Average of three separate experiments, m ± SEM (**), P < 0.01 ns, not significant. BV CAR was a recombinant BV expressing the human CAR glycoprotein, and BV CAR-infected cells released CAR-displaying virions in the extracellular medium. cells expressing irrelevant membrane glycoprotein. Control consisted of BV CAR-infected cells, i.e. 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. 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. Cells were reacted with anti-HA tag monoclonal antibody followed by Alexa Fluor ® 488-labeled complementary antibody. 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). In situ analysis of scFvE2/p17 and scFvG2/p17 proteins in Sf9 cells. Subcellular fractions were analyzed by SDS-PAGE and Western blotting using anti-HA tag antibody. Sf9 cells infected with BV-E2/p17 or BV-G2/p17 as indicated on top of the panel were harvested at 48 h pi and processed for cell fractionation into cytosolic compartment (Cy), membranes (Mb), nuclear compartment (Nu) and cytoskeletal-associated proteins (Sk). M, molecular mass markers, with their apparent molecular masses indicated in kilodaltons (kDa) on the right side of the blots. Lysates of mock-infected cells (mo) or BV-E2/p17- or BV-G2/p17-infected cells harvested at 24, 48 and 72 h pi, as indicated on top of the panel were clarified by centrifugation, and soluble fraction (S) and pelletable material (P) analyzed as above. Whole cell lysates were analyzed by SDS-PAGE and Western blotting using anti-HA tag antibody. Sf9 cells were mock-infected (lanes mo), or infected with BV-E2/p17 or BV-G2/p17, and harvested at 24, 48 and 72 h pi, as indicated on top of the panel. 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.Įxpression, solubility and cellular localization of scFvE2/p17 and scFvG2/p17 in Sf9 cells. 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 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.Įxpression of scFvE2/p17 in insect cells using a BV vector resulted in baculoviral progeny displaying scFvE2/p17. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. 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. 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. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. 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. 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. 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.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |