LETTER TO THE EDITOR
Heparan Sulfate in Baculovirus Binding and Entry of Mammalian Cells Md Nasimuzzaman Department of Hematology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
akkonen 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
April 2014 Volume 88 Number 8
FIG 1 (A) Heparinase III digestion of cell surface HS severely reduced baculovirus transduction. Cells were incubated for 1 h at 37°C with 1.0 and 2.0 milli international units (mIU)/ml of heparinase III (Sigma, St. Louis, MO) and transduced with BacMam vector (Life Technologies, San Diego, CA) for 1 h at 37°C. Unbound virus was removed by washing. (B) HS-deficient CHO-K1 cells were less susceptible to baculovirus vector transduction. BacMam vector was used to transduce wild-type CHO-K1 and mutant pgsD-677 cells. The cells were analyzed for green fluorescent protein (GFP) expression 48 h later. Values are means and standard errors of means (error bars) of three experiments. MOI, multiplicity of infection.
virus (3), but good transduction of baculovirus. In addition to HS, herpes simplex virus 1 uses herpesvirus entry mediator (HveA) and members of the nectin family receptor (HveC) for binding to cells (4, 5). It should be investigated whether baculovirus might use any other binding molecules during entry into 293T cells.
Editor: G. McFadden Address correspondence to [email protected]. For the author reply, see doi:10.1128/JVI.00083-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00024-14
Journal of Virology
p. 4607– 4608
jvi.asm.org
4607
Downloaded from http://jvi.asm.org/ on March 16, 2015 by JAMES COOK UNIVERSITY
M
Letter to the Editor
REFERENCES 1. Makkonen KE, Turkki P, Laakkonen JP, Yla-Herttuala S, Marjomaki V, Airenne KJ. 2013. 6-O- and N-sulfated syndecan-1 promotes baculovirus binding and entry into mammalian cells. J. Virol. 87:11148 –11159. http: //dx.doi.org/10.1128/JVI.01919-13. 2. Lidholt K, Weinke JL, Kiser CS, Lugemwa FN, Bame KJ, Cheifetz S, Massague J, Lindahl U, Esko JD. 1992. A single mutation affects both N-acetylglucosaminyltransferase and glucuronosyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 89:2267–2271. http://dx.doi.org/10.1073 /pnas.89.6.2267.
3. Nasimuzzaman M, Persons DA. 2012. Cell membrane-associated heparan sulfate is a receptor for prototype foamy virus in human, monkey, and rodent cells. Mol. Ther. 20:1158 –1166. http://dx.doi.org/10 .1038/mt.2012.41. 4. Montgomery RI, Warner MS, Lum BJ, Spear PG. 1996. Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87:427– 436. http://dx.doi.org/10.1016/S0092-8674(00)81363-X. 5. Geraghty RJ, Krummenacher C, Cohen GH, Eisenberg RJ, Spear PG. 1998. Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor. Science 280:1618 –1620. http://dx.doi .org/10.1126/science.280.5369.1618.
Downloaded from http://jvi.asm.org/ on March 16, 2015 by JAMES COOK UNIVERSITY
4608
jvi.asm.org
Journal of Virology
Heparan Sulfate in Baculovirus Binding and Entry of Mammalian Cells Md Nasimuzzaman Department of Hematology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
akkonen 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
April 2014 Volume 88 Number 8
FIG 1 (A) Heparinase III digestion of cell surface HS severely reduced baculovirus transduction. Cells were incubated for 1 h at 37°C with 1.0 and 2.0 milli international units (mIU)/ml of heparinase III (Sigma, St. Louis, MO) and transduced with BacMam vector (Life Technologies, San Diego, CA) for 1 h at 37°C. Unbound virus was removed by washing. (B) HS-deficient CHO-K1 cells were less susceptible to baculovirus vector transduction. BacMam vector was used to transduce wild-type CHO-K1 and mutant pgsD-677 cells. The cells were analyzed for green fluorescent protein (GFP) expression 48 h later. Values are means and standard errors of means (error bars) of three experiments. MOI, multiplicity of infection.
virus (3), but good transduction of baculovirus. In addition to HS, herpes simplex virus 1 uses herpesvirus entry mediator (HveA) and members of the nectin family receptor (HveC) for binding to cells (4, 5). It should be investigated whether baculovirus might use any other binding molecules during entry into 293T cells.
Editor: G. McFadden Address correspondence to [email protected]. For the author reply, see doi:10.1128/JVI.00083-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00024-14
Journal of Virology
p. 4607– 4608
jvi.asm.org
4607
Downloaded from http://jvi.asm.org/ on March 16, 2015 by JAMES COOK UNIVERSITY
M
Letter to the Editor
REFERENCES 1. Makkonen KE, Turkki P, Laakkonen JP, Yla-Herttuala S, Marjomaki V, Airenne KJ. 2013. 6-O- and N-sulfated syndecan-1 promotes baculovirus binding and entry into mammalian cells. J. Virol. 87:11148 –11159. http: //dx.doi.org/10.1128/JVI.01919-13. 2. Lidholt K, Weinke JL, Kiser CS, Lugemwa FN, Bame KJ, Cheifetz S, Massague J, Lindahl U, Esko JD. 1992. A single mutation affects both N-acetylglucosaminyltransferase and glucuronosyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 89:2267–2271. http://dx.doi.org/10.1073 /pnas.89.6.2267.
3. Nasimuzzaman M, Persons DA. 2012. Cell membrane-associated heparan sulfate is a receptor for prototype foamy virus in human, monkey, and rodent cells. Mol. Ther. 20:1158 –1166. http://dx.doi.org/10 .1038/mt.2012.41. 4. Montgomery RI, Warner MS, Lum BJ, Spear PG. 1996. Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87:427– 436. http://dx.doi.org/10.1016/S0092-8674(00)81363-X. 5. Geraghty RJ, Krummenacher C, Cohen GH, Eisenberg RJ, Spear PG. 1998. Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor. Science 280:1618 –1620. http://dx.doi .org/10.1126/science.280.5369.1618.
Downloaded from http://jvi.asm.org/ on March 16, 2015 by JAMES COOK UNIVERSITY
4608
jvi.asm.org
Journal of Virology
