MicroRNA Regulation of Human Protease Genes Essential for Influenza Virus Replication Victoria A. Meliopoulos, Lauren E. Andersen, Paula Brooks, Xiuzhen Yan, Abhijeet Bakre, J. Keegan Coleman, S. Mark Tompkins, Ralph A. Tripp* Department of Infectious Diseases, University of Georgia, Athens, Georgia, United States of America Abstract Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-kB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies. Citation: Meliopoulos VA, Andersen LE, Brooks P, Yan X, Bakre A, et al. (2012) MicroRNA Regulation of Human Protease Genes Essential for Influenza Virus Replication. PLoS ONE 7(5): e37169. doi:10.1371/journal.pone.0037169 Editor: Andrew D. Badley, Mayo Clinic, United States of America Received September 13, 2011; Accepted April 16, 2012; Published May 14, 2012 Copyright: 2012 Meliopoulos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases (HHSN266200700006C) (www.niaid.nih. gov). No additional external funding received for this study. 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: ratripp@uga.edu Introduction Influenza A viruses generally cause seasonal epidemics however they have the potential to cause pandemics associated with substantial morbidity and mortality [1,2]. Development of seasonal vaccines is required for influenza virus due to high viral mutation rates that lead to antigenic drift, and also because of periodic antigenic shift which can render vaccines less or ineffective [3]. There are various antiviral drugs that have proven efficacy in the treatment of influenza infections: two M2 ion channel inhibitors (amantadine and rimantadine) and several neuraminidase inhibitors (including zamamivir and oseltamivir) [4,5,6,7,8,9]. Early treatment with these antiviral drugs reduces the duration of symptoms and the time to recovery; however, the use of antiviral drugs is complicated by the emergence of drug resistant viruses [5,6,10,11,12]. In addition, antiviral drug use may come with unwelcome effects that could include an increase in population vulnerability due to lack of seroconversion, as well as driving drug resistance among circulating strains [11]. Thus, it is critical to discover new targets for chemoprophylactics and treatment. Recent advances in our understanding of RNA interference (RNAi) have provided a means to perform genome-wide screens to determine and validate host cell genes that may be required for influenza virus replication [13,14]. RNAi is an efficient mecha- nism for the sequence-specific inhibition of gene expression [15,16], and is mediated by small interfering RNAs (siRNA) incorporated in the RNA-induced silencing complex (RISC) where the antisense or guide strand of the siRNA can suppress protein expression or direct degradation of messenger RNAs that contain homologous sequences [17,18,19]. Synthetic siRNAs can be readily developed to target viral or host genes and have been successfully applied in disease intervention approaches. For example, siRNA targeting respiratory syncytial virus has shown efficacy for silencing virus replication [20,21,22,23,24,25], a feature that has led to RNAi-based clinical trials as a new therapeutic option [23]. In addition, there are promising results from targeting host genes, such as the use of siRNA silencing for the treatment of age-related macular degeneration [26], and in the case of influenza, inhibiting the host gene CAMK2B prevented vRNA transcription in vitro [27], and shRNA inhibition of trypsin also inhibited replication and apoptosis [28]. Recently, several studies employed genome-wide RNAi screens to identify host genes required for influenza virus infection and replication [27,29,30,31,32], and genes have also been identified by random homozygous gene perturbation [33] and by a proteomic screen [34]. Although there were few common genes detected among the studies, meta-analysis revealed that influenza virus was co-opting many of the same host cell pathways [27,29,30,31,32,35]. Thus, the inability to find the same genes among the studies is not unexpected given that multiple genes may be affected in the same host cell pathway, that the tempo of gene expression may vary among the cell lines studied, and that differences can be attributed PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e37169