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<li> Identification of NS1 protein sumoylation sites.<BR> Our preliminary data showed that the NS1 protein is sumoylated in vitro. Sequence analysis has identified two major potential sumoylation site in the NS1 protein. In this aim, we will identify the sumoylation sites of influenza virus NS1 protein. The lysine residues present within the sumoylation consensus sites in NS1 (K70 and K118) will be replaced with arginine (R) residues using site-directed mutagenesis. The mutated gene will be subjected to in vitro and in vivo sumoylation assays to determine if these lysine sites are targeted by sumoylation. <li> Effect of sumoylation on cellular localization of NS1. <BR> Influenza viruses replicate in the nucleus of infected cells and consequently, viral proteins, once synthesized, have to be transported to the nucleus where they function. Although transport of proteins to the nucleus can be mediated by the presence of nuclear localization signals (NLS), sumoylation is also involved in protein movement between cellular compartments. In this aim, we will examine the role of NS1 sumoylation in the nuclear trafficking by immunofluorescence assays using NS1 transfected cells.<li> Study the in vivo role of sumoylation on virus replication and pathogenesis. <BR> Cellular localization and function of viral proteins may not only depend on post-translational modifications, such as sumoylation, but also on their complex host-virus interactions. Influenza virus sumoylation mutants will be generated using reverse genetics (previously established in our laboratory). The role of NS1 sumoylation on down regulation of interferon beta will be examined by the ability of the mutant viruses to replicate in interferon competent and incompetent embryonating eggs. In additions, the role of sumoylation on virus replication in chickens will be examined to determine its role in AI pathogenesis. These studies will allow us to examine the effect of sumoylation under normal physiological conditions in chickens. </ol>
NON-TECHNICAL SUMMARY: Influenza is a major respiratory disease of poultry caused by type A Influenza viruses. To better understand the molecular mechanisms of influenza pathogenesis it is imperative that we understand the role of individual viral proteins in virus replication as well as their interaction with cellular proteins. The purpose of this project is to understand the molecular mechanisms involved in influenza virus pathogenesis.
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APPROACH: Identification of NS1 protein sumoylation sites: The K70 and K118 in NS1 protein, have been identified as potential sumoylation sites. In this aim, we will mutate these lysine residues to argine (R) and determine, in vitro, if these residues are the ones targeted by the sumoylation system. K to R substitution in each of the predicted sumoylation sites of NS1 will be produced using QuickChange Site Directed Mutagenesis kit Mutations will be verified by sequence analysis and the mutated genes in vitro translated and subjected to in vitro sumoylation assay If the K to R mutations introduced fail to block sumoylation of either protein it is possible that further cryptic sites are present and additional lysine residues with lower sumoylation scores will be evaluated. Effect of sumoylation on cellular localization of NS1 protein: Approach: The effect of sumoylation of NS1 in their nucleocytoplasmic localization will be evaluates by transfection of expression plasmid for wild type and K-to-R mutant NS1 proteins, into MDCK and COS-1 cells. At 24 and 48 h post-transfection cells will be fixed and the cellular localization of viral protein will be determined using confocal microscopy after simultaneous staining with antibodies for NS1 and antibodies specific for different cellular subcompartments. In addition, to augment the production of proteins necessary for sumoylation, wild type and mutant plasmids will also be cotransfected with plasmids expressing SUMO1, Ubc9/Ubc9-C93S. At 24 and 48 h post transfection the cells will be fixed and the cellular localization of viral protein will be determined using confocal microscopy after staining with using specific antibodies. Study the in vivo role of sumoylation on virus replication and pathogenesis: Since the cellular localization of wild type and mutant NS1 proteins may vary when expressed in the presence of normal stoichiometric proportions of other influenza proteins and vRNA, wild type and mutant viruses will be generated by reverse genetics and the cellular localization of these proteins evaluated by immunofluorescence and confocal microscopy. In addition, effect of these mutations on virus replication will be determined by comparing the growth kinetics in 7-day (interferon incompetent) and 12-day (interferon competent) old embryonating eggs. The pathogenesis of NS1 sumoylation mutants will be tested using 4 week old chickens. Virus shedding will be determined using tracheal swabs and histological lesions in the trachea and lungs will be compared to parental AI virus.
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PROGRESS: 2007/01 TO 2007/12
OUTPUTS: Our long-term goal is to understand the molecular mechanisms involved in influenza virus pathogenesis. The overall objective in this project was to investigate the role of NS1 protein in the biology of the virus. The central hypothesis was that sumoylation of influenza virus NS1 protein affects protein trafficking and therefore plays a role in viral replication and pathogenesis. Greater understanding of the function of NS1 protein was essential for developing improved strategies against influenza viruses. PARTICIPANTS: Vinayak Brahmakshatriya is a graduate student in Poultry Science at Texas A&M University. The research conducted is in partial fulfillment towards PhD. TARGET AUDIENCES: Poultry industry and researchers PROJECT MODIFICATIONS: Initial experiment showed that SUMO modification of the NS1 protein occurs in-vitro as predicted by computer analysis of the protein. Site-directed mutations experiments showed that there were multiple sites that were modified and it was not possible to mutate them individually, thus we initiated the experiments in which the entire NS1 gene was deleted.
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IMPACT: 2007/01 TO 2007/12
Using reverse genetics we have developed several truncated NS1 mutant avian influenza viruses. The NS1 mutant viruses replicated well in 7 day-old but not in 11 day-old embryonating chicken eggs. This virus was able to replicate in chickens were it showed reduced pathogenicity. We have also made a recombinant virus in which the entire NS1 gene was disrupted. We are currently evaluating the pathogenesis of these mutant viruses in chickens.