Iduronic Acid-Containing Glycosaminoglycans on Target Cells Are Required for Efficient Respiratory Syncytial Virus Infection

Virology 271, 264–275 (2000) doi:10.1006/viro.2000.0293, available online at http://www.idealibrary.com on

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Louay K. Hallak,* Peter L. Collins,‡ Warren Knudson,† and Mark E. Peeples*,‡,1

*Departments of Immunology/Microbiology and †Biochemistry and Pathology, Rush-Presbyterian-St. Luke’s Medical Center, 1653 W. Congress Parkway, Chicago, Illinois 60612; and ‡Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 7 Center Drive MSC 0720, Bethesda, Maryland 20892-0720 Received December 22, 1999; returned to author for revision February 8, 2000; accepted March 6, 2000

Respiratory syncytial virus (RSV) is an important human respiratory pathogen, particularly in infants. Glycosaminoglycans (GAGs) have been implicated in the initiation of RSV infection of cultured cells, but it is not clear what type of GAGs and GAG components are involved, whether the important GAGs are on the virus or the cell, or what the magnitude is of their contribution to infection. We constructed and rescued a recombinant green fluorescent protein (GFP)-expressing RSV (rgRSV) and used this virus to develop a sensitive system to assess and quantify infection by flow cytometry. Evaluation of a panel of mutant Chinese hamster ovary cell lines that are genetically deficient in various aspects of GAG synthesis showed that infection was reduced up to 80% depending on the type of GAG deficiency. Enzymatic removal of heparan sulfate and/or chondroitin sulfate from the surface of HEp-2 cells also reduced infection, and the removal of both reduced infection even further. Blocking experiments in which RSV was preincubated with various soluble GAGs revealed the relative blocking order of: heparin Ͼ heparan sulfate Ͼ chondroitin sulfate B. Iduronic acid is a component common to these GAGs. GAGs that do not contain iduronic acid, namely, chondroitin sulfate A and C and hyaluronic acid, did not inhibit infection. A role for iduronic acid-containing GAGs in RSV infection was confirmed by the ability of basic fibroblast growth factor to block infection, because basic fibroblast growth factor binds to GAGs containing iduronic acid. Pretreatment of cells with protamine sulfate, which binds and blocks GAGs, also reduced infection. In these examples, infection was reduced by pretreatment of the virus with soluble GAGs, pretreatment of the cells with GAG-binding molecules, pretreatment of the cells with GAG-destroying enzymes or in cells genetically deficient in GAGs. These results establish that the GAGs involved in RSV infection are present on the cell rather than on the virus particle. Thus, the presence of cell surface GAGs containing iduronic acid, like heparan sulfate and chondroitin sulfate B, is required for efficient RSV infection in cell culture. © 2000 Academic Press

INTRODUCTION helical conformation by the nucleocapsid (N) protein and associated with three proteins required for transcription Respiratory syncytial virus (RSV) belongs to the Pneu- and replication: the phosphoprotein (P), the large (L) movirus genus of the Paramyxoviridae family. It causes severe pneumonia and bronchiolitis in some infants, mild protein, and the M2-open reading frame 1 (M2-1) protein. upper respiratory tract infections in persons of all ages, The envelope is derived from the host cell, and it con- and serious disease in some normal adults, elderly per- tains a lipid bilayer membrane and the three viral trans- sons, and immunocompromised individuals. In immuno- membrane glycoproteins: the glycoprotein (G), the fusion competent patients, RSV remains limited to the respira- (F) protein, and the small hydrophobic (SH) protein. The tory tract. In cell culture, however, RSV infects many cell G protein has been identified as the attachment protein lines, including human epidermoid HEp-2 and HeLa (Levine et al., 1987). The RSV G protein is an unusual cells, monkey kidney cells (Vero), and rodent cells such attachment protein with mucin-like features, including a as Chinese hamster ovary (CHO) and mouse L cells. In high content of O-linked sugars, and it is distinct from the many cell lines, within 48 h of inoculation, RSV induces attachment protein of other paramyxoviruses. The F pro- characteristic cytopathic effects, including syncytia, loss tein is required for virus-to-cell membrane fusion during of cell integrity, and spicule-like structures on the cell the initiation of infection and cell-to-cell fusion during surface (Walter and Downing, 1993). syncytia formation (Walsh and Hruska, 1983). Expression The virus particle is composed of a nucleocapsid, a of the F protein, alone, in cells causes some fusion, but matrix protein, M1, and an envelope. The nucleocapsid coexpression of either the G or SH proteins, and partic- contains the negative-strand RNA genome wrapped in a ularly coexpression of both, enhances fusion (Pastey and Samal, 1997; Heminway et al., 1994). Interestingly, neither G nor SH is absolutely required for infection, because 1 To whom reprint requests should be addressed. Fax: (312) 942- RSV lacking both genes is able to replicate in Vero cells, 2808. E-mail: [email protected]. although it is seriously impaired in animals (Karron et al.,

1997). There also are two nonstructural proteins encoded cosamine (Backstrom et al., 1979) or C4 sulfation of by the RSV genome: NS1 and NS2. It is not clear whether galactosamine (Silbert et al., 1991). Inhibition of sulfation M2-2 is a structural or nonstructural protein. at these positions has been shown to reduce the idu- GAGs are unbranched polymers of repeating disac- ronic acid content (Backstrom et al., 1979; Silbert et al., charide units that consist of either glucuronic acid or its 1991; Greve et al., 1988). (6) Other modifications include epimer, iduronic acid, linked to glucosamine or galac- sulfation at position C6, C3, or C2. tosamine, with further modifications such as sulfation at Heparin was the first GAG found to affect virus repli- various positions. They are found on the outer face of the cation, when it was shown to limit the growth of herpes cell membrane, in intracellular vesicles, and in the ex- simplex virus (HSV) (Nahmias and Kibrick, 1964). Later, it tracellular matrix. GAGs are expressed at varying levels was shown that herpes virus interacts with cell surface on most animal tissues. Of the seven types of GAGs, five HS to initiate infection (WuDunn and Spear, 1989; Spear might be physiologically relevant for RSV infection be- et al., 1992; Shieh et al., 1992). Other viruses have also cause they are expressed on the surface of most cells: been found to interact with GAGs, including Sindbis virus heparan sulfate (HS), chondroitin sulfate A (chondroitin- (Klimstra et al., 1998; Byrnes and Griffin, 1998), foot-and- 4-sulfate), B (dermatan sulfate), and C (chondroitin-6- mouth disease virus (Mondor et al., 1998), human immu- sulfate) (described herein as CS-A, CS-B, and CS-C, nodeficiency virus type 1 (Jackson et al., 1996), vaccinia respectively) and hyaluronic acid (HA). The other two virus (Hsiao et al., 1999; Hsiao et al., 1998), and dengue types, heparin and keratan sulfate, are found only in virus (Chen et al., 1997). GAG involvement has also been specialized cells, a subset of mast cells and keratino- reported for RSV infection, although there is controversy cytes, respectively, but not on any structural lung cells as to whether the GAGs are on the virus envelope (Bour- (Schmid et al., 1982). geois et al., 1998) or on the target cell (Krusat and All of the cell surface GAGs are covalently linked to Streckert, 1997). transmembrane “core” proteins, except for HA, which is Insertion of the GFP marker gene into the genome of a secreted, although some HA does bind to the cell sur- virus can serve as a useful tracer for following virus face via its receptor, CD44 (Knudson and Knudson, 1993; infection (Baulcombe et al., 1995). By constructing and Underhill, 1992). The GAGs that are linked to core pro- using rgRSV, we have been able to quantitatively study teins are attached covalently via an O-glycosidic linkage the role of GAGs in mediating RSV infection of cultured to Ser within the signature Ser-Gly dipeptide preceded cells. We examined cells in which particular GAGs have by acidic amino acids, but not all such sites are modified been removed by mutation or enzymatic digestion and (Esko and Zhang, 1996). The various GAGs are distin- studied purified GAGs for their ability to block infection. guished both by the composition of their disaccharide We found that both cell surface HS and CS-B are involved subunits and by postsynthetic modifications. The com- in infection of cultured cells and that binding activity position of the five GAGs described in the present report correlates with the presence of iduronic acid in the GAG are as follows: HS consists of glucuronic acid or iduronic disaccharide. acid linked to N-acetylglucosamine; CS-A and CS-C contain glucuronic acid linked to N-acetylgalactosamine RESULTS modified by sulfation on C4 or C6, respectively; CS-B rgRSV contains iduronic acid linked to N-acetylgalactosamine modified by sulfation on C4; and HA contains glucuronic We inserted the GFP gene before the first gene in a acid linked to N-acetylglucosamine but is not sulfated full-length cDNA copy of the RSV genome along with the and, as mentioned above, is not covalently linked to a proper gene start and gene end signals. In this construct, protein. Some GAG chains occur in copolymer forms, For the GFP gene flanked by RSV gene-start and gene-stop example, CS-A is often present on the same chain as signals was inserted into a naturally occurring BstXI site CS-B and/or CS-C, such that most of the CS-B is present (RSV antigenome positions 35–46) that lies at the bound- in copolymers. ary between the 44-nucleotide leader region and the Biosynthesis of cell surface GAGs involves several NS1 gene (Fig. 1A). Insertion of the GFP gene cassette steps: (1) synthesis of the core protein and translocation regenerated the leader region intact, placed the GFP and anchoring in the ER, (2) transfer of xylose from gene as the first gene, inserted a 10-nucleotide inter- UDP-xylose to the hydroxyl group of serine followed by genic, and otherwise left the gene order intact. The the addition of two galactose residues to complete the recombinant rgRSV virus thus expressed one additional trihexose linkage region, (3) sequential addition of glu- mRNA, namely GFP, compared with wild-type RSV. This curonic acid and aminosugars, (4) N-deacetylation and plasmid was transfected into HEp-2 cells along with four N-sulfation, and (5) epimerization of some of the glucu- other plasmids encoding the N, P, L, and M2-1 proteins to ronic acid at the C5 position, generating iduronic acid, by support RSV transcription and replication, as previously the action of uronosyl 5-epimerase (Lindahl et al., 1986; described (Collins et al., 1995). The cells were also in- Lindahl, 1990a). This step requires N-sulfation of glu- fected with vaccinia virus, MVA-T7, to provide T7 RNA 266 HALLAK ET AL.

FIG. 1. (A) Construction of rgRSV, a recombinant RSV expressing the GFP gene as the first, promoter-proximal gene. The upper diagram shows the leader–NS1–NS2–N region of the wild-type RSV antigenome, together with the sequence of the leader–NS1 junction. The sequence is numbered according to the complete antigenome sequence, and the naturally occurring BstXI site is underlined. The lower diagram shows the same region in rgRSV, where the GFP expression cassette was inserted into the BstXI site. The sequence of the leader–GFP and GFP–NS1 junctions is shown. (B) HEp-2 cells infected with rgRSV. After inoculation, cells were incubated for 48 h at 37°C and observed in an inverted fluorescence microscope. Individual infected cells, as well as syncytia, are present. polymerase to drive transcription from each of the plas- to inoculate HEp-2 cells in several rounds of amplifica- mids (Wyatt et al., 1995). Successfully transfected/in- tion to prepare a stock of rgRSV. HEp-2 cells infected fected cells became green as viewed through a fluores- with rgRSV are readily detected by fluorescence micros- cence microscope between 24 and 48 h posttransfec- copy by 20 h postinoculation. Figure 1B shows cells at tion. The supernatant medium was harvested and used 48 h postinoculation. GLYCOSAMINOGLYCANS IN RSV INFECTION 267

cells are approximately three times less susceptible to rgRSV infection.

Treatment of cells with GAG-specific enzymes reduces infection To confirm the results obtained from the mutant CHO cell lines and to attempt to identify the GAG(s) that mediates RSV infection, we used GAG-specific enzymes to remove GAGs from the cell surface and tested the ability of rgRSV to infect these cells. HEp-2 monolayers were treated with enzymes at 37°C for 3 h with occa- sional shaking, washed, and then inoculated with rgRSV [multiplicity of infection (m.o.i.) of 1] for1hat37°C, FIG. 2. Sensitivity of proteoglycan synthesis-deficient CHO cell lines incubated at 37°C for 24 h in complete medium, and to rgRSV. Each cell line was inoculated with the same amount of rgRSV analyzed by flow cytometry (Fig. 3). and analyzed by flow cytometry at 36 h postinoculation. Percentages of All enzymatic treatments reduced the susceptibility of infected cells relative to K1 are noted above each bar. HEp-2 cells to rgRSV infection, indicating that cellular GAGs are involved in rgRSV infection. Only HA lyase (Streptomyces hyalurolyticus) had no effect (Fig. 3A, sam- Infection of GAG-deficient CHO cell lines ple 6). This enzyme specifically cleaves HA at ␤-GlcNAc- If cell surface GAGs are involved in RSV infection, cell [1–4]glycosidic bonds (Ohya and Kaneko, 1970), and its lines deficient in individual enzymes needed to produce lack of effect suggests that HA is not an important GAG the various GAGs might differ in their susceptibility to for rgRSV infection. rgRSV infection. A series of such mutant CHO cell lines Several of the other enzymes used have overlapping have previously been isolated after mutagenesis of the specificities, making it difficult to determine with cer- parental CHO K1, and their defects have been character- tainty the responsible GAG from this experiment. For ized (Esko and Raetz, 1978). They were first used to study instance, sheep testicular hyaluronidase type III (Fig. 3A, the role of GAGs in virus infection by WuDunn and Spear sample 7) randomly cleaves ␤-N-acetyl-hexosamine-[1– (1989) in an examination of HSV infection. We tested 4]glycosidic bonds in HA, CS-A, CS-B, CS-C, and their CHO K1 and several of these mutant cell lines for their copolymers (Borders and Raftery, 1968). Chondroitinase sensitivity to rgRSV infection. Cells were inoculated with AC (sample 3) also reduced infection, but in addition to rgRSV and incubated at 37°C for 36 h. The appearance of removing CS-A and CS-C, this enzyme also releases GFP in CHO cells is delayed compared with HEp-2 cells, CS-B if it is in a copolymer with either CS-A or CS-C requiring a longer incubation before analysis. The per- (Yamagata et al., 1968; Suzuki et al., 1968). Therefore, the centage of infected cells for each CHO cell line was reduction in infection caused by chondroitinase AC could determined by flow cytometry (Fig. 2). be due to the release of CS-A, B, or C from the cell The mutant CHO cell line A-745 is deficient in xylosyl- surface. transferase, the enzyme required for the first sugar trans- CS-B lyase cleaves CS-B at its ␤-D-galactosamine-L- fer reaction in GAG formation. Less than 7% of the enzy- iduronic acid linkage (Michelacci and Dietrich, 1975), matic activity remains in these cells. As a result of this and heparitinase (Flavobacterium heparinum) cleaves deficiency, A-745 does not express HS or CS (Esko et al., HS in poorly sulfated regions (Lohse and Linhardt, 1992) 1985). CHO A-745 cells were five times less susceptible (Fig. 3A, sample 5). CS-B lyase reduced rgRSV infection to rgRSV infection than CHO K1 (Fig. 2). Another mutant to 55% (sample 2), whereas heparitinase reduced infec- cell line, CHO pgsB-761, is deficient (less than 2% en- tion to 37% (sample 5). These results suggest that both zyme activity remaining) in galactosyltransferase I (UDP- CS-B and HS (Godavarti and Sasisekharan, 1998; Fors- D-galactose:xylose ␤-1,4-D-galactosyltransferase), result- berg et al., 1999) are involved in initiating rgRSV infection. ing in deficiencies in both CS and HS expression (Esko Heparinase I cleaves only heparin and HS and reduced et al., 1987). These cells are three times less susceptible infection to 60% (sample 4). Because heparin is not found to rgRSV infection than CHO K1. A third cell line, CHO on the surface of cells, this effect must be due to release pgsD-677, is defective in both N-acetylglucosaminyl- of HS, consistent with the heparitinase results. transferase and glucuronosyltransferase, by 50- and To confirm that both CS-B and HS are involved in 1000-fold, respectively (Lidholt et al., 1992). Both of these rgRSV infection and that heparitinase and CS-B lyase are enzymes are required for the biosynthesis of HS. Inter- removing separate entities rather than copolymers, we estingly, pgsD-677 makes approximately three times compared the effect of each enzyme alone to a combi- more total CS than CHO K1 (Esko et al., 1988). These nation of the two. Figure 3B shows 53% infection (sample 268 HALLAK ET AL.

for rgRSV, then soluble HS should be able to bind to the virus and block infection. In these experiments, we also tested heparin, because it has the same carbohydrate composition as HS except that it contains more iduronic acid, the epimerized form of glucuronic acid; is more highly sulfated; and is less acetylated (Lindahl et al., 1989; Lindahl, 1990b). Bovine lung heparin and bovine kidney HS were serially diluted in medium, rgRSV was added, and the mixture was incubated at 37°C for 1 h. The mixture was then used to inoculate HEp-2 cells. After removing the inoculum, washing the cells, and adding fresh medium, the plates were incubated at 37°C. After 24 h, the cells were analyzed by flow cytometry (Fig. 4A). Heparin strongly inhibited rgRSV infection, but bovine kidney HS did not have an effect at any of the concentrations shown. The poor blocking effect of bovine kidney HS despite the reduction in infection caused

FIG. 3. Treatment of HEp-2 cells with GAG-specific enzymes before inoculation with rgRSV. The 70% confluent cells in 12-well plates were washed and covered with 250 ␮l of OptiMEM I containing the minimal number of enzyme units that gave the highest RSV reduction at 37°C for 3 h (data not shown). Cells were analyzed by flow cytometry at 24 h postinoculation. (A) Each column represents treatment with a different enzyme in the presence of 10% FBS to block the activity of possible contaminating proteases: (1) untreated cells, (2) chondroitin sulfate B lyase (20 mIU/ml), (3) chondoitinase AC (0. 3 mIU/ml), (4) heparinase I (3.3 mIU/ml), (5) heparitinase (3.3 mIU/ml), (6) bacterial HA lyase (20 Sigma units/ml), and (7) testicular HA lyase (20 Sigma units/ml). (B) Pretreatment of HEp-2 cells with the same concentration of enzymes as in A: (1) untreated cells, (2) chondroitin sulfate B lyase, (3) heparitinase, or (4) both chondroitin sulfate B lyase and heparitinase. Percentage of infected cells relative to untreated cells are noted above each bar.

2) in CS-B lyase-treated cells, 35% infection (sample 3) in heparitinase-treated cells, and 14% infection (sample 4) when both enzymes were used together. This result confirms the involvement of both HS and CS-B in mediating rgRSV infection. The finding that the combined effect of the two lyases was greater than either alone indicated that both molecules are involved in RSV infection but that the presence of one could partially, but not completely, substitute for the other. FIG. 4. Effects of soluble heparin or HS on rgRSV infectivity. Dilutions of bovine lung heparin or HS were prepared and mixed with fixed Soluble heparin and HS block infection amounts of rgRSV and incubated 30 min at 37°C. The mixtures were replaced with fresh medium and assessed by flow cytometry at 24 h If enzymatic removal of HS from the HEp-2 cell surface postinoculation. (A) Bovine kidney HS compared with heparin. (B) reduced rgRSV infection by removing an attachment site Bovine kidney HS compared with bovine and porcine intestinal HS. GLYCOSAMINOGLYCANS IN RSV INFECTION 269 by removing cell surface HS (Fig. 3) is puzzling but has been found previously for RSV (Bougeois et al., 1998) and Sindbis virus (Byrnes and Griffin, 1998) infections. How- ever, we also tested HS from bovine and porcine intestinal mucosa for their ability to block rgRSV infection. Unlike the HS from bovine kidney, the HS from both intestinal sources blocked rgRSV infection (Fig. 4B). The difference in activity of HS from different tissues might be due to the degree of sulfation and/or the iduronic acid content, even though the basic building blocks, alternat- ing hexuronic acid (glucuronic or iduronic) and D-glucosamine (N-acetylated or N-sulfated), remain the same (Toida et al., 1997; Tekotte et al., 1994). The vendor reports their total sulfation as 5.62%, 10.2%, and 5.79% for bovine kidney, bovine intestinal, and porcine intestinal HS, respectively, suggesting that total sulfate content is not responsible for the difference in inhibition.

