridae, antiviral agent, drug design /synthesis /production, enzyme inhibitor drug screening /evaluation, enzyme structure, nucleic acid sequence, plasmid, protein structure function X ray crystallography
Project start date: 1999-08-01
Project end date: 2003-07-31
5R01AI046222-04 (2002): $228520
5R01AI046222-03 (2001): $221861
5R01AI046222-02 (2000): $215400
Frederic D Bushman
University Of Pennsylvania
Project start date: 2002-06-01
Project end date: 2013-01-31
Favored Sites For HIV CDNA Integration In The Human Genome
Frederic D Bushman, Professor
Microbiologyuniversity Of Pennsylvania
Grant 5R01AI052845-08 from National Institute Of Allergy And Infectious Diseases IRG: AMCB
Project start date: 2002-06-01
Project end date: 2013-01-31
LENTIVIRAL VECTOR INTEGRATION IN THE CLINIC
Frederic D Bushman, Professor
University Of Pennsylvania, 3451 Walnut Street, Philadelphia, Pa 19104
Grant 3U19AI066290-04S1_0002 from National Institute Of Allergy And Infectious Diseases
Keywords: AIDS; AIDS Virus; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immune Deficiency Syndrome Virus; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Acquired Immunodeficiency Syndrome Virus; Address; Body Tissues; Cells; Cellular Oncogene; Clinic; Cloning; Collaborations; Complement; Complement Proteins; DNA; Data; Deoxyribonucleic Acid; Enrollment; Event; Frequencies (time pattern); Frequency; Gene Transcription; Gene Transfer; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Genes; Genetic Intervention; Genetic Transcription; Genome, Human; Goals; HIV; HIV Infections; HTLV-III; HTLV-III Infections; HTLV-III-LAV Infections; Human; Human Genome; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human, General; Immunologic Deficiency Syndrome, Acquired; Infection; Intervention, Genetic; LAV-HTLV-III; Lentiviral Vector; Lentivirinae; Lentivirus; Lentivirus Vector; Location; Lymphadenopathy-Associated Virus; Man (Taxonomy); Man, Modern; Maps; Measures; Methods; Molecular Biology, Gene Therapy; Patients; Pattern; Population; Principal Investigator; Programs (PT); Programs [Publication Type]; Proto-Oncogenes; Protocol; Protocols documentation; RNA Expression; Rest; Retroviridae; Retroviruses; Safety; Sampling; Site; Subfamily lentivirinae; Survey Instrument; Surveys; T-Cells; T-Lymphocyte; T-Lymphotropic Virus Type III Infections, Human; Testing; Therapy, DNA; Thymus-Dependent Lymphocytes; Tissues; Transcription; Transcription, Genetic; Tumor Suppressor Proteins; Virus-HIV; Virus-Lenti; Virus-Retrovirus; base; c-ONC; cell transduction; cell type; cellular transduction; enroll; experiment; experimental research; experimental study; gene therapy; genetic therapy; genome sequencing; hazard; immuno-gene therapy; immunogene therapy; in vivo; lentiviral integration; lentivirus integration; preference; programs; protooncogene; research study; thymus derived lymphocyte; transduced cells; transfer of a gene; tumor suppressor; vector
Budget start date: 1-AUG-2009
Budget end date: 28-FEB-2010
PFA/PA: PAR-03-138
3U19AI066290-04S1_0002 (2009): $81019
Sponsored Links Excellgen http://Excellgen.com
MODIFYING HIV TO INTEGRATE AT PRESELECTED DNA SITES
Frederic D Bushman, Professor
Salk Institute For Biological Studies La Jolla, Ca 920371099
Grant 5R01AI037489-02 from National Institute Of Allergy And Infectious Diseases IRG: ARRC
Project start date: 1994-12-01
Project end date: 1996-11-30
5R01AI037489-02 (1996): $204693