Baek Kim
University Of Rochester
Project start date: 2009-01-01
Project end date: 2012-12-31
Sponsored Links Excellgen http://Excellgen.com
TARGETING PI3K/AKT CELL SURVIVAL PATHWAY AS A NOVEL ANTI-HIV THERAPY
Baek Kim, Assistant Professor
University Of Rochester, 518 Hylan Bldg., Box 270140, Rochester, Ny 14627
Grant 5R01AI077401-02 from National Institute Of Allergy And Infectious Diseases
Abstract: Persistent HIV-1 infection of tissue macrophage and CNS microglia can be observed throughout pathogenesis, and these cell types have been considered as key long-living HIV-1 reservoirs in the infected patients. We recently observed that human primary macrophages infected with M-tropic HIV-1 display a greatly elevated survival capability upon cellular insults. Considering the various cytotoxic environments caused by HIV-1 infection, which are well explained in the CNS model, it is a logical speculation that HIV-1 may have evolved to display self-protective responses in macrophage and microglia reservoirs against the HIV-1 induced cytotoxic insults and to achieve the long term survival of these HIV-1 infected cell types and persistent HIV-1 production. However, the viral factors responsible for this extended survival phenotype of HIV-1 infected non-dividing target cells and the cellular mechanisms involved in this long-term survival phenotype have never been envisioned. Due to the limited access of human primary microglia, we have recently established a human microglial cell line system that also displays the greatly enhanced survival capability upon HIV-1 infection. This model system enabled us to reveal both the mechanisms underlying HIV-1 induced cell survival and intracellular Tat as a key viral factor responsible for the cytoprotective phenotype of HIV-1. Our preliminary data also suggest that the well characterized PI3K/Akt survival pathway is mechanistically involved in the cytoprotective effect of HIV-1 infection and intracellular Tat expression. Using our model system as well as primary human macrophages, we identified several key host factors regulated by Tat expression, which will guide us in pinpointing the cytoprotective mechanism of action elicited by intracellular HIV-1 Tat. More interestingly, employing several PI3K/Akt inhibitors, which had been developed as anti-cancer agents, we revealed that the inhibition of the PI3K/Akt pathway can lead to the abolishment of the long-term survival phenotype of HIV-1 infected macrophages and eventually to the inhibition of HIV-1 production from the infected macrophages. In this proposal, we seek to 1) understand the cellular mechanisms and host/viral factors involved in the cytoprotective effect of HIV-1 infection in primary human macrophages and microglia, and 2) test the anti-HIV effect of the PI3K/Akt inhibitors by specifically eradicating macrophages and microglia HIV-1 reservoirs. This proposed work will shed light on understanding the molecular and cellular mechanisms involved in the long term survival of HIV-1 infected macrophage and microglia and the establishment of the HIV-1 reservoirs in the infected patients. The goal of the research is to develop new strategies to specifically eradicate the long- living HIV-1 reservoirs. Long term infection of HIV-1 in tissue macrophage and central nerve system reservoirs leads to persistent viral production and clinical complications such as HIV-1 associated dementia. Due to lack of antiviral therapy inhibiting viral production from long-living HIV-1 reservoirs that have been already infected, the viral spreading initiated from the HIV-1 reservoirs is currently beyond control. This application proposes to identify new antiviral strategies to specifically eradicate long-living HIV-1 reservoirs, which can minimize persistent viral production and infection in the HIV-1 infected patients
Keywords: AIDS; AIDS Dementia; AIDS Dementia Complex; AIDS Virus; AIDS with dementia; AIDS-related dementia; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immune Deficiency Syndrome related dementia; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Anti-Cancer Agents; Anti-HIV Therapy; Anti-Tumor Agents; Anti-Tumor Drugs; Antineoplastic Agents; Antineoplastic Drugs; Antineoplastics; Antiproliferative Agents; Antiproliferative Drugs; Antiviral Agents; Antiviral Drugs; Antiviral Therapy; Antivirals; Apoptotic; BZS; Biological Models; Body Tissues; Brain; CD4 Positive T Lymphocytes; CD4 T cells; CD4 lymphocyte; CD4+ T cell; CD4+ T-Lymphocyte; CD4-Positive Lymphocytes; Cancer Drug; Cancer Treatment; Cancers; Cell Communication; Cell Interaction; Cell Line; Cell Lines, Strains; Cell Survival; Cell Viability; Cell-to-Cell Interaction; CellLine; Cells; Cells, CD4; Central Nervous System; Chemotherapeutic Agents, Neoplastic Disease; Clinical; Data; Dementia Complex, AIDS-Related; Dementia Complex, Acquired Immune Deficiency Syndrome; Dementia Due to HIV Disease; Dementia associated with AIDS; Dementia in human immunodeficiency virus (HIV) disease; Development; Elements; Encephalon; Encephalons; Environment; Exposure to; Fatal Outcome; Goals; HIV Dementia; HIV associated dementia; HIV-1; HIV-1 associated dementia; HIV-1 dementia; HIV-Associated Cognitive Motor Complex; HIV-I; HIV-related dementia; HIV1; Health; Hortega cell; Host Factor; Host Factor Protein; Human; Human immunodeficiency virus 1; Human, General; Immunodeficiency Virus Type 1, Human; Immunologic Deficiency Syndrome, Acquired; Infection; Integration Host Factors; Killings; Lead; Length of Life; Life; Light; Longevity; MHAM; MMAC1; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Microglia; Model System; Modeling; Models, Biologic; Molecular; Nerve; Nervous; Nervous System, Brain; Nervous System, CNS; Neuraxis; Neuropathogenesis; Organ; Outcomes Research; PTEN; PTEN gene; PTEN1; Pathogenesis; Pathway interactions; Patients; Pb element; Phenotype; Phosphatase and Tensin Homolog; Photoradiation; Play; Production; Research; Research, Outcomes; Resistance; Role; Series; Stimulus; System; System, LOINC Axis 4; T4 Cells; T4 Lymphocytes; TAT; Testing; Therapeutic Agents; Time; Tissues; Trans-Activation of Transcription Protein; Trans-Activator of Transcription of HIV; Transactivating Regulatory Protein; Tumor-Specific Treatment Agents; Viral; Viral Diseases; Virus Diseases; Work; anti-cancer therapeutic; anticancer agent; anticancer drug; anticancer therapeutic; anticancer therapy; cancer therapy; cell type; chemotherapy; cultured cell line; cytotoxic; design; designing; experiment; experimental research; experimental study; genetic inhibitor; gitter cell; heavy metal Pb; heavy metal lead; helper T cell; human T cell leukemia virus III; human T lymphotropic virus III; inhibitor; inhibitor/antagonist; life span; lifespan; macrophage; malignancy; mesoglia; microglial cell; microgliocyte; neoplasm/cancer; novel; pathway; perivascular glial cell; public health relevance; research study; resistant; response; social role; tat Protein; therapy, AIDS anti-HIV; treatment of viral infectious disease; viral infection; viral resistance; virus infection
Relevance: Health Relevance Long term infection of HIV-1 in tissue macrophage and central nerve system reservoirs leads to persistent viral production and clinical complications such as HIV-1 associated dementia. Due to lack of antiviral therapy inhibiting viral production from long-living HIV-1 reservoirs that have been already infected, the viral spreading initiated from the HIV-1 reservoirs is currently beyond control. This application proposes to identify new antiviral strategies to specifically eradicate long-living HIV-1 reservoirs, which can minimize persistent viral production and infection in the HIV-1 infected patients
Project start date: 2009-01-01
Project end date: 2012-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2010
PFA/PA: PA-07-070
5R01AI077401-02 (2010): $311850
Grants awarded to Baek Kim
TESTING REVERSE TRANSCRIPTASE FIDELITY AS A HIV TARGET
Baek Kim
Microbiology And Immunologyuniversity Of Rochester
517 Hylan Bldg., Box 270140
rochester, Ny 14627
Grant 5R29GM055500-05 from National Institute Of General Medical Sciences IRG: ARRC
