HANTAVIRUS AND ARENAVIRUS HOST-PATHOGEN INTERACTIONS

University Of Vermont & St Agric College, Burlington VT

Investigators

Linked publications & trials

Abstract

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 through April of 2009 have focused my efforts on setting up my laboratory, submitting new grant proposals (see pending funding below), and beginning my Cobre-funded studies. My research program is focused on emerging infectious diseases, particularly the hantaviruses and arenaviruses. My two specific research themes are outlined below. My first research theme is to study the interactions of hantavirus and arenavirus proteins with host cellular proteins to determine how these interactions directly impact viral replication as well as basic cellular processes in host cells. The hantaviruses and arenaviruses each have proteomes that consist of four proteins. At present, very little is known regarding the host cellular proteins that these viral proteins interact with during infection. To identify novel host protein- virus protein interactions, we have generated a library of plasmids that encode the proteomes of the New World hantaviruses Sin Nombre virus (SNV) and Andes virus (ANDV), as well as each of the seven pathogenic arenaviruses (Lassa, lymphocytic choriomeningitis, Junin, Machupo, Guanarito, Sabia, and Whitewater Arroyo viruses). Using these plasmids, we plan to express selected viral proteins in mammalian cells for the purpose of identifying cellular binding partners through protein pull-down assays and mass spectrometry. We hope to identify interactions that are critical for viral replication and/or directly contribute to viral pathogenesis as these interactions would serve as excellent targets for the design of novel antiviral compounds. Over the past year, we used this protein-pull down/mass spectrometry approach to probe for host cellular proteins that interact with the ANDV nucleocapsid protein (N), nonstructural protein of the S segment (NSs), and glycoprotein precursor (GPC). Through two independent runs, we were able to map seven unique host proteins that interact with these ANDV proteins. The NSs protein interacts with the DEAD box RNA helicase DDX21, the N protein interacts with the polyadenylate-binding protein 3 (PABP), and the GPC interacts with the chaperone proteins calreticulin (CALR), calnexin (CALN), calumenin, and BCL-2 associated athanogene 2 (BAG2), as well as the ER Golgi intermediate compartment (ERGIC) cargo protein ERGIC-53. We have high confidence in these results as CALN and CALR have both previously been shown to specifically interact with the GPC from the related Hantaan hantavirus. Additionally, PABP has recently been shown to interact with the related orthobunyavirus N protein, which leads to a selective shutdown of host mRNA translation while favoring viral mRNA translation. Our goals for the next year will be to confirm that these ANDV protein- host protein interactions are real using additional biochemical assays, including mapping the interacting domains between these proteins, and to determine the impact of each interaction on viral replication through targeted host protein knock down. We also plan to test how these interactions may alter normal cellular processes and potentially contribute to pathogenesis. For example, the interaction of GPC with ERGIC-53, a protein which is critical for normal trafficking of coagulation factors, could contribute to the pulmonary edema observed during hantavirus disease. We also plan to expand our screening to include the SNV proteome, as well as selected arenavirus proteomes. From a funding standpoint, there are two potential grants that could be submitted in the next year. The first grant would be an R21 application to screen for novel host proteins that interact with the pathogenic arenavirus proteins. We have already built our library of expression vectors for the arenavirus proteins and can use the above described ANDV studies as preliminary data to demonstrate that our approach is feasible. We would propose to identify novel binding partners, confirm the most interesting candidates, and then focus on these binding partners for hypothesis driven R01 work upon the completion of the R21. The second grant would be an R01 application to test hypotheses regarding the ANDV-host protein interactions. Here, we can choose the most interesting candidate interactions and test hypotheses for how these interactions augment viral replication and/or contribute to pathogenesis. For example, we could focus on the interactions of ANDV GPC with ERGIC-53 to test the hypothesis that this interaction is critical for the formation of viral replication factories during infection. Additionally, we could test the hypothesis that the ANDV GPC-ERGIC-53 interaction impairs the ability of host cells to secrete coagulation factors during infection. My second research theme is to determine the role of T cells in human hantavirus pathogenesis. To generate funding for these studies, I submitted a grant (Burroughs Wellcome Fund) and an NIH contract (Large Scale T cell Epitope Discovery Program) in the Fall of 2008. The BWF application was not funded, and we are waiting for reviews of the contract at present. In support of these applications, we utilized a combination of bioinformatic algorithms and immunogenicity screening in HLA transgenic mice to identify 80 potential HLA-A2 supertype-restricted class I epitopes from both SNV and ANDV. Interestingly, we found a substantial number (n = 48) of immunogenic peptides from the viral polymerase, which has not previously been shown to be immunogenic. Additionally, I was successful in recruiting collaborators in New Mexico and Chile that have access to virtually the only available pool of SNV and ANDV patients. They have agreed to collect lymphocytes from acutely ill and convalescent hantavirus patients for the proposed studies. If the contract is not funded, we plan to apply for funding through additional mechanisms. My final grant application from 2008 was in response to the Ecology of Infectious Disease RFA from NSF. This grant proposes to study SNV transmission among naturally infected deer mice in Utah. This is a collaborative grant with Dr. Denise Dearing at U. of Utah. We are Co-PIs on this application. Publications I have no new publications for 2008, but have three manuscripts that will be submitted over the next several months for review in the Journal of Virology. Mentoring Summaries: Dr. Jonathan Boyson Dr. Boyson has met with Jason Botten, a new Immunobiology faculty member numerous times informally, and once formally to discuss his plans. Dr. Boyson and Dr. Botten discussed solutions to various logistical issues inherent in setting up a laboratory. Dr. Boyson reviewed a grant submission that Jason submitted shortly after he arrived and there has been discussion regarding the merits of rotating Cell and Molecular Biology graduate students. Dr. Boyson and Dr. Botten attend the same weekly Immunobiology lab meeting which affords he opportunity to keep each other apprised of recent developments in the laboratory. There is ongoing discussion regarding potential collaborative projects. Dr. Sally Huber My goal is to meet with Jason Botten monthly to review his progress in writing and submiting manuscripts and in the preparation and submission of grant applications. I will also advise him on University policies regarding promotion, reappointment and tenure. I will strongly encourage him to participate in the COBRE seminar series by inviting two senior investigator speakers in his area of research interest. This has the advantage of making Dr. Botten better known to investigators in his research area and providing input from these senior scientists on Dr. Botten's research and proposed studies.