Research paper Green fluorescent protein – Tagged HCV non-enveloped capsid like particles: Development of a new tool for tracking HCV core uptake Konstantina Katsarou a, 1 , Elisavet Serti a, 1 , Panagiota Tsitoura a , Alexandros A. Lavdas b , Agoritsa Varaklioti c , Angela-Maria Pickl-Herk d , Dieter Blaas d , Devrim Oz-Arslan a , Rong Zhu e , Peter Hinterdorfer e , Penelope Mavromara a , Urania Georgopoulou a, * a Molecular Virology Laboratory, Hellenic Pasteur Institute,11521 Athens, Greece b Laboratory of Cellular and Molecular Neurobiology, Hellenic Pasteur Institute, Greece c 2nd Regional Blood Transfusion and Haemophilia Centre, Laikon General Hospital, Greece d Max F. Perutz Laboratories, Medical University of Vienna, Austria e Institute for Biophysics, J. Kepler University, A-4040 Linz, Austria article info Article history: Received 9 January 2009 Accepted 15 April 2009 Available online 4 May 2009 Keywords: Hepatitis C virus Fluorescent non-enveloped capsid-like particle Cellular uptake abstract Circulating ‘free’ non-enveloped Hepatitis C virus (HCV) core protein has been demonstrated in HCV- infected patients, and HCV subgenomes with deletions of the envelope proteins have been previously identified. Initial studies from our laboratory, previously published, indicated that expression of HCV core in insect cells can direct the formation of capsid-like particles lacking the envelope glycoproteins. These protein nanospheres, morphologically similar to natural capsids, were shown to be taken up by human hepatic cells and to produce cell-signalling events. To follow the intracellular fate of these particles we fused the core protein to eGFP. We demonstrate that the chimeric proteins core 173 -eGFP, eGFP-core 191 and eGFP-core 173 can be efficiently expressed, self-assembled, and form fluorescent non-enveloped capsid-like particles. By using confocal microscopy and FACS analysis, we provide evidence that the fluorescent nanospheres can not only enter human hepatic cells – the main target of HCV – but also human immune cells such as T and B lymphocytes, as well as human myeloid leukaemia cells differ- entiated along the monocyte/macrophage-like pathway. The fluorescent particles might thus be used to trace the intracellular trafficking of naked HCV capsids as showed by live microscopy and to further understand their biological significance. Ó 2009 Elsevier Masson SAS. All rights reserved. 1. Introduction Hepatitis C virus (HCV) is an enveloped virus belonging to the Flaviviridae family [1]. Its nucleocapsid is surrounded by a lipid bilayer containing the glycoproteins E1 and E2. However, different forms of HCV particles have been found in the circulation of infected individuals, among them naked capsids [2,3]. A plausible explanation for the presence of non-enveloped particles in the blood is their release into the circulation by the lysis of infected hepatocytes that accompanies liver inflammation. Non-enveloped particles have also been detected as viral inclusions in the cyto- plasm of liver cells of infected patients [40]. Furthermore, HCV subgenomes with in-frame deletions of both envelope proteins were identified with relatively high abundance in the liver as well as in the serum of HCV infected individuals. The biological signifi- cance of these findings remains unclear although recently it was suggested that defective HCV clones might be associated with poor response to combination therapy [4–6]. Defective RNA viral genomes lacking the envelope coding sequence or possessing a natural stop codon have also been recently reported for other flaviviruses with important implications for their evolutionary dynamics and viral persistence [35]. Recently, Tsitoura et al. [7] reported the generation of recombinant non-enveloped HCV core particles in the absence of other HCV proteins and, more importantly, demonstrated that these naked capsids can be taken up by cells and induce cell-sig- nalling phenomena. These intriguing properties of HCV core protein can be of great interest as a recent report highlights the possibility that subgenomic RNA resembling natural occurring deletion mutants are efficiently trans-packaged into virus-like particles by helper virus or helper cell lines [36]. For these reasons we aimed at developing a strategy for the labelling of the HCV non-enveloped capsid by using enhanced * Corresponding author. Tel.: þ30 210 6478876; fax: þ30 210 6426323. E-mail address: uraniag@pasteur.gr (U. Georgopoulou). 1 Authors contributed equally. Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.04.016 Biochimie 91 (2009) 903–915