Glycosaminoglycans (GAGs) on the surface of cultured cells are important in the first step of efficient respiratory syncytial virus (
RSV) infection. We evaluated the importance of sulfation, the major biosynthetic modification of GAGs, using an improved recombinant
green fluorescent protein-expressing RSV (rgRSV) to assay
infection. Pretreatment of HEp-2 cells with 50 mM
sodium chlorate, a selective inhibitor of sulfation, for 48 h prior to inoculation reduced the efficiency of rgRSV
infection to 40%.
Infection of a CHO mutant cell line deficient in N-sulfation was three times less efficient than
infection of the parental CHO cell line, indicating that N-sulfation is important. In contrast,
infection of a cell line deficient in 2-O-sulfation 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 purified soluble
heparin, the prototype GAG, before inoculation had previously been shown to neutralize its infectivity. Here we tested chemically modified
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
infection, confirming that N-sulfation, but not C6-O- or C2-O-sulfation, is important for
RSV infection. Analysis of
heparin fragments identified the 10-saccharide chain as the minimum size that can neutralize RSV infectivity. Taken together, these results show that, while
sulfate modification is important for the ability of GAGs to mediate
RSV infection, only certain
sulfate groups are required. This specificity indicates that the role of cell surface GAGs in
RSV infection is not based on a simple charge interaction between the virus and
sulfate groups but instead involves a specific GAG structural configuration that includes N-
sulfate and a minimum of 10 saccharide subunits. These elements, in addition to
iduronic acid demonstrated previously (L. K. Hallak, P. L. Collins, W. Knudson, and M. E. Peeples, Virology 271:264-275, 2000), partially define cell surface molecules important for
RSV infection of cultured cells.