Abstract: This is a application to investigate the role of HIV RT fidelity in the genomic hypervariablity of HIV in response to selective pressures such as drug therapy. Presumably, it is the hypervariablity of HIV which leads to the emergence of drug-resistant strains. This genetic diversity has been hypothesized to result from error-prone replication by the viral polymerase. However, direct in vivo evidence relating the infidelity of HIV RT to genomic diversity is lacking. The investigator plans to pursue his investigation by three major objectives First, the investigator will use random mutagenesis of the Beta-9/Beta 10 palm region of the HIV RT followed by genetic selection in a bacterial-based genetic complimentation assay to isolate HIV RT fidelity mutants. The selected mutants, and a mutant of interest already generated from his preliminary work, will then be characterized to determine the extent to which replicational accuracy has changed. Mutants which exhibit a 5-fold change in fidelity will be considered for further study. Second, the investigator will construct proviral clones containing the HIV RTs with mutations of interest and determine the degree of genetic variability in the genome of the HIV species carrying these altered RTs. Third, the investigator plans to measure the capability of HIV with mutant RT genes characterized from the previous aims to escape from the selective pressure of HIV protease inhibitors
Keywords: RNA directed DNA polymerase, gene expression, human immunodeficiency virus, protease inhibitor, protein biosynthesis gene mutation, virus genetics
Project start date: 1997-07-01
Project end date: 2002-06-30
5R29GM055500-05 (2001): $113929
5R29GM055500-04 (2000): $111226
5R29GM055500-03 (1999): $108628
Targeting PI3K/Akt Cell Survival Pathway As A Novel Anti-HIV Therapy
Baek Kim, Associate Professor
Microbiology And Immunologyuniversity Of Rochester
Grant 1R01AI077401-01A2 from National Institute Of Allergy And Infectious Diseases IRG: ADDT
Abstract: Persistent HIV-1 infection of tissue macrophage and CNS microglia can be observed throughout pathogenesis, and these cell types have been considered as key long-living HIV-1 reservoirs in the infected patients. We recently observed that human primary macrophages infected with M-tropic HIV-1 display a greatly elevated survival capability upon cellular insults. Considering the various cytotoxic environments caused by HIV-1 infection, which are well explained in the CNS model, it is a logical speculation that HIV-1 may have evolved to display self-protective responses in macrophage and microglia reservoirs against the HIV-1 induced cytotoxic insults and to achieve the long term survival of these HIV-1 infected cell types and persistent HIV-1 production. However, the viral factors responsible for this extended survival phenotype of HIV-1 infected non-dividing target cells and the cellular mechanisms involved in this long-term survival phenotype have never been envisioned. Due to the limited access of human primary microglia, we have recently established a human microglial cell line system that also displays the greatly enhanced survival capability upon HIV-1 infection. This model system enabled us to reveal both the mechanisms underlying HIV-1 induced cell survival and intracellular Tat as a key viral factor responsible for the cytoprotective phenotype of HIV-1. Our preliminary data also suggest that the well characterized PI3K/Akt survival pathway is mechanistically involved in the cytoprotective effect of HIV-1 infection and intracellular Tat expression. Using our model system as well as primary human macrophages, we identified several key host factors regulated by Tat expression, which will guide us in pinpointing the cytoprotective mechanism of action elicited by intracellular HIV-1 Tat. More interestingly, employing several PI3K/Akt inhibitors, which had been developed as anti-cancer agents, we revealed that the inhibition of the PI3K/Akt pathway can lead to the abolishment of the long-term survival phenotype of HIV-1 infected macrophages and eventually to the inhibition of HIV-1 production from the infected macrophages. In this proposal, we seek to 1) understand the cellular mechanisms and host/viral factors involved in the cytoprotective effect of HIV-1 infection in primary human macrophages and microglia, and 2) test the anti-HIV effect of the PI3K/Akt inhibitors by specifically eradicating macrophages and microglia HIV-1 reservoirs. This proposed work will shed light on understanding the molecular and cellular mechanisms involved in the long term survival of HIV-1 infected macrophage and microglia and the establishment of the HIV-1 reservoirs in the infected patients. The goal of the research is to develop new strategies to specifically eradicate the long- living HIV-1 reservoirs. Long term infection of HIV-1 in tissue macrophage and central nerve system reservoirs leads to persistent viral production and clinical complications such as HIV-1 associated dementia. Due to lack of antiviral therapy inhibiting viral production from long-living HIV-1 reservoirs that have been already infected, the viral spreading initiated from the HIV-1 reservoirs is currently beyond control. This application proposes to identify new antiviral strategies to specifically eradicate long-living HIV-1 reservoirs, which can minimize persistent viral production and infection in the HIV-1 infected patients
