SEQUENTIAL ISOLATION OF PROTEOGLYCAN SYNTHESIS MUTANTS BY USING HERPES-SIMPLEX VIRUS AS A SELECTIVE AGENT - EVIDENCE FOR A PROTEOGLYCAN-INDEPENDENT VIRUS ENTRY PATHWAY

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to h erpes simplex virus (HSV) infection. These cells, termed sog9, were de rived 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 su sceptibility to HSV-1 compared with control L cells. In steady-state l abeling experiments, sog9 cells accumulated almost no [S-35]sulfate-la beled or [6-H-3]glucosamine-labeled glycosaminoglycans, suggesting tha t the initiation of glycosaminoglycan assembly was specifically reduce d in these cells. Despite these defects, sog9 cells were fully suscept ible to vesicular stomatitis virus (VSV) and permissive for both VSV a nd HSV replication, assembly, and egress. HSV plaques formed in the so g9 monolayers in proportion to the amount of input virus, suggesting t he block to infection was in the virus entry pathway. More importantly , HSV-1 infection of sog9 cells was not significantly reduced by solub le heparan sulfate, indicating that infection was glycosaminoglycan in dependent. Infection was inhibited by soluble gD-1, however, which sug gests that glycoprotein go 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 stab ilizing the virus at the sog9 cell surface. Thus, sog9 cells provide d irect genetic evidence for a proteoglycan-independent entry pathway fo r HSV-1, and results with these cells suggest that HSV-1 is a useful r eagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.