2009 — 2010 |
Campbell, Edward M |
K22Activity Code Description: To provide support to outstanding newly trained basic or clinical investigators to develop their independent research skills through a two phase program; an initial period involving and intramural appointment at the NIH and a final period of support at an extramural institution. The award is intended to facilitate the establishment of a record of independent research by the investigator in order to sustain or promote a successful research career. |
Analysis of Restriction Factor Function in Situ @ Loyola University Chicago
DESCRIPTION (provided by applicant): TRIM5? is a cytoplasmic restriction factor that inhibits infection by retroviruses by other species. Specifically, TRIM5? proteins from rhesus monkeys can inhibit infection by HIV-1, although the mechanism by which this inhibition occurs remains poorly understood. This application seeks to examine the interaction between retroviruses and cytoplasmic restriction factors microscopically. The research will expand upon previously developed fluorescent microscopy methods that allow the identification of HIV-1 virions that have productively entered the target cell cytoplasm. Using this system, we have shown an interaction between cytoplasmic HIV-1 virions and TRIM5? protein from rhesus macaque during restriction. This work will examine the interaction between retroviruses, including HIV-1 and MLV, and restriction factors, including TRIM5? and Fv-1, to more precisely understand the events leading to retroviral restriction. This will be accomplished by examining the ability of relevant TRIM5? mutants to interact with cytoplasmic virions and restrict infection. As the interaction between restriction factors and retroviruses occurs between individual mature capsid cores and cytoplasmic restriction factors, this interaction has proven very difficult to examine biochemically. Direct observation of this interaction microscopically therefore represents an extremely promising method by which to examine this process. A better understanding of how restriction factors inhibit infection by retroviruses could lead to the development of pharmaceuticals designed to intervene at this currently unexploited step in the viral life cycle. Lay language description: Recently identified primate proteins called restriction factors have recently been found to inhibit infection by viruses native to other species. The TRIM5? protein from rhesus monkeys has recently been identified as being able to inhibit infection by HIV-1. This research application seeks to better understand the mechanism by which this restriction occurs with the hope that this understanding leads to better treatment of people infected with HIV-1 or methods to prevent the spread of the virus.
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2011 — 2012 |
Campbell, Edward M |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Generating Reagents to Explore the Anitviral Potential of Trim Family Proteins @ Loyola University Chicago
DESCRIPTION (provided by applicant):The TRIM family of proteins mediates a number of recently reported antiviral activities mediated against diverse viruses. The best studied of these activities is the ability of the TRIM51 protein to inhibit the replication of certain retroviruses, including HIV-1, by binding determinants present on the viral capsid core during early stages of infection. Recent data suggests that other TRIM family proteins can affect HIV-1 replication at other steps in the viral replication cycle. Numerous studies suggest that TRIM family proteins can also negatively regulate the replication of a diverse range of viruses, including adenoviruses, herpesviruses and influenza viruses, although these activities remain poorly characterized. The recent finding that the expression of many TRIM family proteins are upregulated in response to interferon treatment also indicates that the TRIM family proteins represent an intracellular element of the innate immune system, acting to inhibit the replication of intracellular pathogens. Because of the tendency of TRIM51 to multimerize into high-order structures in cells, biochemical analysis of this protein has been unusually difficult. This will likely be true of other TRIM family members. My previous studies have utilized quantitative fluorescent microscopy to visualize and quantify the interactions occurring between TRIM51 and HIV-1 viral complexes in the cytoplasm. This project would develop retroviral vectors that induce the expression of epitope tagged TRIM family members and use these vectors to generate cell lines stably expressing these proteins. These vectors and cell lines will be useful in identifying TRIM proteins with antiviral activity against diverse viruses and expose novel interactions occurring in cells between viruses and TRIM family proteins. Once identified, these same vectors and cell lines will allow for the visualization of interactions occurring between TRIM proteins and viral proteins and/or genetic elements affected by TRIM activity. This will catalyze new areas of research for my lab and increase our understanding of the antiviral activity of TRIM family proteins.
