Virus Research 108 (2005) 45–55 Insertion of targeting domains into the envelope glycoprotein of Moloney murine leukemia virus (MoMLV)-based vectors modulates the route of mCAT-1-mediated viral entry A. Viejo-Borbolla a,b,c,∗ , M. Pizzato d , E.D. Blair b,1 , T.F. Schulz a,c a Department of Medical Microbiology and Genitourinary Medicine, University of Liverpool, Liverpool, UK b Department of Applied Diagnostics, Genetics Research, GlaxoSmithKline, Stevenage, UK c Department of Virology, Hannover Medical School, Hannover, Germany d Department of Histology, Microbiology and Medical Biotechnology, University of Padova, Padova, Italy Received 22 April 2004; received in revised form 23 July 2004; accepted 23 July 2004 Available online 22 September 2004 Abstract Several groups have inserted targeting domains into the envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMLV) in an attempt to produce targeted retroviral vectors for human gene therapy. While binding of these modified Envs to the target molecule expressed on the surface of human cells was observed, specific high-titer infection of human cells expressing the target molecule was not achieved. Here we investigate the initial steps in the entry process of targeted MoMLV vectors both in murine and human cells expressing the MoMLV receptor, the mouse cationic amino acid transporter-1 (mCAT-1). We show that insertion of a small ligand targeted to E-selectin and of a single chain antibody (scFv) targeted to folate-binding protein (FBP) into the N-terminus of MoMLV Env results in the reduction of the infectivity and the kinetics of entry of the MoMLV vectors. The use of soluble receptor-binding domain (sRBD), bafilomycin A1 (BafA1) and methyl-- cyclodextrin (MC) increase the infectivity of the MoMLV vectors targeted to FBP (MoMLV-FBP) suggesting that the scFv targeted to FBP increases the threshold for fusion and might re-route entry of the targeted MoMLV-FBP vector towards an endocytic, non-productive pathway. © 2004 Elsevier B.V. All rights reserved. Keywords: MoMLV; Targeted vectors; sRBD; Entry; Fusion 1. Introduction Retroviruses bind to their receptor(s) through the surface (SU) subunit of the envelope glycoprotein (Env). Following specific envelope–receptor interactions, conformational changes take place in Env that result in the formation of an extended trimeric coiled-coil at the N-terminus of the transmembrane (TM) subunit, in a process reminiscent of the spring-loaded mechanism of the hemagglutinin (HA) protein of influenza virus (Carr and Kim, 1993; Bullough et ∗ Corresponding author. Present address: Department of Molecular and Cell Biology, Centro Nacional de Biotecnolog´ ıa, CSIC, C/Darwin 3, 28049 Madrid, Spain. Tel.: +34 91 585 4678; fax: +34 91 585 4506. E-mail address: aviejo@cnb.uam.es (A. Viejo-Borbolla). 1 Present address: Integrated Medicines Ltd., Topfield House, Caxton, Cambs CB3 8PQ, UK. al., 1994; Fass et al., 1996; Weissenhorn et al., 1997, 1999). This process allows the interaction between the viral fusion peptide and the cellular membrane leading to fusion (Hart et al., 1991; Moore et al., 1990; Weissenhorn et al., 1999; Wyatt et al., 1995). Moloney murine leukemia virus’ (MoMLV) fusion event takes place in different cellular compartments depending on the cell system used. Thus, the use of lysosomotropic agents has shown that MoMLV entry in most naturally infected cells requires internalization within a low-pH cellular organelle (Andersen and Nexo, 1983; McClure et al., 1990). The exact nature of the internalized compartment has not been deter- mined yet although a report suggests that caveolae play an important role in MoMLV entry (Lu and Silver, 2000). In contrast, fusion takes place at the cell surface membrane in the transformed XC cell line and in avian DF-1 cell line ex- 0168-1702/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2004.07.012