Combined miRNA and mRNA Signature Identifies Key Molecular Players and Pathways Involved in Chikungunya Virus Infection in Human Cells Tanvi Saxena 1 , Bhavna Tandon 1 , Shivani Sharma 1 , Shibu Chameettachal 2 , Pratima Ray 3 , Alok R. Ray 4,5 , Ritu Kulshreshtha 1 * 1 Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi, India, 2 Department of Textile Technology, Indian Institute of Technology, Delhi, India, 3 Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India, 4 Centre for Biomedical Engineering, Indian Institute of Technology, Delhi, India, 5 All India Institute of Medical Sciences, New Delhi, India Abstract Since its discovery, Chikungunya fever caused by a virus (CHIKV) has ravaged most of Africa and Southeast Asia. Despite there being more than a million reported cases in India alone and the seriousness of the disease in the chronic phase, a clear understanding of the disease pathogenesis and host response remains elusive. Here, we use microarray technology and quantitative PCR method to establish the complete miRNA, snoRNA and mRNA signature of host response upon CHIKV infection in human cell line infection model, HEK293T. The results were further validated in human primary cells (dermal fibroblasts). miRNA expression profiling revealed regulation of 152 miRNAs post CHIKV infection. An interesting overlap in miRNA signature was seen majorly with HCV, HPV and HIV1 virus. The microarray data further validated by qRT-PCR revealed induction of miR-744, miR-638, miR-503 and others among the top upregulated miRNAs. Notably, we found induction of snoRNAs belonging to C/D cluster including close paralogs of U3, U44, U76 and U78 snoRNAs. Genes were found to be differentially expressed along 3 major pathways; TGF-b, endocytosis and the cell cycle pathways. qRT-PCR data confirmed strong induction of TGF-b (SMAD6, JUN, SKIL) and endocytosis pathway (CXCR4, HSPA8, ADRB1) genes while downregulation of cell cycle genes (CDC27 and CDC23). Interestingly, use of TGF-b inhibitor, SB-431542, increased CHIKV mediated cell death. Overall, this study aims at providing the first complete transcriptome signature of host response upon CHIKV infection to aid identification of possible biomarkers and therapeutic targets. Citation: Saxena T, Tandon B, Sharma S, Chameettachal S, Ray P, et al. (2013) Combined miRNA and mRNA Signature Identifies Key Molecular Players and Pathways Involved in Chikungunya Virus Infection in Human Cells. PLoS ONE 8(11): e79886. doi:10.1371/journal.pone.0079886 Editor: Dong-Yan Jin, University of Hong Kong, Hong Kong Received June 11, 2013; Accepted September 26, 2013; Published November 21, 2013 Copyright: ß 2013 Saxena 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: PR and ARR acknowledge support from Department of Biotechnology, GOI for financial support (DBT Grant NO. BT/PR9367/MED/32/09/2007). 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: ritu@dbeb.iitd.ac.in Introduction Chikungunya virus (CHIKV) is an enveloped, positive sense, single stranded RNA virus of the Togaviridae family and Alphavirus genus [1]. It is an insect-borne virus that is transmitted to humans by virus carrying Aedes mosquito though a few cases of maternal- fetus transmissions have been reported [2,3]. The incubation period for CHIKV ranges from 3 to 7 days, in accordance with other alphaviruses. The classic clinical symptoms post CHIKV infection are abrupt febrile illness, polyarthralgia and maculopap- ular rash [4,5]. However, ,5% of CHIKV cases remain asymptomatic [4–8]. Due to similar initial symptoms, Chikungu- nya is often confused with Dengue, but the characteristic arthralgia in its chronic phase differentiates the two [9]. Until now, there is no effective treatment for the disease and the patient management is mainly symptomatic via analgesics and primary anti-inflammatory drugs [10]. This is primarily because of critical lack of knowledge about mechanism of CHIKV pathogenesis and the molecules involved. Viral infections involve active dynamics between the host cell and the virus particle. Virus utilizes host machinery in various ways to its advantage while host cell activates the innate and adaptive responses to block viral replication and thus the spread [11]. Therefore, designing specific viral therapeutics and diagnos- tics requires identification of key viral and host factors that are required for viral entry and infection. Until now there is no comprehensive study on molecular interactions involved in CHIKV: host biology. Recently, cytokines and chemokines were shown to play an important role in CHIKV immunopathology [12]. Although Interferon -c, Interleukin-2 and Interleukin-10 have been shown to be involved in the pathogenesis of CHIKV, their role is still unexplored and limited studies have been conducted on the virus’ capability to induce cell damage and secretion of associated factors [13]. Certain studies implicating Interleukin–1b, Interleukin–6, and RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) as biomarkers of Chikungunya severity were conducted but their roles were not investigated in detail [14]. Many studies concerning the chronic stage of CHIKV infection and the autoimmune nature of the subsequent arthralgia have been conducted [4,5] but the study of the initial host response is surprisingly bleak. The mobilization of the apoptotic machinery by CHIKV for propagation has been PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e79886