HANTAVIRUS AND ARENAVIRUS HOST-PATHOGEN INTERACTIONS

汉坦病毒和沙粒病毒宿主-病原体相互作用

• 批准号: 7959822
• 负责人: Jason W. Botten
• 金额: $ 23.62万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959822
• 关键词: Algorithms; Andes Virus; Antiviral Agents; Applications Grants; Area; Arenavirus; Binding; Binding Proteins; Biochemical; Bioinformatics; Biological Assay; Blood Coagulation Factor; Bolivian Hemorrhagic Fever Virus; Boxing; Calnexin; Cell physiology; Cells; Centers of Research Excellence; Chile; Communicable Diseases; Computer Retrieval of Information on Scientific Projects Database; Contracts; Contracts Review; Data; Deer Mouse; Development; Disease; Ecology; Emerging Communicable Diseases; Epitopes; Faculty; Funding; Genetic Translation; Glycoproteins; Goals; Golgi Apparatus; Grant; Guanarito virus; HLA-A2 Antigen; Hantavirus; Human; Immunobiology; Infection; Institution; Journals; Junin virus; Laboratories; Libraries; Lymphocyte; Lymphocytic Choriomeningitis; Mammalian Cell; Manuscripts; Maps; Mass Spectrum Analysis; Mentors; Molecular Biology; Molecular Chaperones; New Mexico; Nonstructural Protein; Nucleocapsid Proteins; Orthobunyavirus; Pathogenesis; Patients; Peptides; Plasmids; Policies; Poly A; Polymerase; Preparation; Proteins; Proteome; Publications; Pulmonary Edema; RNA Helicase; Recruitment Activity; Research; Research Personnel; Resources; Role; Running; Screening procedure; Senior Scientist; Series; Sin Nombre virus; Solutions; Source; T-Lymphocyte; T-Lymphocyte Epitopes; Testing; Time; Transgenic Mice; United States National Institutes of Health; Universities; Utah; Vermont; Viral; Viral Pathogenesis; Viral Proteins; Virus; Work; Writing; calreticulin; design; expression vector; falls; graduate student; immunogenic; immunogenicity; interest; knock-down; meetings; member; novel; pathogen; programs; response; trafficking; transmission process; virology