Project start date: 2009-01-01
Project end date: 2012-12-31
NOVEL ONCOLYTIC ADENOVIRUS FOR PANCREATIC THERAPY
Baek Kim, Assistant Professor
University Of Rochester, 518 Hylan Bldg., Box 270140, Rochester, Ny 14627
Grant 5R21CA122213-02 from National Cancer Institute
Abstract: Pancreatic cancer accounts for approximately 2% of all new cancer cases in the United States, but almost 6% of cancer deaths. This reflects the very poor prognosis of this disease, and the current lack of effective therapies. As a consequence, there is a major need to explore new treatments, including gene therapy. Oncolytic, conditionally-replicating adenoviruses (CRADs) are presently being explored for their potential efficacy in the context of pancreatic cancer. The current generation of oncolytic CRADs are based on viruses with specific modifications in the E1 locus, including deletion of the E1B gene or transcriptionally-targeted, tumor-cell specific expression of the E1A gene. While initial clinical studies of oncolytic CRADs have shown that these vectors are generally safe and well tolerated, additional improvements will be necessary to make oncolytic CRADs more effective. In this proposal, we will generate new oncolytic CRADs using an entirely novel strategy. These adenovirus vectors will contain mutant DNA polymerases with a high functional dNTP requirement, and are expected to replicate selectively in tumor cells as a result. We anticipate that a powerful opportunity for synergy may exist, by combining our polymerase-based strategy with current E1-locus based oncolytic approaches - resulting in a new generation of CRADs that may have improved safety and efficacy. Overall, these experiments are expected to facilitate the development of novel approaches to the development of improved oncolytic adenovirus vectors for treatment of pancreatic cancer. It is hoped that these improvements will provide safer and more effective treatments for this deadly disease. Pancreatic cancer accounts for approximately 2% of all new cancer cases in the United States (32,180 in total, for 2005), but almost 6% of cancer deaths (31,800 in total, for 2005). Thus, pancreatic cancer (PC) is not only common, but it is also associated with a very poor prognosis. This makes PC an important potential target for new treatment approaches, including gene therapy. This application proposes to develop a novel gene therapy approach employing oncolytic adenovirus replicating specifically in cancer cells
Keywords: Accounting; Address; Adenoviral Vector; Adenoviridae; Adenovirus Vector; Adenoviruses; Animal Model; Animal Models and Related Studies; Athymic Nude Mouse; Biological Models; CRAd; Cancer cell line; Cancers; Cells; Cessation of life; Clinical Research; Clinical Study; Construction; DNA Polymerases; DNA-Dependent DNA Polymerases; DNA-Directed DNA Polymerase; Death; Deoxynucleoside-triphosphate[{..}]DNA deoxynucleotidyltransferase (DNA-directed); Deoxynucleotide-triphosphate[{..}]DNA deoxynucleotidyltransferase (RNA-directed); Development; Disease; Disorder; EC 2.7.7.49; EC 2.7.7.7; Environment; Est2 protein; Facility Construction Funding Category; Forecast of outcome; Gastrointestinal Tract, Pancreas; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Generalized Growth; Generations; Genes; Genes, p53; Genetic Alteration; Genetic Change; Genetic Intervention; Genetic defect; Growth; Human; Human, General; Immunocompetent; In Vitro; Intervention, Genetic; Lentiviral Vector; Lentivirus Vector; Malignant Cell; Malignant Neoplasms; Malignant Pancreatic Neoplasm; Malignant Tumor; Malignant neoplasm of pancreas; Man (Taxonomy); Man, Modern; Mice, Athymic; Mice, Nude; Model System; Models, Biologic; Modification; Molecular Biology, Gene Therapy; Mutate; Mutation; Normal Cell; Nude