Virus Research 225 (2016) 23–32 Contents lists available at ScienceDirect Virus Research j ourna l h o mepa ge: www.elsevier.com/locate/virusres Efficient porcine reproductive and respiratory syndrome virus entry in MARC-145 cells requires EGFR-PI3K-AKT-LIMK1-COFILIN signaling pathway Rui Wang a , Xin Wang a , Jia-qiang Wu c , Bo Ni a , Li-bing Wen d , Li Huang a , Ying Liao b , Guang-zhi Tong b , Chan Ding b , Xiang Mao a,b,∗ a College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu Province, China b Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China c Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan, China d Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Science, Nanjing, China a r t i c l e i n f o Article history: Received 3 February 2016 Received in revised form 16 July 2016 Accepted 8 September 2016 Available online 9 September 2016 Keywords: Porcine reproductive and respiratory syndrome virus Entry Epidermal growth factor receptor Cofilin a b s t r a c t Viruses have evolved diverse strategies to take over cellular machinery to facilitate their infection. In our studies presented here, we first demonstrated that Src kinase was involved in PRRSV entry in MARC- 145 cells. Further studies demonstrated epidermal growth factor receptor (EGFR) was activated by the currently unknown mechanism(s) during PRRSV entry, which subsequently initiated EGFR downstream signal pathways, such as PI3K/AKT/LIMK1. Through these pathways, the virus entry signal was ultimately transferred to cofilin, which might regulate the actin fragmentation and reorganization to facilitate the virus penetration and cytoplasmic trafficking. © 2016 Published by Elsevier B.V. 1. Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative pathogen for porcine reproductive and respiratory syndrome (PRRS), an infectious disease that causes major eco- nomic impact in the swine industry worldwide (Neumann et al., 2005). PRRSV is a positive-sense RNA virus that belongs to the fam- ily of Arteriviridae and the order of Nidovirales (Cavanagh, 1997; Conzelmann et al., 1993; Meulenberg et al., 1993). The viral genome is about 15 kb in length, which consists of the 5 ′ un-translated region (UTR), ten open reading frames (ORF1a, ORF1b, ORF2a, ORF2b, and ORFs 3 through 7, including ORF5a), and the 3 ′ UTR (Johnson et al., 2011). The ORFs 1a and 1b account for 75% of the viral genome and encode two long polypeptides (pp), pp1a and pp1ab; after enzymatic cleavage, the polypeptides produce 14 non- structural proteins (nsps) that are responsible for viral replication. The ORFs 2–7 encode eight structural proteins, including GP2a, GP2b, GP3, GP4, GP5, GP5a, matrix protein (M) and nucleocapsid ∗ Corresponding author at: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu Province, China. E-mail addresses: xmao@njau.edu.cn, xmao@shvri.ac.cn (X. Mao). protein (N) (Allende et al., 1999; Firth et al., 2011; Johnson et al., 2011), which are mainly associated with virus entry, assembling, and release. Viruses have evolved diverse strategies to take over cellular machinery to facilitate their infection. It has been demonstrated that PRRSV infects the target cells through receptor-mediated endocytosis (Nauwynck et al., 1999). Heparin sulfate, sialoadhesin (Sn), CD163, CD151 and vimentin have been identified as PRRSV receptors, and they play different roles in PRRSV infection. Heparan sulfate glycosaminoglycans (GAGs) on the macrophage surface are assumed to function as PRRSV attachment factors that concentrate virions on the cell surface, hence allowing a more efficient infec- tion. Sialic acids on the viral envelope proteins interact with the porcine Sn receptor on the macrophage surface, thereby trigger- ing internalization of the virus. Upon binding to sialoadhesin, the virus is internalized via clathrin-mediated endocytosis (Delputte and Nauwynck, 2004; Nauwynck et al., 1999). CD163 is respon- sible for uncoating virus particles and releasing the viral genome (Van Gorp et al., 2008, 2010; Welch and Calvert, 2010). An acidic pH is required to trigger a fusion event between the viral envelope and the endosomal membrane to facilitate viral genome release (Kreutz and Ackermann, 1996; Nauwynck et al., 1999). Further identifica- http://dx.doi.org/10.1016/j.virusres.2016.09.005 0168-1702/© 2016 Published by Elsevier B.V.