Altering the surface properties of baculovirus Autographa californica NPV by insertional mutagenesis of the envelope protein gp64

Abstract

The envelope protein gp64 of the baculovirus Autographa californica nuclear polyhedrosis virus is essential for viral entry into insect cells, as the glycoprotein both mediates pHdependent membrane fusion and binds to host cell receptors. Surface modification of baculovirus particles by genetic engineering of gp64 has been demonstrated by various strategies and thus has become an important and powerful tool in molecular biology. To improve further the presentation of peptides on the surface of baculovirus particles, several insertion sites within the gp64 envelope protein were selected by their theoretical maximum surface probability and investigated for efficient peptide presentation. The ELDKWA peptide of the gp41 of HIV-1, specific for the human mAb 2F5, was inserted into 17 different positions of the glycoprotein gp64. Propagation of viruses was successful in 13 cases, mutagenesis at four positions did not result in production of intact virus particles. Western blotting, FACS analysis and ELISA were used for characterization of the different binding properties of the mutants. Insertion of this peptide into the native envelope protein resulted in high avidity display on the surface of baculovirus particles. This approach offers the possibility of effective modification of surface properties in regard to host range specificity and antigen display.

A pH-Sensitive Heparin-Binding Sequence from Baculovirus gp64 Protein Is Important for Binding to Mammalian Cells but Not to Sf9 Insect Cells

“…AcV1, another monoclonal antibody against an epitope extensively overlapping with the heparin-binding region, does not inhibit virus binding to the cell surface (41). Lastly, this heparinbinding region can tolerate insertions of and substitutions by a variety of foreign sequences without affecting virus titers, suggesting that the heparin-binding function is dispensable for infection of Sf9 cells (10,37,51). Our results for the effects of GT24 and heparin on virus infectivity also support this hypothesis.…”

Virus-Coated Layer-by-Layer Colloids as a Multiplex Suspension Array for the Detection and Quantification of Virus-Specific Antibodies

“…A multiplex influenza-coated suspension array could be used to detect and quantify relevant virus-specific antibody titers in patient blood and to subtype influenza virus infections. Moreover, fusing existing recombinant baculoviral HIV-1 gp120 surface libraries (16,27 ) to our composite could facilitate a more precise and reliable determination of early-phase HIV infections.…”

Preferential Gene Transfer of Lentiviral Vectors to Liver-Derived Cells, Using a Hepatitis B Peptide Displayed on GP64

“…In the baculoviral vector system this site was previously described for the introduction and surface display of peptides ranging in size from 6 to 12 amino acids, with the resulting baculovirus particles having an approximately 5-fold reduction in viral infectivity compared with GP64 (Ernst et al, 2000;Spenger et al, 2002). Characterization of the PreS1-GP64 protein showed that it was indeed expressed and resulted in surface display of the PreS1 peptide as determined by immunostaining and flow cytometric measurements ( Fig.…”

“…At present, the structure of GP64 has not yet been determined, the domains of the protein involved in cell binding have not been characterized, and the mammalian cell receptor(s) or general cell surface motifs used for cell binding have not been described (Duisit et al, 1999;Tani et al, 2001;Ghosh et al, 2002). Because previous reports of peptide insertions into the GP64 envelope protein did not show alteration of baculoviral tropism (Ernst et al, 2000;Spenger et al, 2002) and because no modifications were made to ablate the native tropism, the observed loss in gene transfer to HeLa cells was not expected. This suggests that insertion of the PreS1 peptide may alter the structure of the GP64 protein, in particular the domains involved in recognizing mammalian cell surface molecules, as the PreS1-GP64 protein appears to retain other functions (Fig.…”