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2011 — 2015 |
Campbell, Edward M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Cell Biology of Trim5alpha @ Loyola University Chicago
DESCRIPTION (provided by applicant): This application will seek to better understand the mechanism by which the TRIM5alpha protein from rhesus macaques (rhTRIM5alpha) inhibits HIV-1 infection by defining the molecular and cellular biology of rhTRIM5alpha during restriction. We have identified a number of cellular proteins that associate with TRIM5alpha in cells. We have also developed powerful new methods to quantitatively characterize the association of TRIM5alpha with these cellular proteins. In the first aim, we will examine the connection between the ability of rhTRIM5alpha to self-associate and restrict retroviral infection. Specifically, we will characterize the effects of specific rhTRIM5alpha variants that have lost the ability to localize to cytoplasmic bodies due to specific mutations we have introduced into the linker2 region of the protein. We will identify the determinants in this region that mediate ability of rhTRIM5alpha to self-associate, measuring this ability with a novel, imaging based self-association assay we have recently developed. We will utilize the knowledge generated in these studies to develop rhTRIM5alpha variants with increased ability to restrict HIV-1 infection. In the second aim, we will define the role of the protein p62/sequestosome1 in regulating the degradation of TRIM5alpha. The known biological function of this protein, a scaffolding protein that mediates the shuttling of ubiquitylated cargo proteins to the proteasome, makes it an interesting candidate to play a role in the biology of TRIM5alpha mediated retroviral restriction. We will test the hypothesis that p62 conditionally regulates the ability of different cellular degradative pathways to degrade TRIM5alpha. In the third aim, we will use quantitative imaging methods to define the events that occur in cells when restriction sensitive virions enter the cytoplasm of target cells. By monitoring the presence of biologically relevant cellular proteins in TRIM5alpha cytoplasmic bodies during restriction, we will be able g restriction, we will be able to dissect and develop an understanding of this critical biological process. PUBLIC HEALTH RELEVANCE: Primates, including humans, possess proteins, such as TRIM5alpha that have evolved for millions of years to effectively combat viral infection. Understanding the mechanisms by which TRIM5alpha mediates its antiviral effects could allow this knowledge to be harnessed in the form of antiviral therapy for people infected with HIV-1
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2013 — 2014 |
Campbell, Edward M |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Exploring the Role of Microtubules in Hiv-1 Uncoating @ Loyola University Chicago
DESCRIPTION (provided by applicant): Following human immunodeficiency virus (HIV) membrane fusion with the target cell membrane, a series of events occur to establish an infection. Specifically, early in the life cycle, the virus must reverse transcribe its RNA genome and induce the transport of this genome to the nuclear envelope for subsequent nuclear import and integration. At some point prior to the nuclear import of the viral genome, the virus undergoes the poorly understood process of uncoating, defined as the disassembly of the assembled capsid structure from the viral ribonucleoprotein complex. However, the precise mechanism of uncoating remains elusive. The foundation of this application is the hypothesis that uncoating is influenced by the interaction between the mature, assembled viral capsid lattice and microtubule motor proteins and/or microtubule- associated proteins (MAPs). We provide evidence that MT disruption inhibits HIV-1 uncoating and propose to utilize assays to measure uncoating, live cell imaging assays and capsid binding assays to identify the MAPs which are involved in facilitating HIV-1 uncoating.
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2015 — 2019 |
Campbell, Edward M Diaz-Griffero, Felipe (co-PI) [⬀] Pornillos, Owen (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Defining the Microtubule Motors Which Drive the Uncoating and Trafficking of Hiv @ Loyola University Chicago
? DESCRIPTION (provided by applicant): HIV-1 uncoating is a poorly understood field and strengthening the idea behind this mechanism will add a strong base to the viral life cycle and interesting targets for antiviral therapy. Our previous work on the role of microtubules during HIV-1 uncoating has provided valuable information to the field of trafficking and HIV-1 uncoating. Our preliminary work supports a strong dependency of several microtubule associated proteins (MAPs) during HIV-1 uncoating and involvement of a variety of other host factors in this process. With this, we propose three specific aims to explore the role of these MAPs during HIV-1 uncoating. Our first aim would be to decipher a mechanism by which these MAPs control viral uncoating. In the second aim, we would further characterize how the viral core trafficking is controlled by these MAPs and in the last aim, we would define the direct interactions between MAPs and the viral core which contribute to HIV-1 uncoating and trafficking. Our expertise and collaborations would help achieve these specific aims and show how the microtubule trafficking machinery plays an important role during HIV-1 uncoating. Findings from these proposed aims will provide critical understanding to the molecular interactions which drive HIV-1 uncoating and trafficking and would help identify better candidates for future antiviral therapy.
