Heparan Sulfate in Baculovirus Binding and Entry of Mammalian Cells

Abstract

Makkonen and his colleagues published an article in the Journal of Virology entitled "6-O-and N-Sulfated Syndecan-1 Promotes Baculovirus Binding and Entry into Mammalian Cells" (1). Some parts of the data and conclusions seem misleading and not fully justified.Heparan sulfate (HS) alone can bind baculovirus to cells during entry and transduction. Syndecan-1 (SDC-1) contains a protein core and HS chains. Therefore, it is called heparan sulfate proteoglycan (HSPG). I have examined the ability of baculovirus to enter mammalian cells after treatment with heparinase III (EC 4.2.2.8), which specifically digests HS chains from the protein core. For HT1080 cells, heparinase III could significantly reduce baculovirus entry compared with untreated control cells (Fig. 1A). The reduced susceptibility of cells with the baculovirus vector after enzymatic removal of HS indicates that HS plays an important role in baculovirus entry. The protein core of syndecan-1 does not have any role in baculovirus binding.The pgsD-677 cell has a single mutation affecting both N-acetylglucosaminyltransferase and glucuronosyltransferase, which are necessary for polymerization of HS chains, and does not synthesize HS but does produce three times more chondroitin sulfate (CS) than the wild-type CHO-K1 cell does (2). A baculovirus vector was used to evaluate entry into the CHO-K1 cell and its mutant, pgsD-677. The lack of HS expression severely impaired the ability of baculovirus vector to enter into the mutant cell (Fig. 1B). Despite overproduction of CS in pgsD-677 cells, poor transduction in mutant cells further demonstrated the specificity of baculovirus for HS.The authors masked SDC-1 with various concentrations of anti-SDC-1 antibody and tested whether baculovirus binding could be inhibited. They showed that anti-SDC-1 antibody was able to decrease baculovirus binding and transduction of cells. The anti-SDC-1 antibody (sc-5632; Santa Cruz Biotechnology, CA) used in this study was raised against the core protein of SDC-1, but not against HS. Therefore, an explanation should be given for how blocking of SDC-1 (not HS) with its antibody can block the binding of baculovirus.The authors also showed that increasing concentrations of recombinant SDC-1 (ab83609; Abcam, Cambridge, MA) could decrease baculovirus entry and transduction in a dose-dependent manner. Abcam produces recombinant SDC-1 protein in Escherichia coli which does not efficiently glycosylate SDC-1. As a result, recombinant SDC-1 cannot compete with mammalian cell surface SDC-1. Therefore, an explanation of how recombinant SDC-1 can inhibit baculovirus entry into cells is needed.The authors further mentioned that baculovirus could bind 293T cells despite low-level expression of SDC-1. They argued that this could be due to significant differences in the disaccharide composition of HS chains of SDC-1. I found that expression of HS in 293T cells was low and resulted in poor transduction of foamy virus (3), but good transduction of baculovirus. In addition to HS, herpes simplex virus 1 ...

Purification of baculovirus vectors using heparin affinity chromatography

“…A similar level of inhibition was detected at a multiplicity of infection of 1 (Figure 2a) and 2 (Figure 2b). Thus, inhibition of baculovirus infection by heparin confirms our previously published data that baculovirus binds heparan sulfate to enter cells, 22 and suggested that heparin columns could likely be used for purification.…”

“…21,22 We first tested whether heparin could bind and inhibit baculovirus infection of mammalian cells. Increasing concentrations of heparin from 0.5 µg/ml to 10.0 µg/ml were added to baculovirus supernatants, incubated for 1 hour at room temperature, and added to HT1080 cells at a multiplicity of infection of 1 and 2 in separate experiments.…”

“…Based on the finding that baculovirus infection of HT1080 cells is inhibited by heparin, and the report that baculovirus binds to cell surface heparan sulfate, 21,22 we tested whether baculovirus could be purified using heparin affinity chromatography. Prior to chromatography, baculovirus supernatants were treated with 50 Units/ml of Benzonase nuclease for 2 hours at room temperature.…”

“…21,22 The heparin-binding domains of the baculovirus envelope are responsible for receptor-ligand interaction. Here, we demonstrate that baculovirus vector particles can be purified using scalable heparin affinity column chromatography and can be effectively concentrated by ultracentrifugation without loss of infectivity.…”

Baculovirus-Derived Vectors for Immunization and Therapeutic Applications

“…It is suggested that the mechanism by which the baculoviruses ingress mammalian cells is clathrin and dynamin-dependent endocytosis and macropinocytosis. Makkonen showed that this process also requires the host cell proteins heparan sulfate proteoglycans and syndecan-1 that serves as a receptor (Long et al, 2006a,b;Kataoka et al, 2012;Makkonen et al, 2013Makkonen et al, , 2014Nasimuzzaman, 2014). Once inside the cell, the baculovirus virions are transported into the endosome, where acidic conditions promote membrane fusion mediated by GP64, and the genetic material is thus liberated into the cytoplasm.…”