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. ? ? ?
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