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. I joined the faculty at UVM in July of 2008, and am currently completing my second year of COBRE-supported research. In the past year, I have focused my efforts on advancing my research program, preparing manuscripts for publication, and writing grant proposals to obtain extramural funding for future studies. My research program is focused on emerging infectious diseases, particularly the hantaviruses and arenaviruses. In the previous year of funding, we had utilized a cutting edge proteomics approach to identify human ER-Golgi intermediate compartment-53 kDa protein (ERGIC-53) as a potential interacting partner of the glycoprotein precursor (GPC) encoded by Andes hantavirus (ANDV). Accordingly, we studied this interaction in great detail during the current funding period. To independently validate the interaction, we affinity purified, from human cells, the ANDV GPC and found that ERGIC-53 was co-immunoprecipated via Western blot. We also performed the reciprocal experiment and found that affinity purification of ERGIC-53 co-immunoprecipiated the ANDV GPC. To determine whether this interaction is highly conserved, we tested whether GPCs encoded by additional hantaviruses, as well as arenaviruses, also interact with ERGIC-53. Interestingly, we found that GPCs from Sin Nombre hantavirus, as well as several pathogenic arenaviruses (Lassa virus (LASV), Junin virus (JUNV), lymphocytic choriomeningitis virus (LCMV), Machupo virus, and Whitewater Arroyo virus), also interact with ERGIC-53. To determine the importance of ERGIC-53 for viral replication, we silenced ERGIC-53 expression in human cells via siRNA and then challenged these cells with JUNV and LCMV to determine how the absence of ERGIC-53 would impact the ability of each virus to undergo productive replication. Compared to control cells that were transfected with a scrambled siRNA, we observed a significant reduction in viral titer following ERGIC-53 knock-down for both JUNV and LCMV. Inversely, we found that overexpression of ERGIC-53 in cells prior to JUNV challenge led to a significant increase in viral titer. ERGIC-53 was originally discovered for its important role as a cargo receptor for the blood coagulation factors V and VIII;individuals with mutations in ERGIC-53 have bleeding disorders due to an inability to secrete factors V and VIII. It may be that ERGIC-53 is a cargo receptor required for the efficient transport of the arenavirus and hantavirus GPCs from the ER to the Golgi. The results of our studies suggest that ERGIC-53 plays an important role in arenavirus replication and may therefore represent a valuable target for the development of broad-spectrum antivirals to target the pathogenic arenaviruses and, potentially, the hantaviruses as well. Another interesting hypothesis is that ERGIC-53's interaction with the GPCs encoded by JUNV, LASV, or ANDV may disrupt its normal chaperone function for the blood coagulation factors V and VIII, leading to the hemorrhagic manifestations seen following infection with these viruses. We are currently preparing a manuscript describing our results for submission to PLoS Pathogens. In the next year, we plan to study several aspects of the ERGIC-53 interaction with viral GPCs. Specifically, we plan to define the molecular basis for the interaction, determine how the interaction contributes to GPC morphogenesis and the formation of viral factories in the ERGIC, and to determine whether ERGIC-53's normal cargo function for cellular proteins, including the factors V and VIII, is impaired via interaction with arenavirus and hantavirus GPCs. These proposed studies will be the subject of an RO1 application that will be assembled for submission to NIAID. In the past year of funding, I submitted five grants for review: an NSF grant to study the ecology of SNV transmission among naturally infected deer mice in Utah;a developmental grant and a grant to the NERCE to study arenavirus-host pathogen interactions;and two R21 applications (an A0 and A1) to NIAID to identify host proteins that interact with the proteins encoded by the pathogenic arenaviruses. The NSF grant is still pending, the NERCE grants were not funded (no scores or reviews given), and the A0 and A1 R21 applications received impact scores of 30 and 29, respectively. We are hopeful that one of the R21 applications will be funded as the impact scores for both applications meet the interim NIAID payline, which has been set at 30 as of April 7th, 2010. Publications since the previous progress report (including submitted manuscripts) One deficiency that I am currently trying to address is a lack of publications from my laboratory here at UVM. Both my senior mentors at UVM, as well as reviewers of my NIAID R21 grant proposals, have indicated that to be more competitive for extramural funding, I need to publish more papers describing my work here at UVM. Accordingly, as mentioned above, I am currently writing a manuscript describing our ERGIC-53 studies and plan to write an additional paper describing a novel quantitative RT-PCR assay for measuring arenavirus RNA species. I plan to submit both for review in the spring/summer of 2010. I also have a first author manuscript that is currently under review at the Journal of Virology (see below). 1.Botten, J., Whitton, J. L., Barrowman, P., Sidney, J., Whitmire, J. K., Alexander, J., Sette, A., and Buchmeier, M. A universal human vaccine for pathogenic Old World arenaviruses. Submitted to Journal of Virology 2.Kotturi, M. F., Botten, J., Maybeno, M., Sidney, J., Glenn, J., Bui, H. H., Oseroff, C., Crotty, S., Peters, B., Grey, H., Buchmeier, M. J., and Sette A. Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage. Submitted to Immunome Research 3.Alexander, J., Bilsel, P., del Guercio, M., Marinkovic-Petrovic, A., Southwood, S., Stewart, S., Ishioka, G., Kotturi, M., Botten, J., Newman, M., Sidney, J., Sette, A. Identification of broad binding Class I HLA supertype epitopes to provide universal coverage of Influenza A virus. Human Immunology, In press 4.Botten, J., Sidney, J., Mothe, B., Peters, B., Sette, A., and Kotturi, M. Coverage of related pathogenic species by conserved versus multivalent epitope design. Arenaviruses as a model system. Microbiology and Molecular Biology Reviews, In press 5.Alexander, J., Bilsel, P., del Guercio, M., Stewart, S., Marinkovic-Petrovic, A., Southwood, S., Crimi, C., Vang, L., Walker, L., Ishioka, G., Chitnis, V., Sette, A., Assarsson, E., Hannaman, D., Botten, J., Newman, M. (2010) Universal Influenza DNA Vaccine Encoding Conserved CD4+ T Cell Epitopes Protects Against Lethal Viral Challenge in HLA-DR Transgenic Mice. Vaccine;28, 664-672. 6.Kotturi, M. F., Botten, J., Sidney, J., Bui, H. H., Giancola, L., Maybeno, M., Babin, J., Oseroff, C., Pasquetto,. V., Greenbaum, J. A., Peters, B., Ting, J., Do, D., Vang, L., Alexander, J., Grey, H., Buchmeier, M. J., and Sette A. (2009) A multivalent and cross-protective vaccine strategy against arenaviruses associated with human disease. PLoS Pathogens, 5:e1000695.
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