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 我于2008年7月加入UVM的教职员工，直到2009年4月，我一直致力于建立实验室，提交新的赠款提案（请参阅下面的资金），并开始我的Cobre资助的研究。我的研究计划的重点是新兴的传染病，尤其是汉坦病毒和体育症病毒。我的两个具体研究主题在下面概述。 我的第一个研究主题是研究汉塔病毒和体育症病毒蛋白与宿主细胞蛋白的相互作用，以确定这些相互作用如何直接影响宿主细胞中的病毒复制以及基本细胞过程。汉坦病毒和体育症病毒各有四种蛋白质的蛋白质组。目前，关于这些病毒蛋白在感染过程中与宿主细胞蛋白相互作用的宿主细胞蛋白知之甚少。 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病毒）。使用这些质粒，我们计划在哺乳动物细胞中表达选定的病毒蛋白，以通过蛋白质下拉的测定法和质谱法鉴定细胞结合伴侣。我们希望确定对病毒复制至关重要的相互作用和/或直接导致病毒发病机理，因为这些相互作用将是设计新型抗病毒化合物的绝佳靶标。 在过去的一年中，我们使用了这种蛋白质向下/质谱方法来探测与ANDV Nucleocapsid蛋白（N）相互作用的宿主细胞蛋白，S段（NSS）的非结构性蛋白（NSS）和糖蛋白蛋白前体（GPC）。通过两次独立运行，我们能够绘制七个与这些ANDV蛋白相互作用的独特宿主蛋白。 NSS蛋白与死盒RNA解旋酶DDX21相互作用，N蛋白与聚腺苷酸结合蛋白3（PABP）相互作用，GPC与伴侣蛋白钙蛋白钙蛋白钙蛋白钙蛋白膜产量（CALR）（CALR）（CALR），CALNEXIN（CALNEXIN），CALNEXIN（CALN），CALUMENIN和BCL-2相关的Athanogen and ersed Athanogen以及golgi and ersed Athanogen uss 2（calnexin used Athanoge and 2（calnexin）以及gpci Intred Ass 2（Calnexin），以及（ERGIC）货物蛋白ERGIC-53。我们对这些结果具有很高的信心，因为CALN和CALR以前都被证明与相关的Hantaan Hantavirus的GPC特别相互作用。此外，最近已显示PABP与相关的原肺炎病毒N蛋白相互作用，该蛋白会导致选择性关闭宿主mRNA翻译，同时又有利于病毒mRNA翻译。 我们明年的目标是确认这些ANDV蛋白质 - 宿主蛋白相互作用是实际使用其他生化测定方法的，包括绘制这些蛋白质之间的相互作用结构域，并确定每种相互作用通过靶向宿主蛋白敲低的影响病毒复制的影响。我们还计划测试这些相互作用如何改变正常的细胞过程，并有可能导致发病机理。例如，GPC与ERGIC-53的相互作用是一种对正常运输凝血因子至关重要的蛋白质，可能有助于在汉塔病毒疾病期间观察到的肺水肿。我们还计划将筛选扩展到包括SNV蛋白质组以及选定的体育症病毒蛋白质组。 从资金的角度来看，明年可以提交两项潜在的赠款。第一个赠款将是R21应用程序，用于筛选与致病性竞技病毒蛋白相互作用的新型宿主蛋白。我们已经为体育病毒蛋白构建了表达媒介库，并且可以将上述和V研究用作初步数据，以证明我们的方法是可行的。我们建议识别新颖的绑定伙伴，确认最有趣的候选者，然后专注于这些结合伙伴在完成R21后的假设驱动R01工作。第二个赠款将是R01应用程序，用于测试有关和V-Host蛋白相互作用的假设。在这里，我们可以选择最有趣的候选相互作用，并检验这些相互作用如何增强病毒复制和/或有助于发病机理的假设。例如，我们可以专注于ANDV GPC与ERGIC-53的相互作用，以检验以下假设：这种相互作用对于感染过程中病毒复制工厂的形成至关重要。此外，我们可以检验以下假设：ANDV GPC-53相互作用会损害宿主细胞在感染过程中分泌凝血因子的能力。 我的第二个研究主题是确定T细胞在人汉塔病毒发病机理中的作用。为了为这些研究产生资金，我在2008年秋季提交了一笔赠款（Burroughs Wellcome Fund）和NIH合同（大规模T细胞表位发现计划）。BWF申请尚未资助，我们正在等待目前的合同审查。为了支持这些应用，我们利用了HLA转基因小鼠中生物信息学算法和免疫原性筛查的组合，以鉴定来自SNV和ANDV的80个潜在的HLA-A2超型限制性I类表位。有趣的是，我们发现病毒聚合酶中有大量的免疫原性肽数量（n = 48），以前尚未证明是免疫原性的。此外，我还成功地招募了新墨西哥州和智利的合作者，这些合作者实际上可以使用SNV和ANDV患者唯一可用的库。他们已同意从急性疾病和康复的汉塔病毒患者中收集淋巴细胞，以进行拟议的研究。如果合同没有资助，我们计划通过其他机制申请资金。 我从2008年开始的最终赠款申请是对NSF感染性疾病RFA的生态的回应。该赠款建议研究犹他州自然感染的鹿小鼠之间的SNV传播。这是与犹他州美国丹尼斯·迪林（Denise Dearing）博士的合作赠款。我们是此应用程序的共同理谈。 出版物 我没有2008年的新出版物，但是有三个手稿将在接下来的几个月中提交，以供《病毒学杂志》审查。 指导摘要： 乔纳森·博伊森博士 博伊森博士与杰森·博顿（Jason Botten）会面，杰森·博顿（Jason Botten）是一位新的免疫生物学教师，无数次，曾经正式讨论他的计划。博伊森博士和博顿博士讨论了建立实验室固有的各种后勤问题的解决方案。 博伊森博士审查了杰森到达后不久提交的一份赠款，并就旋转细胞和分子生物学研究生的优点进行了讨论。 Boyston博士和Botten博士参加了每周一次的一次免疫生物学实验室会议，他有机会互相了解实验室的最新发展。关于潜在协作项目的讨论正在进行。 萨莉·休伯博士 我的目标是每月与Jason Botten会面，以审查他在写作和提交手稿以及赠款申请的准备和提交方面的进度。我还将就晋升，连任和任期的大学政策提供建议。我将强烈鼓励他通过邀请两位高级调查员在研究感兴趣的领域参加COBRE研讨会系列。这具有使Botten博士在其研究领域中广为人知的，并向Botten博士的研究和拟议研究提供了这些高级科学家的意见。