Abstract
Cell surface heparan sulfate serves as an initial receptor for a number of herpesviruses including pseudorabies virus (PrV). It has been demonstrated that the heparan sulfate-binding domain of PrV glycoprotein C is composed of three discrete clusters of basic residues corresponding to amino acids 76-RRKPPR-81, 96-HGRKR-100, and 133-RFYRRGRFR-141, respectively, and that these clusters are functionally redundant, i.e. each of them could independently support PrV attachment to cells (Flynn, S. J., and Ryan, P. (1996) J. Virol. 70, 1355-1364). To evaluate the functional significance of each of these clusters we have used PrV mutants in which, owing to specific alterations in glycoprotein C, the heparan sulfate-binding site is dominated by a single specific cluster. These mutants exhibited different patterns of susceptibility to selectively N-, 2-O-, and 6-O-desulfated heparin preparations in virus attachment/infectivity assay. Moreover PrV mutants differed as regard to efficiency of their attachment to and infection of cells pretreated with relatively low amounts of heparan sulfate-degrading enzymes. Furthermore glycoprotein C species, purified from respective mutants, bound heparin oligosaccharide fragments of different minimum size. These differences suggest that specific clusters of basic amino acids of the heparan sulfate-binding domain of glycoprotein C may support PrV binding to different structural features/stretches within the heparan sulfate chain.
Highlights
Cell surface heparan sulfate serves as an initial receptor for a number of herpesviruses including pseudorabies virus (PrV)
In PrV580C-III, PrV581C-II, and PrV582C-I two out of the three clusters of basic residues that compose the Heparan sulfate (HS)/heparin-binding domain were disrupted by simultaneous replacement of two pairs of basic amino acid residues with neutral amino acids
It has been demonstrated that the HS-binding domain of PrV glycoprotein C (gC) is composed of three discrete clusters of basic amino acid residues and that each of these clusters could independently support PrV attachment to cells [22]
Summary
Materials—Chondroitinase AC I Flavo (chondroitin AC lyase; EC 4.2.2.5) was obtained from the Seikagaku Co. (Tokyo, Japan). Dense monolayers of MDBK (3-dayold) cells in six-well plates, precooled for 20 min at room temperature and for 20 min at 4 °C, were washed with 2 ml of Hanks’ medium and 1-ml portions of the virus/heparin mixture were added. PrV Attachment to Heparinase- or Heparitinase-treated Cells—Confluent monolayers of 2-day-old MDBK cells in six-well plates were washed two times with 2 ml of Hanks’ medium, and 1-ml portions of the same medium containing the indicated number of heparinase or heparitinase units were added. Binding of Glycosaminoglycans to PrV gC—Purified gC (1.5 g) in 0.2 ml of PBS supplemented with 0.05% bovine serum albumin (BSA) was mixed with ϳ5000 cpm of MDBK cell-specific [35S]HS and incubated for 2 h at room temperature. The eluted fractions were diluted in 1 ml of water and transferred to liquid scintillation vials for quantification of radioactivity
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