Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection

Abstract

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.

FIG. 1

Effects of dextran (Dx) and dextran sulfate (DxSO₄) on the efficiency of rgRSV infection of HEp-2 cells. Virus (MOI = 2) was mixed with the indicated concentrations of DxSO₄-5K (average molecular mass, 5 kDa), DxSO₄-10K, or Dx-10K, incubated for 45 min, and used to inoculate cells. Cells were analyzed for GFP expression at 24 h postinoculation.

FIG. 2

Effects of sodium chlorate on rgRSV infection. HEp-2 cells were incubated in sulfate-free medium, without (samples 1 and 2) or with (sample 3) 50 mM sodium chlorate and with (sample 1) or without (samples 2 and 3) 0.8 mM MgSO₄. After 48 h, cells were inoculated with rgRSV (MOI = 1), washed, and incubated in complete medium. At 24 h postinoculation, the medium was removed and cells were analyzed for GFP expression.

FIG. 3

Sensitivity of sulfate-deficient CHO cell lines to rgRSV infection. CHO pgsE-606, deficient in N-sulfation, and pgsF-17, deficient in 2-O-sulfation, were inoculated with rgRSV (MOI = 1) and analyzed for GFP expression at 36 h postinoculation. The average percent infected cells relative to the parental CHO K1 is shown above each bar.

FIG. 4

Neutralization activity of chemically modified full-length soluble heparin chains against rgRSV. Virus (MOI = 1) was mixed with various modified heparins, as indicated, incubated for 45 min, and used to inoculate HEp-2 cells. Cells were analyzed for GFP expression at 24 h postinoculation.

FIG. 5

Effects of heparin chain length on its ability to neutralize rgRSV. Virus (MOI = 1) was mixed with heparin chains of increasing length, as indicated, incubated for 45 min, and used to inoculate HEp-2 cells. Cells were analyzed for GFP expression at 24 h postinoculation.