Targeted delivery of human neurofibromin and c-Raf-1 mutants to the cytoplasmic membrane by use of the influenza virus hemagglutinin.
Mutants of human neurofibromin and c-Raf-1 genes were fused to the 3' end of the hemagglutinin (HA) gene of influenza A virus by oligonucleotide-directed polymerase chain reaction (PCR). The two resulting chimeric genes, HA (1-534)/NF1 (1441-1518) and HA (1-534)/Raf-1 (51-132) which we designated HN and HR, respectively, were cloned in a vaccinia virus expression vector (pTMI) under the control of a T7 RNA polymerase promoter. The clones were 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 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.
Nur-E-Kamal
M S
MS
Institut für Immunologie und Molekularbiologie, Fachbereich Veterinämedizin der Freien Universität Berlin, Germany.
Reverey
H
H
Ponimaskin
E
E
Schroth-Diez
B
B
Herrmann
A
A
Schmidt
M F
MF
D000818:Animals; D002451:Cell Compartmentation; D002459:Cell Fusion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003593:Cytoplasm; D005434:Flow Cytometry; D006031:Glycosylation; D006389:Hemagglutinins, Viral / Q000737:chemistry*; D006801:Humans; D025542:Neurofibromin 1; D009975:Orthomyxoviridae; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins
D006389:Hemagglutinins, Viral; D025542:Neurofibromin 1; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf