New World Hantaviruses Activate IFNl Production in Type I IFN-Deficient Vero E6 Cells Joseph Prescott1, Pamela Hall2, Mariana Acuna-Retamar3, Chunyan Ye3, Marc G. Wathelet3,6, Hideki Ebihara1, Heinz Feldmann1*, Brian Hjelle3,4,5 1 Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana, United States of America, 2 Research Service (151), New Mexico Veterans Affairs Health Care System, Albuquerque, New Mexico, United States of America, 3 Department of Pathology, Center for Infectious Diseases and Immunity, School of Medicine, University of New Mexico, Albuquerque, New Mexico, United States of America, 4 Department of Biology, Center for Infectious Diseases and Immunity, School of Medicine, University of New Mexico, Albuquerque, New Mexico, United States of America, 5 Department of Molecular Genetics and Microbiology, Center for Infectious Diseases and Immunity, School of Medicine, University of New Mexico, Albuquerque, New Mexico, United States of America, 6 Infectious Disease Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico, United States of America Abstract Background: Hantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS). These viruses induce a strong interferon-stimulated gene (ISG) response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN), rendering them unable to mount an efficient innate immune response to virus infection. Interferon l, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner. Methodology/Principal Findings: We show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNl. Three New World hantaviruses were similarly able to induce IFNl expression in this cell line. The IFNl contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7) without inducing ISGs. Conclusions/Significance: Herein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNl. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNl production in these cells might increase their utility for virus propagation. Citation: Prescott J, Hall P, Acuna-Retamar M, Ye C, Wathelet MG, et al. (2010) New World Hantaviruses Activate IFNl Production in Type I IFN-Deficient Vero E6 Cells. PLoS ONE 5(6): e11159. doi:10.1371/journal.pone.0011159 Editor: Bradley S. Schneider, Institut Pasteur, France Received April 9, 2010; Accepted May 23, 2010; Published June 17, 2010 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported by U.S Public Services grants U01 AI56618 (BH) and Division of Intramural Research (DIR) of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: feldmannh@niaid.nih.gov Introduction Vero E6 cells are epithelioid cells derived from the kidney of African green monkeys and, with their permissiveness for high viral productivity, are preferred for the isolation and/or propagation of the members of several virus families, including hantaviruses (a genus of the family Bunyaviridae) [1,2,3,4,5,6,7,8]. The utility of these cells for virus propagation is at least three-fold. First, they are infectable by several diverse viruses, perhaps due to an abundant receptor repertoire, or non-specific uptake of virus particles. Second, they have sustained a genetic deletion that ablated the type I IFN locus [9]. Third, Vero E6-encoded interferon regulatory factor 3 (IRF3), a transcription factor necessary for generating responses to virus infection, is relatively inefficient, resulting in a muted initial response to virus infection [10]. For experimental purposes, hantavirus infectious stocks typically consist of the supernatants of infected Vero E6 cells and are used for in vivo and in vitro experiments. At the cellular level, virus infection is detected by interactions between host-encoded pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs). Receptor bind- ing activates several well-characterized signaling cascades and results in the activation of type I IFN. More recently, type III IFNs (IFNl) have been described [11]. These IFNs are comprised of PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11159