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2016 |
Campbell, Edward M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular and Cellular Determinants of Trim5alpha Restriction of Hiv-1 @ Loyola University Chicago
Abstract TRIM5? is a restriction factor which targets the retroviral capsid during infection which inhibits infection by inducing the abortive disassembly of the viral capsid core. The mechanism by which this occurs is poorly understood. We provide data demonstrating that dynamic conformational changes in TRIM5? correlate with the ability to inhibit viral infection, and propose to better define these conformational changes to understand the molecular interactions that drive capsid disassembly. In aim 1, we will define the conformational changes that occur in rhesus TRIM5?, human TRIM5?, and a panel of naturally occurring and structurally guided mutations to define the molecular interactions that drive these conformational changes in the context of the recombinant TRIM5? dimeric unit comprising the coiled coil domain and linker 2 region. In Aim 2, we will expand these studies to define how these conformational changes translate to neighboring domains, including the capsid binding SPRY domain of TRIM5?, and also determine how SPRY binding to assembled CA influences these conformational changes. In aim 3, we propose functional validation of the results obtained in the first 2 aims in experiments which will determine how the biophysical and biochemical proteins translate to the ability to perform the individual, measurable steps in the restriction process.
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2017 — 2019 |
Campbell, Edward M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular and Cellular Determits of Trim5alpha Restriction of Hiv-1 @ Loyola University Chicago
Abstract TRIM5? is a restriction factor which targets the retroviral capsid during infection which inhibits infection by inducing the abortive disassembly of the viral capsid core. The mechanism by which this occurs is poorly understood. We provide data demonstrating that dynamic conformational changes in TRIM5? correlate with the ability to inhibit viral infection, and propose to better define these conformational changes to understand the molecular interactions that drive capsid disassembly. In aim 1, we will define the conformational changes that occur in rhesus TRIM5?, human TRIM5?, and a panel of naturally occurring and structurally guided mutations to define the molecular interactions that drive these conformational changes in the context of the recombinant TRIM5? dimeric unit comprising the coiled coil domain and linker 2 region. In Aim 2, we will expand these studies to define how these conformational changes translate to neighboring domains, including the capsid binding SPRY domain of TRIM5?, and also determine how SPRY binding to assembled CA influences these conformational changes. In aim 3, we propose functional validation of the results obtained in the first 2 aims in experiments which will determine how the biophysical and biochemical proteins translate to the ability to perform the individual, measurable steps in the restriction process.
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2017 — 2019 |
Campbell, Edward M |
T35Activity Code Description: To provide individuals with research training during off-quarters or summer periods to encourage research careers and/or research in areas of national need. |
Summer Research Experience For Medical Students in Inflammation and Infectious Diseases @ Loyola University Chicago
PROJECT SUMMARY We propose to introduce early stage medical students to the challenges and excitement of research by providing a mentored summer research experience with the ultimate goal of inspiring trainees toward academic careers that incorporate basic biomedical and clinical research. The T35 Research Training in Immunology and Infectious Diseases (REIID) program will exist as a dedicated, NIAID-focused research and career development program that leverages the successful administrative framework of the Student Training In Approaches to Research (STAR) Program at Loyola University Chicago Stritch School of Medicine. The T35- REIID program will provide students between their first and second year of medical school with a focused eight-week mentored training experience pursuing NIAID-related research resulting in completion of a research project and formal presentation of their work. The Stritch School of Medicine is a rich academic environment and a leading institution in immunology and infectious disease-related research and clinical care. We have assembled a team of twenty-four experienced, extramurally funded mentors whose research covers epidemiology; genetics; molecular, cellular and pathophysiological mechanisms of disease; translational research; and patient-oriented clinical research. The student?s full-time research training will be supplemented with didactic conferences on topics including experimental design, data management, responsible conduct of research, professional development, and presentation skills. Trainees will be chosen through a competitive process based on their proposed research plan and goals for the summer experience. The T35-REIID program includes student and mentor assessments to evaluate the quality of the research experience and didactic offerings as well as our success at encouraging students to pursue academic careers that include research.
