doi: 10.1128/JVI.06357-11

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PMID: 22072779

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Abstract

Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.

INTRODUCTION

The baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) is a large enveloped insect virus widely used for recombinant protein production (). After packing its double-stranded DNA genome into a rod-shaped nucleocapsid, the budded virus (BV) is released from the infected insect cells with gp64 glycoprotein forming peplomers at one end of the virus particle (, , ). In recent years, BV has also been exploited for gene therapy applications, as it can efficiently transduce many types of mammalian cells with little cytotoxicity (, , , ). Heparan sulfate, the highly negatively charged polysaccharide on the surface of mammalian cells, serves as the major binding molecule during baculovirus transduction ().

gp64 of baculovirus mediates both the initial binding to insect cells and the subsequent endosome escape step (, , ). As a class III fusion protein, it exists as trimers in both the pre- and postfusion states and does not undergo proteolytic cleavage during the pH-dependent conformational change (). The postfusion crystal structure of gp64 has been solved, and it closely resembles those of vesicular stomatitis virus G glycoprotein (VSVG) and glycoprotein B of herpes simplex virus 1 (HSV-1) in the same class, with 5 domains similarly positioned in the elongated molecule (, , ). gp64 has been used to pseudotype lentivirus, and the resulting virus can efficiently transduce a wide variety of mammalian cells (, ). This suggests that gp64 also mediates virus binding to the surface of mammalian cells. Further study on a soluble form of gp64 without the transmembrane domain demonstrated that gp64 binds to heparin in a pH-dependent manner, with a stronger binding at pH 6.2 than at pH 7.4 ().

Heparin-binding sites are rich in basic amino acids (, , , ). Examining the sequence of gp64, we found that the central region of gp64 from residues 271 to 292 is also rich in basic amino acids. In the crystal structure of gp64, this central region consists of a disordered segment and surrounding loop regions which form a linker between domains II and III (). B12D5, a monoclonal antibody against gp64, has a binding epitope of KKRPPTWRHNV which lies within the disordered segment (). These pieces of evidence suggest that the central basic region is exposed on the surface of gp64 protein and may be involved in binding to heparin. In this study, we demonstrated the heparin-binding activity of this central region and mapped the sequence that closely resembles the binding pattern of soluble gp64 protein. The difference in requirement for heparan sulfate between mammalian and insect cells during virus attachment was also investigated.

MATERIALS AND METHODS

Cell lines.

Spodoptera frugiperda (Sf9) insect cells were maintained in spinner flasks using Sfm-900 II serum-free medium (SFM; Invitrogen, Carlsbad, CA). Human U251 glioma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 μg/ml). Jurkat T cells were grown in suspension in RPMI 1640 medium containing 10% FBS, 100 units/ml of penicillin, and 100 μg/ml of streptomycin.

Construction of recombinant baculoviruses.

CMV-Luc and CMV-EGFP baculoviruses with a luciferase or enhanced green fluorescent protein (EGFP) reporter gene, respectively, driven by the human cytomegalovirus (CMV) early promoter have been described previously (). VSVG-CMV-EGFP baculovirus, with the vesicular stomatitis virus G glycoprotein (VSVG) gene under the control of the strong viral polyhedrin promoter (pPH) and EGFP under the CMV promoter, was constructed using the BAC-to-BAC baculovirus expression system according to the supplier's manual (Invitrogen, Carlsbad, CA). In brief, a CMV-EGFP expression cassette was inserted downstream of the p10 promoter in the pFastBac Dual plasmid (). The VSVG gene was cut out from pLP/VSVG plasmid (Invitrogen, Carlsbad, CA) using EcoRI restriction enzyme and inserted into the modified pFastBac Dual vector. Bacmid DNA generated from this transfer vector was used to transfect Sf9 cells. The resulting recombinant baculovirus was further amplified in Sf9 cells at a multiplicity of infection (MOI) of 0.1.

Heparin-agarose bead binding assay.

Three fluorescein isothiocyanate (FITC)-labeled peptides encompassing the central region of gp64 from residues 272 to 287, GV18 (GGVEHRVKKRPPTWRHNV), GT24 (GGKVEHRVKKRPPTWRHNVRAKYT), and KT24 (KRKVEHRVKKRPPTWRHNVRAKYT), were purchased from GL Biochem (Shanghai, China). One milliliter of heparin-agarose beads (Sigma-Aldrich, St. Louis, MO) was equilibrated with 10 ml of 0.3× phosphate-buffered saline (PBS), pH 6.2. Fifteen micrograms of fluorescently labeled peptides was diluted in 500 μl of 0.3× PBS, pH 6.2, and loaded onto the column. After being washed with 6 ml of 0.3× PBS, pH 6.2, the peptides were eluted sequentially with 6 ml of 1× PBS, pH 6.2; 1× PBS, pH 7.4; and 2× PBS, pH 7.4. Eric whitacre cloudburst youtube. The eluted fractions were collected and measured for fluorescence intensity using a Tecan GENios Pro (Tecan Group Ltd., Männedorf, Switzerland).

Transduction assay.

U251 cells were seeded into 24-well plates at 2 × 10⁵ cells per well. One day later, normal cell culture medium was replaced with a mixture of 50% FBS and 50% 1× PBS, pH 7.4 or 6.2.

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