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In JoVE (1)
Other Publications (5)
Articles by Joel W. Graff in JoVE
In vivo Imaging of Transgenic Leishmania Parasites in a Live Host
Colin J. Thalhofer1, Joel W. Graff2, Laurie Love-Homan3, Suzanne M. Hickerson4, Noah Craft5, Stephen M. Beverley4, Mary E. Wilson6,7
1Interdisciplinary Immunology Program, University of Iowa, and the VA Medical Center, 2Department of Biochemistry, University of Iowa, and the VA Medical Center, 3Department of Internal Medicine, University of Iowa, 4Department of Molecular Microbiology, Washington University School of Medicine, 5Division of Dermatology, Harbor-UCLA Medical Center, Hanley-Hardison Research Center, 6Interdisciplinary Immunology Program, Iowa City VA Medical Center, 7Departments of Internal Medicine, Microbiology and Epidemiology, University of Iowa
An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.
Other articles by Joel W. Graff on PubMed
Journal of Virology. Sep, 2002 | Pubmed ID: 12186937
The rotavirus nonstructural protein NSP1 is the least conserved protein in the rotavirus genome, and its function in the replication cycle is not known. We employed NSP1 as bait in the yeast two-hybrid interaction trap to identify candidate cellular partners of NSP1 that may provide clues to its function. Interferon regulatory factor 3 (IRF-3) was identified as an NSP1 interactor. NSP1 synthesized in rotavirus-infected cells bound IRF-3 in a glutathione S-transferase pull-down assay, indicating that the interaction was not unique to the two-hybrid system. NSP1 of murine rotavirus strain EW also interacted with IRF-3. NSP1 deletion and point mutants were constructed to map domains important in the interaction between NSP1 and IRF-3. The data suggest that a binding domain resides in the C terminus of NSP1 and that the N-terminal conserved zinc finger is important but not sufficient to mediate binding to IRF-3. We predict that a role for NSP1 in rotavirus-infected cells is to inhibit activation of IRF-3 and diminish the cellular interferon response.
Epitopes in the P2 Domain of Norovirus VP1 Recognized by Monoclonal Antibodies That Block Cell Interactions
The Journal of General Virology. Oct, 2005 | Pubmed ID: 16186235
Noroviruses cause the majority of epidemic outbreaks of acute viral gastroenteritis worldwide. Human norovirus strains do not grow in cell culture, but recent carbohydrate binding, sequence and structural analyses have begun to define functional domains in the norovirus capsid that may be conserved among multiple antigenic types. The purpose of this study was to localize domains and define sequences in the major capsid protein VP1 that are important for cell interactions. Monoclonal antibodies to genogroups GI.1 and GII.2 reference strains Norwalk virus and Snow Mountain virus, respectively, were generated that blocked binding of recombinant virus-like particles to Caco-2 intestinal cells and inhibited haemagglutination. Peptides that mimicked the mAb binding epitopes were selected from a phage-displayed random nonapeptide library. Anti-recombinant Norwalk virus mAb 54.6 and anti-recombinant Snow Mountain virus mAb 61.21 recognized epitopes located in the protruding P2 domain of VP1. The epitope recognized by mAb 61.21 contained amino acids that are completely conserved among norovirus strains across genogroups, including strains isolated from swine, bovine and murine species. This study identifies the first epitope involved in inhibition of norovirus-cell interactions and supports increasing evidence that interactions between noroviruses and host cells rely on structures in the P2 domain of VP1.
Natural Products That Reduce Rotavirus Infectivity Identified by a Cell-based Moderate-throughput Screening Assay
Virology Journal. 2006 | Pubmed ID: 16948846
There is widespread interest in the use of innate immune modulators as a defense strategy against infectious pathogens. Using rotavirus as a model system, we developed a cell-based, moderate-throughput screening (MTS) assay to identify compounds that reduce rotavirus infectivity in vitro, toward a long-term goal of discovering immunomodulatory agents that enhance innate responses to viral infection.
Zinc-binding Domain of Rotavirus NSP1 is Required for Proteasome-dependent Degradation of IRF3 and Autoregulatory NSP1 Stability
The Journal of General Virology. Feb, 2007 | Pubmed ID: 17251580
Interferon regulatory factor 3 (IRF3) is a key transcription factor involved in the induction of interferon (IFN) in response to viral infection. Rotavirus non-structural protein NSP1 binds to and targets IRF3 for proteasome degradation early post-infection. Mutational analysis of cysteine and histidine residues within the conserved N-terminal zinc-binding domain in NSP1 of bovine rotavirus strain B641 abolished IRF3 degradation in transfected cells. Thus, the integrity of the zinc-binding domain in NSP1 is important for degradation of IRF3. In contrast to bovine strain B641, IRF3 was stable in cells infected with porcine rotavirus strain OSU and OSU NSP1 bound only weakly to IRF3. Both B641 NSP1 and OSU NSP1 were stabilized in cells or cell-free extracts in the presence of the proteasome inhibitor MG132 and when the zinc-binding domain was disrupted by site-directed mutagenesis. Data from the B641 analyses that show IRF3 degradation is dependent on the presence of NSP1 and the integrity of the N-terminal zinc-binding domain, coupled with the regulated stability of IRF3 and NSP1 by the proteasome, collectively support the hypothesis that NSP1 is an E3 ubiquitin ligase.
Rotavirus NSP1 Inhibits NFkappaB Activation by Inducing Proteasome-dependent Degradation of Beta-TrCP: a Novel Mechanism of IFN Antagonism
PLoS Pathogens. Jan, 2009 | Pubmed ID: 19180189
Mechanisms by which viruses counter innate host defense responses generally involve inhibition of one or more components of the interferon (IFN) system. Multiple steps in the induction and amplification of IFN signaling are targeted for inhibition by viral proteins, and many of the IFN antagonists have direct or indirect effects on activation of latent cytoplasmic transcription factors. Rotavirus nonstructural protein NSP1 blocks transcription of type I IFNalpha/beta by inducing proteasome-dependent degradation of IFN-regulatory factors 3 (IRF3), IRF5, and IRF7. In this study, we show that rotavirus NSP1 also inhibits activation of NFkappaB and does so by a novel mechanism. Proteasome-mediated degradation of inhibitor of kappaB (IkappaBalpha) is required for NFkappaB activation. Phosphorylated IkappaBalpha is a substrate for polyubiquitination by a multisubunit E3 ubiquitin ligase complex, Skp1/Cul1/F-box, in which the F-box substrate recognition protein is beta-transducin repeat containing protein (beta-TrCP). The data presented show that phosphorylated IkappaBalpha is stable in rotavirus-infected cells because infection induces proteasome-dependent degradation of beta-TrCP. NSP1 expressed in isolation in transiently transfected cells is sufficient to induce this effect. Targeted degradation of an F-box protein of an E3 ligase complex with a prominent role in modulation of innate immune signaling and cell proliferation pathways is a unique mechanism of IFN antagonism and defines a second strategy of immune evasion used by rotaviruses.