Anti viral function of interferon alpha induced human beta defensin 3

描述(申请人提供)：人类副流感病毒3型(HPIV-3)是一种呼吸道副粘病毒，感染肺上皮细胞，导致婴儿和儿童的高发病率。到目前为止，还没有针对HPIV-3的有效疫苗或抗病毒疗法，因此，阐明该病毒在感染的上皮细胞中诱导的先天免疫抗病毒反应对于开发有效的抗病毒疗法具有重要的潜力。干扰素是一种重要的抗病毒细胞因子，它激活JAK/STAT途径诱导抗病毒蛋白。因此，干扰素的抗病毒作用是宿主先天免疫的重要组成部分。最近我们报道(Basu等人)J·维罗尔将军。87：2653-2662,2006)干扰素通过JAK/STAT独立方式激活可溶性分泌的抗病毒蛋白的能力。为了研究JAK/STAT非依赖途径的特征，我们的初步研究表明，干扰素可在人肺上皮细胞中诱导人β-防御素-3(HBD3)，而纯化的HBD3可抑制这些细胞中的HPIV-3感染。有趣的是，干扰素诱导的HBD3依赖于细胞外信号调节激酶[ERK，丝裂原活化蛋白激酶(MAPK)途径的一个组成部分]的激活，并且不依赖于JAK/STAT。HBD3基因的进一步鉴定显示存在两个共同的AP-1结合位点。根据这些结果和干扰素激活AP-1转录因子的能力，我们推测HBD3是一种新的干扰素诱导的抗病毒蛋白，它的产生可能不依赖于JAK/STAT信号通路，可能是通过激活ERK/AP1信号通路来实现的。1.研究人β防御素3(HBD3)作为干扰素诱导的抗病毒蛋白在人肺上皮细胞中的作用。基于我们的初步结果，我们计划建立HBD3作为细胞外干扰素诱导的抗病毒蛋白。将利用HBD3过表达的细胞和HBD3内源性表达被抑制的细胞(通过siRNA技术实现)来研究HBD3在干扰素抗病毒作用中的作用。2.研究干扰素非依赖JAK/STAT诱导HBD3的机制：ERK/AP-1途径的作用。ERK/AP-1通路在HBD3基因表达中的重要性将通过a)显性负性和结构性活性的MAPKs/AP-1，b)针对MAPKs/AP-1的siRNA来分析。我们还将通过研究HBD3等抗病毒因子的反式激活机制，强调干扰素诱导的ERK/AP-1通路在JAK/STAT非依赖性抗病毒信号转导中的作用。相关性：目前的应用旨在研究HBD3对高致病性人类呼吸道病毒HPIV-3的抗病毒功能。此外，还旨在研究干扰素诱导的JAK/STAT非依赖性抗病毒途径。这些研究对于开发针对HPIV-3的新型抗病毒疗法以及潜在地针对其他呼吸道副粘病毒具有重要意义。人副流感病毒3型(HPIV-3)是一种感染婴儿和儿童的高致病性呼吸道病毒。目前，还没有疫苗或抗病毒疗法来对抗HPIV-3的相互作用。我们的研究重点是阐明针对HPIV-3的新的抗病毒天然免疫反应机制。从拟议的研究中获得的知识将有助于开发有效的抗病毒疗法来对抗HPIV-3感染。

DESCRIPTION (provided by applicant): Human parainfluenza virus type 3 (HPIV-3) is a respiratory paramyxovirus that infects lung epithelial cells to cause high morbidity among infants and children. To date, no effective vaccine or anti-viral therapy exists for HPIV-3 and therefore, elucidation of innate immune anti-viral response induced by this virus in infected epithelial cells hold significant potential for development of effective anti-viral therapies. Interferon-a (IFN) is a critical anti-viral cytokine that activates Janus kinases/signal transducers and activators of transcription (JAK/STAT) pathway to induce anti-viral proteins. Therefore, anti-viral action of IFN constitutes an important arm of host-derived innate immunity. Recently we reported (Basu et al. J. Gen. Virol. 87:2653-2662, 2006) the ability of IFN to activate soluble secreted anti-viral proteins via JAK/STAT independent fashion. In an attempt to characterize the JAK/STAT independent pathway, our preliminary studies have revealed that IFN induces human beta defensin-3 (HBD3), a soluble secreted anti-microbial protein in human lung epithelial cells and purified HBD3 inhibited HPIV-3 infection in these cells. Interestingly, HBD3 induction by IFN was dependent on extracellular signal-regulated kinase [ERK, a component of mitogen-activated protein kinase (MAPK) pathway] activation and was JAK/STAT independent. Further characterization of HBD3 gene revealed presence of two consensus AP-1 binding sites. Based on these results and the ability of IFN to activate AP-1 transcription factors, we hypothesize that HBD3 is a novel IFN induced anti-viral protein which is produced independent of JAK/STAT pathway probably via activation of ERK/AP1 signaling cascade. We plan to characterize this novel JAK/STAT independent pathway in details by the following specific aims: 1. Study the role of human beta defensin-3 (HBD3) as an IFN induced anti-viral protein in human lung epithelial cells. Based on our preliminary results, we plan to establish HBD3 as an extracellular IFN induced anti-viral protein. The contribution of HBD3 during anti-viral action of IFN will be studied using HBD3 over- expressing cells and cells suppressed for endogenous expression of HBD3 (achieved via siRNA technology). 2. Characterize the mechanism of JAK/STAT independent induction of HBD3 by IFN: role of ERK/AP- 1 pathway. The importance of ERK/AP-1 pathway in HBD3 gene expression will be analyzed by utilizing a) dominant-negative and constitutively active MAPKs/AP-1, b) siRNA against MAPKs/AP-1. We will also emphasis on the contribution of IFN induced ERK/AP-1 pathway during JAK/STAT independent anti-viral signaling by studying mechanism of trans-activation of anti-viral factors like HBD3. Relevance: The current application aims to study the anti-viral function of HBD3 against highly pathogenic human respiratory virus, HPIV-3. In addition, it aims at characterization of lesser-known JAK/STAT independent anti-viral pathway induced by IFN. These studies are important for development of novel anti- viral therapies against HPIV-3 and potentially for other respiratory paramyxoviruses. Human parainfluenza virus type 3 (HPIV-3) is a highly pathogenic respiratory virus that infects infants and children. Currently, no vaccine or anti-viral therapy exists to counteract HPIV-3 interaction. Our proposed research is focused on elucidating novel anti-viral innate immune response mechanism against HPIV-3. The knowledge derived from the proposed studies will be helpful in development of efficient anti-viral therapeutics against HPIV-3 infection.