Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumours (exostoses) that develop from the growth plate of endochondral bone. This condition can lead to skeletal abnormalities, short stature and malignant transformation of exostoses to chondrosarcomas or osteosarcomas. Linkage analyses have identified three different genes for HME, EXT1 on 8q24.1, EXT2 on 11p11-13 and EXT3 on 19p (refs 6-9). Most HME cases have been attributed to missense or frameshift mutations in these tumour-supressor genes, whose functions have remained obscure. Here, we show that EXT1 is an ER-resident type II transmembrane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). Two EXT1 variants containing aetiologic missense mutations failed to alter cell-surface glycosaminoglycans, despite retaining their ER-localization.

In a previous study, a mouse L cell mutant was isolated which is 90% resistant to HSV-1 infection (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro. J. Virol. 67, 93-100, 1993). This cell line, termed gro2C, failed to express heparan sulfate (HS)-glycosaminoglycans on the cell surface, which normally act as initial receptors for HSV-1 attachment to cultured cells. In this report, we extended the characterization of gro2C cells to explore the possibility that cell-surface chondroitin sulfate (CS) facilitates virus attachment to gro2C cells in the absence of HS. We found that soluble CS types A, B, and C strongly interfere with adsorption of HSV-1 to the surface of gro2C cells in a dose-dependent manner, and CS type B (dermatan sulfate) inhibited adsorption to parental (control) L cells by up to 10%. Moreover, gro2C cell infection was hypersensitive to inhibition by HS in comparison to control L cell infection. In all cases, a decrease in adsorption resulted in a decrease in infection. By contrast, the highly-sulfated glycosaminoglycan analog dextran sulfate was a relatively poor inhibitor of gro2C cell infection, indicating that the inhibitory effects of CS were related to its carbohydrate structure and not solely to its strong negative charge. By using a mutant virus strain which does not express the heparin-binding glycoprotein gC, we show that gC was not required for infection of gro2C cells, and was not required for the inhibition by HS or CS. Thus, the characterization of gro2C cell infection has revealed that one or more components of the HSV-1 particle can interact with cell-surface CS as well as HS to mediate infection of susceptible cells.

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to herpes simplex virus (HSV) infection. These cells, termed sog9, were derived from mutant parental gro2C cells, which are themselves defective in heparan sulfate biosynthesis and 90% resistant to HSV type 1 (HSV-1) infection compared with control L cells (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro, J. Virol. 67:93-100, 1993). In this report, we show that sog9 cells exhibit a 3-order-of-magnitude reduction in susceptibility to HSV-1 compared with control L cells. In steady-state labeling experiments, sog9 cells accumulated almost no [ 35 S]sulfate-labeled or [6-3 H]glucosamine-labeled glycosaminoglycans, suggesting that the initiation of glycosaminoglycan assembly was specifically reduced in these cells. Despite these defects, sog9 cells were fully susceptible to vesicular stomatitis virus (VSV) and permissive for both VSV and HSV replication, assembly, and egress. HSV plaques formed in the sog9 monolayers in proportion to the amount of input virus, suggesting the block to infection was in the virus entry pathway. More importantly, HSV-1 infection of sog9 cells was not significantly reduced by soluble heparan sulfate, indicating that infection was glycosaminoglycan independent. Infection was inhibited by soluble gD-1, however, which suggests that glycoprotein gD plays a role in the infection of this cell line. The block to sog9 cell infection by HSV-1 could be eliminated by adding soluble dextran sulfate to the inoculum, which may act by stabilizing the virus at the sog9 cell surface. Thus, sog9 cells provide direct genetic evidence for a proteoglycan-independent entry pathway for HSV-1, and results with these cells suggest that HSV-1 is a useful reagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.

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