Ž . Biochimica et Biophysica Acta 1338 1997 233–243 Targeted delivery of human neurofibromin and c-Raf-1 mutants to the cytoplasmic membrane by use of the influenza virus hemagglutinin Mohammad S.A. Nur-E-Kamal b , Helmut Reverey a , Evgeni Ponimaskin a , Britta Schroth-Diez c , Andreas Herrmann c , Michael F.G. Schmidt a, ) a Institut fur Immunologie und Molekularbiologie, Fachbereich Veterinamedizin der Freien UniÕersitat Berlin, Luisenstrasse 56, D-10117 ¨ ¨ ¨ Berlin, Germany b Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE UniÕersity, Al Ain 17666, United Arab Emirates c Institut fur Biologie r Biophysik, Mathematisch-Naturwissenschaftliche Fakultat I der Humboldt UniÕersitat Berlin, InÕalidenstraße 42, ¨ ¨ ¨ D-10115 Berlin, Germany Received 2 September 1996; revised 12 November 1996; accepted 13 November 1996 Abstract X Ž . Mutants of human neurofibromin and c-Raf-1 genes were fused to the 3 end of the hemagglutinin HA gene of Ž . influenza A virus by oligonucleotide-directed polymerase chain reaction PCR . The two resulting chimeric genes, HA Ž . Ž . Ž . Ž . 1–534 rNF1 1441–1518 and HA 1–534 rRaf-1 51–132 which we designated HN and HR, respectively, were cloned Ž . in a vaccinia virus expression vector pTM1 under the control of a T7 RNA polymerase promoter. The clones were Ž . expressed in a monkey cell line CV-1 and the resulting chimeric proteins analysed. We found that expression levels of the chimeric proteins were similar to that of wild-type HA protein. Comparative endoglycosidase treatment revealed that the expressed chimeric proteins HN and HR were processed as wild-type HA, and FACS-analysis showed that both chimeric expression products localised in the cell membrane as the wild-type control. HN and HR expressing cells showed similar fusogenic activity as CV-1 cells transfected with wild-type HA indicating the correct topology of the fusion inducing Ž . Ž . portion HA of these chimera in the membrane. These findings show that the influenza virus hemagglutinin HA is a suitable vehicle to target foreign proteins with therapeutical potential into the cell membrane. In this respect HN and HR could potentially be used to block the abnormal signals generated by particular proteins in the cell membrane that lead to cell transformation. q 1997 Elsevier Science B.V. Keywords: Hemagglutinin; Neurofibromin; c-Raf-1; Chimera; Target; Palmitoylation Abbreviations: HA, hemagglutinin; PCR, polymerase chain reaction; GTP, guanosine triphosphate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DMEM, Dul- becco’s modified Eagle medium; RBC, red blood cells; PNGase F, peptide N-glycosidase F ) Corresponding author. Fax: q49 30 20936171. 1. Introduction The use of chimeric proteins to target foreign polypeptides to the cell membrane is a new approach in the important area of signaling and growth control. In these respects the cytoplasmic face of the plasma membrane is the most promising target, since this is one main location where growth is controlled by Ž . proteins as for instance Ras and Raf see below . 0167-4838r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0167-4838 96 00206-3