Mice; Numbers; Oncolytic; P53; Pancreas; Pancreas Cancer; Pancreas Neoplasms; Pancreatic; Pancreatic Cancer; Pancreatic Tumor; Polymerase; Prognosis; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); RNA Transcriptase; RNA-Dependent DNA Polymerase; RNA-Directed DNA Polymerase; Rate; Reverse Transcriptase; Revertase; Safety; Specificity; Standards; Standards of Weights and Measures; TERT protein; TP53; TP53 gene; TRP53; Therapy, DNA; Tissue Growth; Transcription Regulation; Transcriptional Control; Transcriptional Regulation; Tumor Cell; Tumor Protein p53 Gene; Tumor of the Pancreas; United States; Viral; Virus; Virus Replication; Viruses, General; adeno vector; adenovector; base; cancer cell; conditionally replicating adenovirus; conditionally replicative adenovirus; disease/disorder; ever shorter teleomeres protein 2; experiment; experimental research; experimental study; gene therapy; genetic therapy; genome mutation; improved; interest; malignancy; model organism; mutant; neoplasm/cancer; neoplastic cell; new approaches; novel; novel approaches; novel strategies; novel strategy; ontogeny; outcome forecast; pancreatic neoplasm; research study; telomerase catalytic subunit; telomerase reverse transcriptase; telomerase reverse transcriptase catalytic subunit; therapeutic gene; tumor; vector; virus multiplication
Project start date: 2007-04-03
Project end date: 2010-03-31
Budget start date: 1-APR-2008
Budget end date: 31-MAR-2010
PFA/PA: PA-06-303
5R21CA122213-02 (2008): $0
1R21CA122213-01A1 (2007): $123200
HIV-1 Vpr Induced DNTP Biosynthesis And DNA Gap Repair
Baek Kim
University Of Rochester 517 Hylan Bldg., Box 270140 Rochester, Ny 14627
Grant 5R21AI058774-02 from National Institute Of Allergy And Infectious Diseases IRG: AMCB
Abstract: HIV-1 viral protein R (Vpr) has been shown to induce cell cycle arrest, and this has been suggested to play an important to role in viral replication and pathogenesis. Our recent studies have revealed that Vpr activates ATR, suggesting that it may tigger cell cycle arrest as a consequence of the induction of the DNA damage pathway. In yeast, the ATR homolog Mec1 has been shown to not only regulate cell cycle progression, but also to control the biosynthesis of cellular dNTP in response to DNA damage, by modulating the activity of ribonucleotide reductase. In preliminary experiments, we have found that cellular dNTP levels also increase upon DNA damage in several human cell lines. This presumably allows repair DNA polymerases, which are also induced by DNA damage, to execute efficient DNA gap repair. Numerous studies have demonstrated that non-dividing cells contain lower dNTP levels than actively dividing cells. However, the dNTP level of human macrophages has not been determined, due to the lack of a highly sensitive dNTP assay. Recently, we established an enzymatic dNTP assay that has allowed the determination of dNTP levels in human macrophages. This analysis revealed that human macrophages contain much a lower intracellular concentration of dNTPs (20 approximately 40nM) than either activated or resting T cells (approximately 2 mu M). In fact, the dNTP concentration in human macrophages is considerably lower than the known dNTP binding affinity of the host DNA polymerases that execute the repair of the 5-nucleotide DNA gaps generated during HIV-1 proviral DNA integration. This suggests that dNTP availability may represent a rate-limiting step in HIV- 1 infection of macrophages. In this proposal, we hypothesize that, like DNA damage, HIV-1 Vpr elevates cellular dNTP levels, and that this has a stimulatory effect on HIV- 1 replication in macrophages, which normally contain very low levels of dNTPs. To test this hypothesis, we will examine the effect of HIV-1 Vpr on cellular dNTP levels and HIV-1 gap repair activity in macrophages; we will also directly test whether the elevation of cellular dNTP levels by deoxynucleosides (dNs) results in an enhancement of the proviral DNA integration and proviral DNA synthesis kinetics of HIV-1 in macrophages. We will also identify host repair proteins essential for Vpr to alter dNTP biosynthesis and gap repair. This study will 1) elucidate a mechanism that HIV-1 employs to achieve its unique ability to infect non-dividing cells and 2) identify host repair proteins that may be potential antiviral targets.
Keywords: DNA repair, biosynthesis, deoxyribonucleoside, human immunodeficiency virus 1, virus protein, HIV infection, T lymphocyte, enzyme activity, green fluorescent protein, macrophage, virus infection mechanism, HeLa cell, clinical research, enzyme linked immunosorbent assay, flow cytometry, human tissue, polymerase chain reaction, tissue /cell culture, western blotting
Project start date: 2004-04-01
Project end date: 2006-11-30
5R21AI058774-02 (2005): $236250
1R21AI058774-01A1 (2004): $236250
Replication Fidelity And Lentiviral Pathogenesis
Baek Kim
University Of Rochester 517 Hylan Bldg., Box 270140 Rochester, Ny 14627
Grant 5R01AI049781-04 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Biochemical studies have demonstrated that lentiviral RTs are highly error prone DNA polymerases, and their infidelity is a presumptive source of viral genomic hypervariability which allows viral escape from host anti-viral immune pressure. The hypothesis that we test in this proposal is that if fidelity of viral RTs is a genetic determinant of viral diversity essential for viral escape, then RT fidelity may also change during the course of viral infection. We have found that one simian immunodeficiency virus (SIV) RT allele, obtained from a pig- tailed macaque during the late symptomatic phase of infection (SIVMNE170), exhibits a greatly elevated level of fidelity, when compared to a RT allele isolated from the same animal at the initial infection stage (SIVMNECL8). This finding supports our hypothesis that viral RT fidelity may change in response to alterations in external selective pressure (i.e. changes in host immune function). Recently, we also found that RTs of SIVMNE35 and 84 from asymptomatic phase of infection has low fidelity, like the CL8 RT. This further supports our prediction that RT fidelity remains low when the host has relatively intact immune capability. Indeed, this 170 RT is the first in vivo high fidelity (hi-fi) RT isolated from natural course of viral infection. We also identified RT variants containing mutations that decrease fidelity at the early infection phase, supporting our hypothesis that RT fidelity changes over time. In this proposed work, we will delineate the evolutionary relationship between RT infidelity, host anti-viral immune capability and disease progression. First, we will systematically determine the fidelity of RT derivatives obtained from representative clones of SIVMNECL8 and SIVmac239 strains isolated at four different stages of viral infection. Secondly, we will use the in vivo altered fidelity RT mutants to understand novel structural and mechanistic determinants of RT infidelity. These proposed studies will enhance our knowledge of the genetic role of RT in the evolution, host immune selection and pathogenesis of immunodeficiency viruses.
