Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection

Glycosaminoglycans (GAGs) on the surface of cultured cells are important in the first step of efficient respiratory syncytial virus (RSV) infection. W e evaluated the importance of sulfation, the major biosynthetic modificatio n of GAGs, using an improved recombinant green fluorescent protein-expressi ng RSV (rgRSV) to assay infection. Pretreatment of HEp-2 cells with 50 mM s odium chlorate, a selective inhibitor of sulfation, for 48 h prior to inocu lation reduced the efficiency of rgRSV infection to 40%. Infection of a CHO mutant cell line deficient in N-sulfation was three times less efficient t han infection of the parental CHO cell line, indicating that N-sulfation is important. In contrast, infection of a cell line deficient in 2-O-sulfatio n was as efficient as infection of the parental cell line, indicating that 2-O-sulfation is not required for RSV infection. Incubating RSV with the pu rified soluble heparin, the prototype GAG, before inoculation had previousl y been shown to neutralize its infectivity. Here we tested chemically modif ied heparin chains that lack their N-, C6-O-, or C2-O-sulfate groups. Only heparin chains lacking the N-sulfate group lost the ability to neutralize i nfection, confirming that N-sulfation, but not C6-O- or C2-O-sulfation, is important for RSV infection. Analysis of heparin fragments identified the 1 0-saccharide chain as the minimum size that can neutralize RSV infectivity. Taken together, these results show that, while sulfate modification is imp ortant for the ability of GAGs to mediate RSV infection, only certain sulfa te groups are required. This specificity indicates that the role of cell su rface GAGs in RSV infection is not based on a simple charge interaction bet ween the virus and sulfate groups but instead involves a specific GAG struc tural configuration that includes N-sulfate and a minimum of 10 saccharide subunits. These elements, in addition to iduronic acid demonstrated previou sly (L. K. Hallak, P. L. Collins, W Knudson, and M. E. Peeples, Virology 27 1:264-275, 2000), partially define cell surface molecules important for RSV infection of cultured cells.