title: Baculovirus display of single chain antibody (scFv) using a novel signal peptide.
abstract: BACKGROUND
  Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication.
  
  
  RESULTS
  Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6.
  
  
  CONCLUSIONS
  Expression of scFvE2/p17 in insect cells using a BV vector resulted in baculoviral progeny displaying scFvE2/p17. The function required for BV envelope incorporation was carried by the N-terminal octadecapeptide of scFvE2/p17, which acted as a signal peptide for BV display. Fusion of this peptide to the N-terminus of scFv molecules of interest could be applied as a general method for BV-display of scFv in a GP64- and VSV-G-independent manner.
authors.affiliation: University Lyon 1, INRA UMR-754, Retrovirus & Comparative Pathology, 50, avenue Tony Garnier, 69366 Lyon Cedex 07, France.
authors.forename: Kuntida
authors.initials: K
authors.lastname: Kitidee
authors.forename: Sawitree
authors.initials: S
authors.lastname: Nangola
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Gonzalez
authors.forename: Pierre
authors.initials: P
authors.lastname: Boulanger
authors.forename: Chatchai
authors.initials: C
authors.lastname: Tayapiwatana
authors.forename: Saw-See
authors.initials: SS
authors.lastname: Hong
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D005822:Genetic Vectors; D015488:HIV Antigens / Q000276:immunology; D007313:Insecta / Q000166:cytology; D021382:Protein Sorting Signals / Q000235:genetics*; D057127:Single-Chain Antibodies / Q000096:biosynthesis*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000276:immunology
chemical_list: D015488:HIV Antigens; D021382:Protein Sorting Signals; D057127:Single-Chain Antibodies; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1