Keywords: Lentivirus, RNA directed DNA polymerase, biochemical evolution, gene mutation, host organism interaction, simian immunodeficiency virus, virus cytopathogenic effect, virus genetics, cytotoxic T lymphocyte, pathologic process, virus replication, Macaca mulatta, molecular cloning
Project start date: 2002-05-15
Project end date: 2006-12-14
5R01AI049781-04 (2005): $275625
5R01AI049781-03 (2004): $275625
Sponsored Links Excellgen http://Excellgen.com
5R01AI049781-02 (2003): $275625
Replicational Fidelity And Lentiviral Pathogenesis
Baek Kim
University Of Rochester 517 Hylan Bldg., Box 270140 Rochester, Ny 14627
Grant 2R01AI049781-05A2 from National Institute Of Allergy And Infectious Diseases IRG: AMCB
Abstract: Among retroviruses, lentiviruses such as HIV-1 display two distinct virological features genomic hypervariation and infection of nondividing/terminally differentiated cells (i.e. macrophage). These traits contribute to viral evolution and pathogenesis, respectively. Our recent research suggests that these two unique virological characteristics of HIV-1 are mechanistically related and require a novel enzymatic property of the viral reverse transcriptase (RT) high binding affinity to dNTP substrates. During the previous funding period of this award, we identified a unique enzymatic property of HIV-1 RT, compared to MuLV RT. Indeed, we found that HIV-1 RT binds to the dNTP substrate with much higher affinity than MuLV RT. Interestingly, due to this unique dNTP binding property, HIV-1 RT efficiently catalyzes both DNA synthesis and mismatch extension (which contributes to its low enzyme fidelity), even at low dNTP concentration. We also found that two HIV-1 RT mutants with reduced dNTP binding affinity, V148I and Q151N, show reduced DNA synthesis efficiency at low dNTP concentrations and increased fidelity. Indeed, HIV-1 variants harboring these mutant RTs failed to infect macrophage containing low dNTP concentrations even though these viruses retain their ability to infect dividing cells containing high dNTP concentrations. In addition, due to their low dNTP binding affinity, these mutants display frequent stalling and elevated RNA template switching even at physiological dNTP concentrations. In fact, the Q151N mutant virus showed the highest viral recombination frequency among all RT mutants ever tested. The central hypothesis of this proposal is that the relatively high dNTP binding affinity of lentiviral RTs may be essential for two of the most important (and seemingly unrelated) features of the lentivirus family their abilities to infect terminally differentiated cells and to undergo high rates of genomic mutation. First, we will perform a series of biochemical and virological experiments to elucidate the unique structural and mechanistic architecture of the HIV-1 RT active site, which is responsible for its high dNTP binding affinity. In this aim, both currently available HIV-1 RT mutants with altered dNTP binding affinity and in vivo HIV-1 RT variants that have been isolated from the HIV-1 infected patients will be employed. Second, we will test whether mutations of HIV-1 RT reducing dNTP binding affinity also reduces proviral DNA synthesis kinetics at low cellular dNTP concentrations, leading to loss of viral infectivity to macrophages. Third, we will test the effect of dNTP binding affinity of HIV-1 RT on the viral recombination frequency. This proposed work is expected to provide insight into the impact of HIV-1 RT on a range of key biological features of HIV- 1 cell tropism, genomic mutability, recombination and pathogenesis.
Project start date: 2000-12-01
Project end date: 2011-11-30
2R01AI049781-05A2 (2007): $351000
TESTING REVERSE TRANSCRIPTASE FIDELITY AS A HIV TARGET
Baek Kim
University Of Washington Office Of Sponsored Programs Seattle, Wa 98105
Grant 1R29GM055500-01A1 from National Institute Of General Medical Sciences IRG: ARRC
Abstract: Adapted from Investigator s ) This is a application to investigate the role of HIV RT fidelity in the genomic hypervariablity of HIV in response to selective pressures such as drug therapy. Presumably, it is the hypervariablity of HIV which leads to the emergence of drug-resistant strains. This genetic diversity has been hypothesized to result from error-prone replication by the viral polymerase. However, direct in vivo evidence relating the infidelity of HIV RT to genomic diversity is lacking. The investigator plans to pursue his investigation by three major objectives First, the investigator will use random mutagenesis of the Beta-9/Beta 10 palm region of the HIV RT followed by genetic selection in a bacterial-based genetic complimentation assay to isolate HIV RT fidelity mutants. The selected mutants, and a mutant of interest already generated from his preliminary work, will then be characterized to determine the extent to which replicational accuracy has changed. Mutants which exhibit a 5-fold change in fidelity will be considered for further study. Second, the investigator will construct proviral clones containing the HIV RTs with mutations of interest and determine the degree of genetic variability in the genome of the HIV species carrying these altered RTs. Third, the investigator plans to measure the capability of HIV with mutant RT genes characterized from the previous aims to escape from the selective pressure of HIV protease inhibitors.
Keywords: RNA directed DNA polymerase, gene expression, human immunodeficiency virus, protease inhibitor, protein biosynthesis, gene mutation, virus genetics
Project start date: 1997-07-01
Project end date: 1998-06-30
1R29GM055500-01A1 (1997): $92216
7R29GM055500-02 (1998): $106128
Baek Kim
University Of Rochester
Project start date: 2000-12-01
Project end date: 2017-03-31
REPLICATIONAL FIDELITY AND LENTIVIRAL PATHOGENESIS
Baek Kim, Assistant Professor
University Of Rochester, 518 Hylan Bldg., Box 270140, Rochester, Ny 14627
Grant 5R01AI049781-08 from National Institute Of Allergy And Infectious Diseases
Keywords: AIDS Virus; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Active Sites; Affinity; Architecture; Assay; Award; Binding; Binding (Molecular Function); Bioassay; Biochemical; Biologic Assays; Biological; Biological Assay; CD4 Positive T Lymphocytes; CD4 T cells; CD4 lymphocyte; CD4+ T cell; CD4+ T-Lymphocyte; CD4-Positive Lymphocytes; Cells; Cells, CD4; Characteristics; DNA; DNA Recombination; DNA Replication; DNA Synthesis; DNA biosynthesis; DNA recombination (naturally occurring); Deoxynucleotide-triphosphate[{..}]DNA deoxynucleotidyltransferase (RNA-directed); Deoxyribonucleic Acid; EC 2.7.7.49; Engineering / Architecture; Environment; Enzymes; Evolution; Exhibits; Family; Frequencies (time pattern); Frequency; Funding; Gene Products, RNA; Genetic Alteration; Genetic Change; Genetic Recombination; Genetic defect; Genetics-Mutagenesis; Genomics; HIV; HIV-1; HIV-I; HIV1; 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; Human immunodeficiency virus 1; Immunodeficiency Virus Type 1, Human; Infection; Interphase Cell; Investigators; Kinetic; Kinetics; LAV-HTLV-III; Lentivirinae; Lentivirus; Lymphadenopathy-Associated Virus; Molecular Biology, Mutagenesis; Molecular Interaction; Mutagenesis; Mutation; Nature; Non-dividing Cell; Pathogenesis; Patients; Physiologic; Physiological; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; RNA; RNA Transcriptase; RNA, Non-Polyadenylated; RNA-Dependent DNA Polymerase; RNA-Directed DNA Polymerase; Recombination; Recombination, Genetic; Reporting; Research; Research Personnel; Researchers; Resting Cell; Retroviridae; Retroviruses; Reverse Transcriptase; Reverse Transcription; Revertase; Ribonucleic Acid; Series; Sites, Active; Subfamily lentivirinae; T4 Cells; T4 Lymphocytes; Testing; Time; Tropism; Variant; Variation; Viral; Viral Pathogenesis; Virus; Virus-HIV; Virus-Lenti; Virus-Retrovirus; Viruses, General; Work; base; design; designing; experiment; experimental research; experimental study; genome mutation; helper T cell; human T cell leukemia virus III; human T lymphotropic virus III; in vivo; insight; macrophage; mutant; novel; polymerization; programs; research study; trait
Project start date: 2000-12-01
Project end date: 2011-11-30
Budget start date: 1-DEC-2009
Budget end date: 30-NOV-2010
5R01AI049781-08 (2010): $309899