This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Exploratory Grants (P20)
Project #
2P20RR021905-06
Application #
8360777
Study Section
Special Emphasis Panel (ZRR1-RI-B (01))
Project Start
2011-07-20
Project End
2012-06-30
Budget Start
2011-07-20
Budget End
2012-06-30
Support Year
6
Fiscal Year
2011
Total Cost
$248,464
Indirect Cost
Name
University of Vermont & St Agric College
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
King, Benjamin R; Hershkowitz, Dylan; Eisenhauer, Philip L et al. (2017) A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR). J Virol 91:
King, Benjamin R; Kellner, Samuel; Eisenhauer, Philip L et al. (2017) Visualization of the lymphocytic choriomeningitis mammarenavirus (LCMV) genome reveals the early endosome as a possible site for genome replication and viral particle pre-assembly. J Gen Virol :
Nock, Adam M; Wargo, Matthew J (2016) Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators. J Bacteriol 198:2503-14
Ziegler, Christopher M; Eisenhauer, Philip; Bruce, Emily A et al. (2016) The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles. PLoS Pathog 12:e1005501
Massilamany, Chandirasegaran; Koenig, Andreas; Reddy, Jay et al. (2016) Autoimmunity in picornavirus infections. Curr Opin Virol 16:8-14
Bonney, Elizabeth A; Krebs, Kendall; Saade, George et al. (2016) Differential senescence in feto-maternal tissues during mouse pregnancy. Placenta 43:26-34
Sateriale, Adam; Miller, Peter; Huston, Christopher D (2016) Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells. Infect Immun 84:1045-53
Ziegler, Christopher M; Eisenhauer, Philip; Bruce, Emily A et al. (2016) A novel phosphoserine motif in the LCMV matrix protein Z regulates the release of infectious virus and defective interfering particles. J Gen Virol 97:2084-9
Klaus, Joseph P; Botten, Jason (2016) Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment. J Vis Exp :e53682
Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas et al. (2016) Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity. FEBS Lett 590:1042-52

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