Explorer Ablation of the Regulatory IE 1 Protein of Murine Cytomegalovirus Alters In Vivo Pro-inflammatory TNF-alpha Production during Acute Infection

Little is known about the role of viral genes in modulating host cytokine responses. Here we report a new functional role of the viral encoded IE1 protein of the murine cytomegalovirus in sculpting the inflammatory response in an acute infection. In time course experiments of infected primary macrophages (MWs) measuring cytokine production levels, genetic ablation of the immediate-early 1 (ie1) gene results in a significant increase in TNFa production. Intracellular staining for cytokine production and viral early gene expression shows that TNFa production is highly associated with the productively infected MW population of cells. The ie1dependent phenotype of enhanced MW TNFa production occurs at both protein and RNA levels. Noticeably, we show in a series of in vivo infection experiments that in multiple organs the presence of ie1 potently inhibits the pro-inflammatory cytokine response. From these experiments, levels of TNFa, and to a lesser extent IFNb, but not the anti-inflammatory cytokine IL10, are moderated in the presence of ie1. The ie1mediated inhibition of TNFa production has a similar quantitative phenotype profile in infection of susceptible (BALB/c) and resistant (C57BL/6) mouse strains as well as in a severe immuno-ablative model of infection. In vitro experiments with infected macrophages reveal that deletion of ie1 results in increased sensitivity of viral replication to TNFa inhibition. However, in vivo infection studies show that genetic ablation of TNFa or TNFRp55 receptor is not sufficient to rescue the restricted replication phenotype of the ie1 mutant virus. These results provide, for the first time, evidence for a role of IE1 as a regulator of the pro-inflammatory response and demonstrate a specific pathogen gene capable of moderating the host production of TNFa in vivo. Citation: Rodrı́guez-Martı́n S, Kropp KA, Wilhelmi V, Lisnic VJ, Hsieh WY, et al. (2012) Ablation of the Regulatory IE1 Protein of Murine Cytomegalovirus Alters In Vivo Pro-inflammatory TNF-alpha Production during Acute Infection. PLoS Pathog 8(8): e1002901. doi:10.1371/journal.ppat. 1002901 Editor: William J. Britt, University of Alabama at Birmingham, United States of America Received February 7, 2012; Accepted July 27, 2012; Published August 30, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by: The Wellcome Trust (WT066784/Z/02/Z), and the BBSRC/EPSRC to SynthSys, a Centre for Integrative Systems Biology (CISB) reference BB/D019621/1 to PG. Deutsche Forschungsgemeinschaft, research fellowship KR-3890/1-1 to KK. BBSRC studentship, and University of Edinburgh Principal’s Fellowship to SRM. The work of HT was supported by grants from the Wellcome Trust and the European Research Council (233457-SCG) to Mike Tyers. Deutsche Forschungsgemeinschaft, SFB 587, individual project A13 to MM. Deutsche Forschungsgemeinschaft, SFB 490, individual projects E2 and E4 to VW and MJR. Ministerio de Ciencia e Innovacion (SAF2008-00382 and SAF2011-25155) to AA. SULSA (Scottish Universities Life Sciences Alliance) to MA. Helmholtz Society through collaborative research program VISTRIE to SJ. British Heart Foundation reference FS/05/022 and University of Edinburgh Alumni Fund to MB. IF is funded by National Institutes of Health. The funders had no role in 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: p.ghazal@ed.ac. uk . These authors contributed equally to this work.