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2018 — 2019 |
Campbell, Edward M |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Viral and Cellular Determits of Hiv-1 Nuclear Import Kinetics @ Loyola University Chicago
Abstract The ability to infect non-dividing cells is what separates lentiviruses, including HIV-1, from other retroviruses which require the breakdown of the nuclear envelope during cell division to access the genome of target cells. Lentiviruses, in contrast, have evolved the ability to selectively translocate their genome across the nuclear pore complex (NPC). Unfortunately, the mechanism by which HIV-1 translocates through the NPC remains one of the least well understood aspect of the viral lifecycle, in large part because there is no direct, definitive method to monitor HIV-1 nuclear import. In this application, we describe a method to monitor HIV-1 nuclear import kinetics directly through the ability to selectively and potently inhibit HIV-1 nuclear import at various times post-infection. This method prevents infection of any viruses which have not yet traversed the NPC and entered the nucleus, and thereby allows us to directly monitor the kinetics of nuclear import by measuring the fraction of the viral inoculum which has cleared the NPC at times following a synchronized infection. As such, this assay provides a unique opportunity to appreciate nuclear translocation of the viral genome in target cells, including primary cells, and to determine how specific viral determinants and cellular factors influence the nuclear translocation of the viral genome.
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2019 — 2021 |
Campbell, Edward M Gallagher, Thomas Miller (co-PI) [⬀] Shepherd, Jason D [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Virus-Like Intercellular Communication in the Nervous System
Abstract Recent studies by our group have revealed that the neuronal gene Arc, a master regulator of synaptic plasticity and information storage in the brain, acts as a repurposed retroviral Gag protein that forms capsids with the capacity to transmit genetic information between cells. These findings lead to a paradigm shift in the way we view both mechanisms of cognition and more generally how cells can signal to each other. This transformative R01 application will address these questions using a synergistic team of neuroscientists and virologists who will apply their expertise to Arc, intercellular gene transmission, and neuronal development. We will determine what genetic messages are transferred between neurons in Arc particles, how these particles enter ?target? neurons to deliver their RNA cargo to cell cytoplasm, and how delivery of this cargo influences the neuronal and synaptic processes that underlie memory and cognition. The methodologies to address these questions, as well as the potential impact of the answers, make this application ideally suited to the transformative R01 mechanism.
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0.957 |
2021 |
Campbell, Edward M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Defining the Nuclear Import Pathways of Hiv-1 @ Loyola University Chicago
Abstract Human Immunodeficiency virus (HIV-1), like all primate lentiviruses possesses the ability to infect non-dividing cells by engaging with components of the nuclear pore complex and mediating the nuclear translocation of the viral ribonucleoprotein complex (RNP) for subsequently integration into the host cell genome. The viral capsid protein (CA) interacts with numerous host factors involving constituents of the nuclear pore complex (NPC) to accomplish the process of nuclear import. Unfortunately, the exact mechanism by which HIV-1 translocates through the nuclear pore complex remains one of the least understood steps of the viral life cycle. In addition, recent evidences supporting the notion of NPCs being more heterogeneous than previously thought further confounds this situation. We have developed an inducible nuclear pore blockade that allows the rate of nuclear import of functional, infectious viral genomes to be monitored in any relevant cells types. Using this technique, we observe that certain CA mutants are insensitive to a Nup62 mediated nuclear pore blockade in cells which potently block infection by wild type CA, demonstrating that HIV-1 can utilize distinct nuclear import pathways during infection. In this application, we will determine the degree to which NPC are heterogeneous and map the specific nuclear pore constituents that makeup the NPCs utilized by HIV-1 during nuclear entry. With our nuclear pore blockade, we now have the capability to block NPCs using different nuclear pore constituents. As such, this application aims to define and visualize the specific nuclear pore constituents that interact and mediate the nuclear import of HIV-1 and how specific NPC usage affects viral integration in target cells. Collectively this application would close critical gaps to our understanding in to the nuclear import of HIV-1.
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2021 |
Campbell, Edward M |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Tissue Specific Detection of Inflammation in Vivo. @ Loyola University Chicago
Abstract The development and resolution of inflammation is central to the pathogenesis of numerous human diseases. This is true of bacterial infections, in which host inflammatory responses promote pathogen clearance, as well as inflammatory disorders, such as colitis, in which chronic inflammatory responses drive pathogenesis in local tissue environments. In both cases, a critical mediator of cellular inflammatory responses which drive tissue inflammation is the inflammasome, a multiprotein complex, which leads to the activation of caspase-1 and the cleavage and release of inflammatory mediators, such as IL-1?. To monitor cellular inflammatory responses in vivo, we have developed caspase-1 biosensors that allow the detection of inflammatory responses in the context of mouse models of bacterial infection (S. aureus) and colitis. In this application, we propose to develop mice expressing this novel biosensor in a tissue specific fashion. This mouse model will allow us to define the cell types driving inflammation in the context of these established disease models as well as develop an animal model which will be a valuable tool to monitor tissue specific inflammatory responses in the context of numerous mouse models of human disease.
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