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.Conclusion: 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. © 2010 Kitidee et al; licensee BioMed Central Ltd.
Figures
![Figure 2 In situ analysis of scFvE2/p17 and scFvG2/p17 proteins in Sf9 cells. (A), Immunofluorescence (IF) microscopy. Sf9 cells expressing scFvE2/p17 alone (i, ii), or coexpressing scFvE2/p17 and Pr55Gag (iii), were harvested at 48 h pi and nonpermeabilized (i), or permeabilized with Triton X-100 (ii, iii). Cells were reacted with anti-HA tag monoclonal antibody followed by Alexa Fluor® 488-labeled complementary antibody. (B), Flow cytometry. Nonpermeabilized Sf9 cells expressing scFvE2/p17, scFvG2/p17 or the scFv-N18E2/M6 chimera were harvested at 48 h pi, reacted with antibodies as in (A), and analyzed by flow cytometry. Results shown were the proportion of HA tag-positive cells, expressed as the fold ratio over the values of control cells, attributed the value of 1. Control consisted of BVCAR-infected cells, i.e. cells expressing irrelevant membrane glycoprotein. BVCAR was a recombinant BV expressing the human CAR glycoprotein, and BVCAR-infected cells released CAR-displaying virions in the extracellular medium [41]. Average of three separate experiments, m ± SEM; (**), P < 0.01; ns, not significant.](https://s3-eu-west-1.amazonaws.com/com.mendeley.prod.article-extracted-content/images/3a1651b9-f282-3eb9-85db-5de984fe16eb/thumbnail-f14e1631-1fb4-4f29-947c-9d292dcd21d9-1.png)
![Figure 7 Topology and functionality of BV-displayed scFvE2/p17 molecules. Baculoviral progeny recovered from the culture medium of Sf9 cells infected with BV-E2/p17 was isolated by ultracentrifugation. (a), BV-E2/p17 virions were immobilized on ELISA plate and reacted with antiHA tag monoclonal antibody and peroxidase-labeled complementary antibody. Controls were from left to right, respectively: uncoated well, well coated with antigen with no BV addition, and well coated with antigen with addition of irrelevant baculovirus particles (CAR-displaying BVCAR; [41]). (b), Schematic model of scFvE2/p17 molecule displayed at the surface of a BV particle. GP64, baculoviral envelope major glycoprotein. BVE2/p17 virions were reacted in ELISA with immobilized antigen, (c) H6MA-CA-embedded p17 epitope, or (d) synthetic peptide p17.1. Average of three separate experiments, m ± SEM; (*), P < 0.05); (**), P < 0.01).](https://s3-eu-west-1.amazonaws.com/com.mendeley.prod.article-extracted-content/images/3a1651b9-f282-3eb9-85db-5de984fe16eb/thumbnail-8cc94ec9-8c70-4efe-a70d-e44059d71cdf-7.png)
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CITATION STYLE
Kitidee, K., Nangola, S., Gonzalez, G., Boulanger, P., Tayapiwatana, C., & Hong, S. S. (2010). Baculovirus display of single chain antibody (scFv) using a novel signal peptide. BMC Biotechnology, 10. https://doi.org/10.1186/1472-6750-10-80
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