Effects of soluble CS-A, B, and C and HA on infection In similar blocking experiments, we tested the effect of preincubation of soluble CS-A, B, and C with rgRSV (Fig. 5). Only CS-B reduced rgRSV infectivity, suggesting that it contains an important component not present in the other two. CS-A and CS-B have the same structure and position (C4) of sulfation, differing only in that CS-A contains glucuronic acid, whereas CS-B contains its epimer, iduronic acid. CS-B from human milk has been shown by others to have an inhibitory effect on RSV infection (Portelli et al., 1998). Preincubation of rgRSV with dilutions of HA before inoculation of cells did not inhibit rgRSV infection (Fig. FIG. 5. Effects of preincubation with soluble CS-A, CS-B, CS-C, or HA 5B). The slight reduction seen at high concentrations on rgRSV infection. Serial dilutions of each GAG were prepared and may be due to the viscosity of HA in aqueous solution, mixed with equal amounts of rgRSV. The mixture was incubated 30 min which might have retarded diffusion. This is consistent at 37°C and then used to inoculate HEp-2 cells for 2 h. The cells were with the results in Fig. 3, where enzymatic digestion of incubated 24 h, and the percentage of infected cells was quantified by flow cytometry. (A) Effects of CS-A, CS-B, and CS-C on rgRSV infectivity. HA with bacterial HA lyase did not affect rgRSV infection. (B) Effects of HA on rgRSV infectivity.

Iduronic acid is a critical GAG component for mediating infection with bFGF (10 ␮g/ml) for 1 h before, during, or after rgRSV In the work described above, three GAGs, namely, inoculation. As shown in Fig. 6B, bFGF did not affect RSV heparin, HS, and CS-B, inhibited RSV infection, whereas replication when it was added after inoculation, indicat- CS-A, CS-C, and HA had no effect. Interestingly, heparin, ing that its inhibitory activity is not due to an alteration in HS, and CS-B all contain iduronic acid, whereas CS-A, the metabolic state of the cells. However, bFGF did block CS-C, and HA do not (Table 1). infection when it was added before and washed out, or To confirm the importance of iduronic acid in rgRSV with the virus, indicating that bFGF acted on the cell infection, we tested the effect of pretreatment of virus or surface to interfere with an initial step of binding/entry of cells with basic fibroblast growth factor (bFGF), which RSV. binds specifically to GAGs containing iduronic acid Effect of protamine sulfate on infection (Chintala et al., 1994; Westergren-Thorsson et al., 1993; Turnbull et al., 1992). First, virus was preincubated with Protamine sulfate is highly basic polyanion used clin- bFGF, and the mixture was used to inoculate cells. Cells ically as an antagonist for heparin. It binds to heparin, were harvested 24 h later and analyzed by flow cytom- reversing its anticoagulant properties (Byun et al., 1999). etry. As shown in Fig. 6A, bFGF did inhibit infection. To In addition to neutralizing heparin, protamine sulfate can determine whether this effect was due to bFGF binding neutralize HS (Hubbard and Jennings, 1985) and CS-B to the cells or to the virus, we treated cell monolayers (Sie et al., 1989). If HS and CS-B on the cell surface are 270 HALLAK ET AL.

TABLE 1 tion (Table 1), and treatment of cells with heparinase a reduced infection. Heparin, a molecule secreted by mast Iduronic Acid Presence in GAG Polymers and Their IC50 Values cells, is a convenient experimental surrogate for GAGs ␮ GAG Iduronic acid IC50 ( g/ml) but is not itself found on the cell surface and therefore would not be involved in virus entry. However, heparin ϩb Heparin 1.0 released by mast cells during RSV infection might have HS ϩc 12.5d CS-A Ϫe Ͼ400.0 antiviral activity. Both soluble CS-B and HS (from intesti- CS-B ϩf 200.0 nal sources) were able to block infection. Both HS CS-C Ϫe Ͼ400.0 (Nackaerts et al., 1997; Lories et al., 1987) and CS-B (van HA Ϫe Ͼ400.0 Kuppevelt et al., 1984, 1985) are found in the lungs with different distribution patterns. It is interesting to note that a Inhibitory concentration of each GAG that prevents infection of 50% of the HEP-2 cells. Data from Figs. 4 and 5. there is a switch from CS-B to CS-C in the lungs with b Lindahl et al., 1986; Perlin, 1979; ϩ represents the presence of increasing age (Schmid et al., 1982). This switch seems iduronic acid. to correlate roughly with the prevalence of severe RSV c Maccarana et al., 1996. d respiratory tract infection in infants rather than adults. Bovine intestine HS data from Fig. 4B. Several studies on the role of cell surface proteogly- e Champe and Harvey, 1987; Ϫ represents the absence of iduronic acid. cans in viral infection have reported the effectiveness of f Bossennec et al., 1990. heparinases and the ability of soluble heparin to block infection but could not explain the apparent discrepancy that soluble HS does not block infection (Bourgeois et al., important in initiating rgRSV infection, as indicated by the experiments described, protamine pretreatment of the cells should bind and block these GAGs, which in turn should reduce the efficiency of infection. Protamine pretreatment of the cells did partially block infection (Fig. 7). In contrast, protamine had no effect on infection when incubated with the virus for 1 h and then diluted fivefold, along with the virus, before inoculation, nor did protamine have an effect when added 1 h after inoculation. Conversely, pretreatment of cells with CS-B or heparin did not inhibit infection, whereas preincubation with rgRSV did. These results are consistent with an important role for cell-associated HS and CS-B in RSV infection.

DISCUSSION Efficient infection of cultured cells by rgRSV, and wtRSV by extension, requires GAGs, most likely as an initial binding step. Four lines of evidence support this conclusion: (1) reduced infection of GAG-deficient cell lines by up to 80%, depending on the defective enzyme involved; (2) neutralization of RSV by soluble GAGs; (3) reduction in RSV infection in cells pretreated with protamine, which neutralizes GAGs (Byun et al., 1999), or pretreatment with bFGF, which binds iduronic acid-containing GAGs (Sie et al., 1989; Hubbard and Jennings, 1985); and (4) reduced infection after enzymatic removal of cellular GAGs. Enzymatic removal of either HS or CS-B decreases infection efficiency, and removal of both fur- FIG. 6. Effect of bFGF on rgRSV infection. Equal amounts of rgRSV ther decreases infection, indicating that RSV uses both of were mixed with serial dilutions of bFGF. (A) HEp-2 cells were inocu- these GAGs for efficient infection of host cells. lated with the mixtures and incubated 24 h before assessment of The surface of most cells is decorated with several infection by flow cytometry. (B) HEp-2 cells were incubated with bFGF (10 ␮g/ml) 1 h before inoculation or during the 1-h adsorption period or GAG types, including HS, CS-A, CS-B, CS-C, and HA 1 h postinoculation. After each exposure, bFGF was removed and cells bound to CD44. In our blocking experiments, soluble were washed three times before the addition of virus or fresh medium. heparin had the strongest blocking effect on RSV infec- Cells were incubated for 24 h before analysis. GLYCOSAMINOGLYCANS IN RSV INFECTION 271

(Whitfield et al., 1992). In this report, we have shown that only iduronic acid-containing soluble GAGs, heparin, HS, and CS-B, reduced RSV infection. Consistent with the role of iduronic acid in RSV binding, treatment of cells with bFGF, which binds GAGs containing iduronic acid, inhibited RSV infection. Although iduronic acid seems to be a requirement for RSV binding, it is clearly not the only necessary GAG component. The amount of each GAG needed to inhibit rgRSV infection by 50% varied, from 1 ␮g/ml for heparin to 12.5 ␮g/ml for some HS to 200 ␮g/ml for CS-B (Table 1). It is likely that other aspects of these GAGs, such as FIG. 7. The effect of protamine, HS, and CS-B on rgRSV infection the type of aminosugar (i.e., glucosamine versus galac- relative to the time of inoculation. HEp-2 cells were treated with pro- tosamine) or sulfation, are important for binding. tamine (200 ␮g/ml) heparin (10 ␮g/ml) or CS-B (500 ␮g/ml) for 1 h, the medium removed and rinsed once with PBS before inoculation (cell). Krusat et al. (1997) have shown an interaction between rgRSV was pretreated with these agents and then diluted five times the RSV attachment protein G and heparin by affinity before inoculation (virus), or the cells were incubated for 1 h with the chromatography. The G protein contains a positively agents after inoculation (post). Cells were incubated 24 h before as- charged lysine-rich region (Langedijk et al., 1996). Pep- sessing the level of infection by flow cytometry. Medium was used as a control. tides from this region of both RSV subgroups bind to heparin, and pretreatment of cells with these peptides inhibits infection (Feldman et al., 1999). It seems likely, 1998; Krusat and Streckert, 1997; Byrnes and Griffin, then, that the virion G proteins binds to cell surface 1998). We obtained the same results when using bovine GAGs. kidney HS, but surprisingly we found that purified HS Bourgeois et al. (1998) concluded that the RSV G from intestinal sources has strong inhibitory effects on protein contains a heparin-like structure (not a heparin- RSV infection. It would be interesting to determine the binding structure) responsible for RSV-cell attachment. structural differences between the HS from these two Their conclusion was based on the idea that one or more sources, because this might identify structural GAG re- of the many O-linked carbohydrates on the G protein quirements for binding RSV. might be a GAG chain, and this is the GAG that enhances Soluble CS-A and CS-C and HA were not inhibitory. RSV infection. This possibility is unlikely because half of Nevertheless, chondroitinase AC treatment of cells be- the reported RSV G protein sequences lack the Ser-Gly fore inoculation reduced the number of infected cells. These findings are reconciled by the fact that most, if not signal necessary for GAG addition (Chopra et al., 1985). all, of the CS-B on the cell surface is in a copolymer form Furthermore, only a fraction of the Ser-Gly sequences in with CS-A and CS-C (Karamanos et al., 1995). Cleavage known proteoglycan core proteins are used for GAG within a CS-A or CS-C region would release the entire linkage (Kokenyesi and Bernfield, 1994). Other require- chain, including the CS-B components. CS-B lyase might ments for the utilization of the Ser-Gly dipeptide in GAG also remove the CS-A and CS-C in copolymers with linkage include the presence of acidic amino acids N CS-B. However, among the soluble chondroitin sulfates, terminal to the Ser-Gly dipeptide (Zhang and Esko, 1994; only CS-B inhibited rgRSV infection, indicating that CS-B Bourdon et al., 1987) and repetition of this dipeptide is the only one of these molecules that binds RSV. (Zhang et al., 1995). These requirements are not found in This is the first report to identify iduronic acid as an the G protein. Therefore, it is unlikely that any GAG is important GAG component in initiation of viral infection. linked to the RSV G protein. Unlike glucuronic acid or other GAG subunits, iduronic Our experiments with the GAG-deficient CHO cell acid allows greater rotation and flexibility of the GAG lines, in which defects in GAG assembly decreased chains required for biological functions and binding (Lin- infection by as much as 80%, are very strong evidence hardt et al., 1991; Casu et al., 1988). For instance, (1) that the important GAGs are on the cell. This finding is synthetically modified HS that contains a higher amount supported by reduced efficiency of RSV infection of cells of iduronic acid than glucuronic acid exhibits higher that have been pretreated with protamine. Because pro- binding activity for bFGF (Kovensky et al., 1999); (2) the tamine neutralizes GAGs (Byun et al., 1999; Sie et al., heparin pentasaccharide that binds to antithrombin III 1989; Hubbard and Jennings, 1985), the important GAGs contains iduronic acid (Piepkorn et al., 1978; Jacobsson must be on the cell. et al., 1986); (3) only iduronic acid-containing GAGs in- Like Krusat et al. (1997), we found that heparin blocks hibit the proliferation of fibroblasts (Westergren-Thorsson RSV infection only when it is present during inoculation. et al., 1993, 1991); and (4) binding of heavy metal ions to We found that pretreatment of cells with heparin or CS-B GAGs is dependent on the presence of iduronic acid or the addition of these reagents after inoculation did not 272 HALLAK ET AL. affect infection. These results again indicate that the clone D46 to generate MP125. This plasmid was used to virus binds cell surface GAGs to initiate infection. rescue virus (Collins et al., 1995) by cotransfecting HEp-2 Finally, we attempted to label the RSV G protein with cells with pTM-1 plasmids expressing the RSV N, P, L, 35 SO4, a specific method to label GAGs, because they are and M2-1 proteins from a T7 promoter and infecting with the major sulfated biomolecules. In two attempts, we the recombinant vaccinia virus MVA-T7 (Wyatt et al., 35 purified virus from cells labeled with SO4 for 24 h but 1995) to provide the T7 polymerase. were unable to detect any 35S-labeled viral proteins or cellular proteoglycans in the virions (data not presented). Cells and media GAG–RSV interaction on the cell surface is necessary HEp-2 cells were maintained in OptiMEM I (GIBCO for 80% of RSV infection (Fig. 2). It is possible that HS BRL, Grand Island, NY) medium supplemented with 2% and/or CS-B is a specific receptor for RSV, as recently FBS. Mutant CHO cell lines lacking different enzymatic shown for a particular sulfated form of HS and HSV type activities required for GAG production or sulfation were 1 (Shukla et al., 1999). It is also possible that this inter- kindly provided by Jeff Esko (Department of Biochemistry, action is not the only interaction between RSV and the Schools of Medicine and Dentistry, University of Ala- cell surface, because rgRSV still infects 20% of CHO bama, Birmingham, AL). CHO cells were maintained in A-745 cells despite their severe GAG deficiency. Perhaps DMEM/F-12 (GIBCO BRL), 10% FBS. the G protein has a second binding step that can pro- ceed at 20% efficiency without GAG binding. It may be Infection experiments that during infection, RSV binds to HS and/or CS-B chains on the cell surface. The bound RSV is thereby Stocks of rgRSV were prepared in HEp-2 cells by concentrated on the plasma membrane, allowing it to inoculation with an m.o.i. of 0.3, incubating for 3 days at search more efficiently for its receptor in two dimensions 37°C and 5% CO2, harvesting by scraping cells from the rather than in three. The presence of a highly conserved monolayer with a rubber policeman, vortexing, and cen- region in the G protein has been suggested to be the trifuging at 1000 g for 10 min to remove debris. Stocks Ϫ binding site that might interact with a cellular receptor were aliquoted and stored at 70°C. (Johnson et al., 1987). For experiments, cells were seeded onto 12-well tis- Others have reported the propagation of RSV lacking sue culture plates in the medium described above and the G and SH genes (Karron et al., 1997). This evidence incubated overnight at 37°C and 5% CO2. The 60–70% would suggest that the only remaining viral glycoprotein, confluent monolayers were washed once with Dulbec- F, has the ability to bind to cells. In wtRSV, perhaps the G co’s PBS (GIBCO BRL) and inoculated with rgRSV at an protein binds to cell surface GAGs, and then the F pro- approximate m.o.i. of 1. The virus stock was diluted in tein binds to a receptor before initiating fusion. OptiMEM I when required. Plates were incubated to allow virus binding at 37°C for 2 h unless otherwise MATERIALS AND METHODS noted. The inoculum was removed, and cells were washed once with PBS; then fresh medium was added, Construction and rescue of rgRSV and the plates were reincubated at 37°C for 24 or 36 h The Green Lantern Protein (Life Technologies, Grand for HEp-2 and CHO cells, respectively. Island, NY) gene was amplified by PCR with Vent poly- Flow cytometry and cell count merase, using a positive sense primer (5Ј-CTGCA- GAACC AAAAAAATGG GGCAAATACC CGGGTCACCA rgRSV-infected cells were washed with PBS and de- TGAGCAAGGG CGAGGAACTG) that contained a BstXI tached from tissue culture dishes using either Versene site (italics) and the RSV gene start sequence (under- 1:5000 (GIBCO BRL) or 1ϫ Trypsin-EDTA (GIBCO BRL). lined) followed by the initial nucleotides from the GFP Cells were pipetted up and down several times to gen- ORF (bold) and a negative sense primer (5Ј-CTGCA- erate a single-cell suspension. Cells were spun at 2000 GAACC ATTTTTTTGG TTTTTTATTA ACTCTCCCGG rpm for 5 min in a Brinkmann Instruments (Westbury, NY) GTACCGCTCA CTTGTACAGC TCGTCCATGC C) that con- Eppendorf Desktop Microfuge 5415. The supernatant tained a BstXI site (underlined), the RSV gene end se- was removed, and cells were suspended in 300 ␮lof quence (italics), and the end of the GFP ORF (bold). The PBS and fixed by the addition of 600 ␮l of 3% parafor- BstXI sequences in these primers matched the BstXI site maldehyde solution (15 g paraformaldehyde, 28 mM in the RSV genome leader. Because the BstXI site is not KH2PO4,36mMNa2HPO4, and H2O to 500 ml; heated to unique in the full-length RSV cDNA sequence, the PCR 60°C until dissolved, filtered, and stored at 4°C). Sam- fragment was inserted into the leader of the partial RSV ples were vortexed briefly before loading them in the flow plasmid D13, containing the leader, NS1, NS2, N, P, M, cytometry system (Ortho Cytoronabsolute; Ortho Diag- and SH genes, to generate plasmid MP91. The MP91 nostic Systems, Raritan, NJ). The fluorescence of 5000 AatII/AvrII fragment containing the GFP gene was then cells for each sample were measured and plotted as a used to replace that fragment in the full-length genome percentage of infected cells. GLYCOSAMINOGLYCANS IN RSV INFECTION 273

Chemicals and enzymes proteodermatan sulphate: Demonstration that most molecules pos- sess only one glycosaminoglycan chain and comparison of amino All soluble GAGs, GAG-specific enzymes, bFGF, and acid sequences around glycosylation sites in different proteogly- protamine were obtained from Sigma Chemical Co. (St. cans. Biochem. J. 232, 277–279. Louis, MO): bovine lung heparin (H-9133), porcine intes- Collins, P. L., Hill, M. G., Camargo, E., Grosfeld, H., Chanock, R. M., and tinal mucosa HS (H-9902), bovine intestinal mucosa HS Murphy, B. R. (1995). Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the (H-5393), bovine kidney HS (H-7640), chondroitin sulfate transcription elongation factor from the 5Ј proximal open reading C (C-4384), chondroitin sulfate B (C-3788), chondroitin frame of the M2 mRNA in gene expression and provides a capability sulfate A (C-9819), human recombinant bFGF (F-0291), for vaccine development. Proc. Natl. Acad. Sci. USA 92, 11563–11567. and salmon protamine sulfate (P-4020). Esko, J. D., and Raetz, C. R. (1978). Replica plating and in situ enzymatic assay of animal cell colonies established on filter paper. Proc. Natl. Acad. Sci. USA 75, 1190–1193. ACKNOWLEDGMENTS Esko, J. D., and Zhang, L. (1996). Influence of core protein sequence on glycosaminoglycan assembly. Curr. Opin. Struct. Biol. 6, 663–670. We would like to thank Greg Spear and Alan Landay for use of the Esko, J. D., Rostand, K. S., and Weinke, J. L. (1988). Tumor formation flow cytometer, Jeff Esko and Patricia Spear for providing the CHO cell dependent on proteoglycan biosynthesis. Science 241, 1092–1096. lines, Ada Cole for use of the inverted fluorescence microscope, and Esko, J. D., Stewart, T. E., and Taylor, W. H. (1985). Animal cell mutants Barb Newton for general laboratory support. This work was supported defective in glycosaminoglycan biosynthesis. Proc. Natl. Acad. Sci. by U.S. Public Health Service Grant AI25586 and by a grant from the USA 82, 3197–3201. Rush University Committee on Research. Esko, J. D., Weinke, J. L., Taylor, W. H., Ekborg, G., Roden, L., Ananthara- maiah, G., and Gawish, A. (1987). Inhibition of chondroitin and hepa- REFERENCES ran sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I. J. Biol. Chem. 262, 12189–12195. Backstrom, G., Hook, M., Lindahl, U., Feingold, D. S., Malmstrom, A., Feldman, S. A., Hendry, R. M., and Beeler, J. A. (1999). Identification of Roden, L., and Jacobsson, I. (1979). Biosynthesis of heparin: Assay a linear heparin binding domain for human respiratory syncytial virus and properties of the microsomal uronosyl C-5 epimerase. J. Biol. attachment glycoprotein G. J. Virol. 73, 6610–6617. Chem. 254, 2975–2982. Forsberg, E., Pejler, G., Ringvall, M., Lunderius, C., Tomasini-Johansson, Baulcombe, D. C., Chapman, S., and Santa, C. S. (1995). Jellyfish green B., Kusche-Gullberg, M., Eriksson, I., Ledin, J., Hellman, L., and fluorescent protein as a reporter for virus infections. Plant J. 7, Kjellen, L. (1999). Abnormal mast cells in mice deficient in a heparin- 1045–1053. synthesizing enzyme [In Process Citation]. Nature 400, 773–776. Borders, C. L., Jr., and Raftery, M. A. (1968). Purification and partial Godavarti, R., and Sasisekharan, R. (1998). Heparinase I from Flavobac- characterization of testicular hyaluronidase. J. Biol. Chem. 243, 3756– terium heparinum: Role of positive charge in enzymatic activity. 3762. J. Biol. Chem. 273, 248–255. Bossennec, V., Petitou, M., and Perly, B. (1990). 1H-n.m.r. investigation of Greve, H., Cully, Z., Blumberg, P., and Kresse, H. (1988). Influence of naturally occurring and chemically oversulphated dermatan sul- chlorate on proteoglycan biosynthesis by cultured human fibroblasts. phates. Identification of minor monosaccharide residues. Biochem. J. J. Biol. Chem. 263, 12886–12892. 267, 625–630. Heminway, B. R., Yu, Y., Tanaka, Y., Perrine, K. G., Gustafson, E., Bern- Bourdon, M. A., Krusius, T., Campbell, S., Schwartz, N. B., and Ruoslahti, stein, J. M., and Galinski, M. S. (1994). Analysis of respiratory syncy- E. (1987). Identification and synthesis of a recognition signal for the tial virus F, G, and SH proteins in cell fusion. Virology 200, 801–805. attachment of glycosaminoglycans to proteins. Proc. Natl. Acad. Sci. Hsiao, J. C., Chung, C. S., and Chang, W. (1998). Cell surface proteo- USA 84, 3194–3198. glycans are necessary for A27L protein-mediated cell fusion: Iden- Bourgeois, C., Bour, J. B., Lidholt, K., Gauthray, C., and Pothier, P. (1998). Heparin-like structures on respiratory syncytial virus are involved in tification of the N-terminal region of A27L protein as the glycosami- its infectivity in vitro. J. Virol. 72, 7221–7227. noglycan-binding domain. J. Virol. 72, 8374–8379. Byrnes, A. P., and Griffin, D. E. (1998). Binding of Sindbis virus to cell Hsiao, J. C., Chung, C. S., and Chang, W. (1999). Vaccinia virus envelope surface heparan sulfate. J. Virol. 72, 7349–7356. D8L protein binds to cell surface chondroitin sulfate and mediates Byun, Y., Singh, V. K., and Yang, V. C. (1999). Low molecular weight the adsorption of intracellular mature virions to cells. J. Virol. 73, protamine: A potential nontoxic heparin antagonist. Thromb. Res. 94, 8750–8761. 53–61. Hubbard, A. R., and Jennings, C. A. (1985). Neutralisation of heparan Casu, B., Petitou, M., Provasoli, M., and Sinay, P. (1988). Conformational sulphate and low molecular weight heparin by protamine. Thromb. flexibility: A new concept for explaining binding and biological prop- Haemost. 53, 86–89. erties of iduronic acid-containing glycosaminoglycans. Trends Bio- Jackson, T., Ellard, F. M., Ghazaleh, R. A., Brookes, S. M., Blakemore, chem. Sci. 13, 221–225. W. E., Corteyn, A. H., Stuart, D. I., Newman, J. W., and King, A. M. Champe, P. C., and Harvey, R. A. (1987). Glycosaminoglycans. In “Bio- (1996). Efficient infection of cells in culture by type O foot-and-mouth chemistry” (D. Barnes, S. Robinson, and L. E. Hoeltzel, Eds.), pp. disease virus requires binding to cell surface heparan sulfate. J. Virol. 81–89. J. B. Lippincott Company, Philadelphia. 70, 5282–5287. Chen, Y., Maguire, T., Hileman, R. E., Fromm, J. R., Esko, J. D., Linhardt, Jacobsson, K. G., Lindahl, U., and Horner, A. A. (1986). Location of R. J., and Marks, R. M. (1997). Dengue virus infectivity depends on antithrombin-binding regions in rat skin heparin proteoglycans. Bio- envelope protein binding to target cell heparan sulfate [see com- chem. J. 240, 625–632. ments]. Nat. Med. 3, 866–871. Johnson, P. R., Spriggs, M. K., Olmsted, R. A., and Collins, P. L. (1987). Chintala, S. K., Miller, R. R., and McDevitt, C. A. (1994). Basic fibroblast The G glycoprotein of human respiratory syncytial viruses of sub- growth factor binds to heparan sulfate in the extracellular matrix of groups A and B: Extensive sequence divergence between antigeni- rat growth plate chondrocytes. Arch. Biochem. Biophys. 310, 180– cally related proteins. Proc. Natl. Acad. Sci. USA 84, 5625–5629. 186. Karamanos, N. K., Vanky, P., Syrokou, A., and Hjerpe, A. (1995). Identity Chopra, R. K., Pearson, C. H., Pringle, G. A., Fackre, D. S., and Scott, of dermatan and chondroitin sequences in dermatan sulfate chains P. G. (1985). Dermatan sulphate is located on serine-4 of bovine skin determined by using fragmentation with chondroitinases and ion- 274 HALLAK ET AL.

pair high-performance liquid chromatography. Anal. Biochem. 225, ficiency virus type 1 attachment to HeLa CD4 cells is CD4 indepen- 220–230. dent and gp120 dependent and requires cell surface heparans. Karron, R. A., Buonagurio, D. A., Georgiu, A. F., Whitehead, S. S., J. Virol. 72, 3623–3634. Adamus, J. E., Clements-Mann, M. L., Harris, D. O., Randolph, V. B., Nackaerts, K., Verbeken, E., Deneffe, G., Vanderschueren, B., Demedts, Udem, S. A., Murphy, B. R., and Sidhu, M. S. (1997). Respiratory M., and David, G. (1997). Heparan sulfate proteoglycan expression in syncytial virus (RSV) SH and G proteins are not essential for viral human lung-cancer cells. Int. J. Cancer 74, 335–345. replication in vitro: Clinical evaluation and molecular characterization Nahmias, A. J., and Kibrick, S. (1964). Inhibitory effect of heparin on of a cold-passaged, attenuated RSV subgroup B mutant. Proc. Natl. herpes simplex virus. J. Bacteriol. 87, 1060–1066. Acad. Sci. USA 94, 13961–13966. Ohya, T., and Kaneko, Y. (1970). Novel hyaluronidase from streptomy- Klimstra, W. B., Ryman, K. D., and Johnston, R. E. (1998). Adaptation of ces. Biochim. Biophys. Acta 198, 607–609. Sindbis virus to BHK cells selects for use of heparan sulfate as an Pastey, M. K., and Samal, S. K. (1997). Analysis of bovine respiratory attachment receptor. J. Virol. 72, 7357–7366. syncytial virus envelope glycoproteins in cell fusion. J. Gen. Virol. 78, Knudson, C. B., and Knudson, W. (1993). Hyaluronan-binding proteins in (Pt 8), 1885–1889. development, tissue homeostasis, and disease. FASEB J. 7, 1233– Perlin, A. S. (1979). Recent Structural Studies on Heparin. In “Heparin: 1241. Structure, Cellular Functions, and Clinical Applications” (N. M. Mc- Kokenyesi, R., and Bernfield, M. (1994). Core protein structure and Duffie, Ed.), pp. 25–37. Academic Press, San Diego. sequence determine the site and presence of heparan sulfate and Piepkorn, M. W., Lagunoff, D., and Schmer, G. (1978). Heparin binding to chondroitin sulfate on syndecan-1. J. Biol. Chem. 269, 12304–12309. antithrombin III: Variation in binding sites and affinity. Biochem. Kovensky, J., Duchaussoy, P., Bono, F., Salmivirta, M., Sizun, P., Herbert, Biophys. Res. Commun. 85, 851–856. J. M., Petitou, M., and Sinay, P. (1999). A synthetic heparan sulfate Portelli, J., Gordon, A., and May, J. T. (1998). Effect of compounds with pentasaccharide, exclusively containing L-iduronic acid, displays antibacterial activities in human milk on respiratory syncytial virus higher affinity for FGF-2 than its D-glucuronic acid-containing iso- and cytomegalovirus in vitro. J. Med. Microbiol. 47, 1015–1018. mers. Bioorg. Med. Chem. 7, 1567–1580. Schmid, K., Grundboeck-Jusco, J., Kimura, A., Tschopp, F. A., Zollinger, Krusat, T., and Streckert, H. J. (1997). Heparin-dependent attachment of R., Binette, J. P., Lewis, W., and Hayashi, S. (1982). The distribution of respiratory syncytial virus (RSV) to host cells. Arch. Virol. 142, 1247– the glycosaminoglycans in the anatomic components of the lung and 1254. the changes in concentration of these macromolecules during de- Langedijk, J. P., Schaaper, W. M., Meloen, R. H., and van Oirschot, J. T. velopment and aging. Biochim. Biophys. Acta 716, 178–187. (1996). Proposed three-dimensional model for the attachment protein Shieh, M. T., WuDunn, D., Montgomery, R. I., Esko, J. D., and Spear, P. G. G of respiratory syncytial virus. J. Gen. Virol. 77, 1249–1257. (1992). Cell surface receptors for herpes simplex virus are heparan Levine, S., Klaiber-Franco, R., and Paradiso, P. R. (1987). Demonstration sulfate proteoglycans. J. Cell Biol. 116, 1273–1281. that glycoprotein G is the attachment protein of respiratory syncytial Shukla, D., Liu, J., Blaiklock, P., Shworak, N. W., Bai, X., Esko, J. D., virus. J. Gen. Virol. 68, 2521–2524. Cohen, G. H., Eisenberg, R. J., Rosenberg, R. D., and Spear, P. G. Lidholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., (1999). A novel role for 3-O-sulfated heparan sulfate in herpes sim- Cheifetz, S., Massague, J., Lindahl, U., and Esko, J. D. (1992). A single plex virus 1 entry. Cell 99, 13–22. mutation affects both N-acetylglucosaminyltransferase and glucu- Sie, P., Cremers, B., Dupouy, D., Caranobe, C., Dol, F., and Boneu, B. ronosyltransferase activities in a Chinese hamster ovary cell mutant (1989). Neutralization of dermatan sulfate in vitro and in vivo by defective in heparan sulfate biosynthesis. Proc. Natl. Acad. Sci. USA protamine sulfate and polybrene. Thromb. Res. 54, 63–74. 89, 2267–2271. Silbert, C. K., Humphries, D. E., Palmer, M. E., and Silbert, J. E. (1991). Lindahl, U. (1990a). Approaches to the synthesis of heparin. Haemo- Effects of sulfate deprivation on the production of chondroitin/der- stasis 20, Suppl 1, 146–153. matan sulfate by cultures of skin fibroblasts from normal and diabetic Lindahl, U. (1990b). Biosynthesis of heparin. Biochem. Soc. Trans. 18, individuals. Arch. Biochem. Biophys. 285, 137–141. 803–805. Spear, P. G., Shieh, M. T., Herold, B. C., WuDunn, D., and Koshy, T. I. Lindahl, U., Feingold, D. S., and Roden, L. (1986). Biosynthesis of (1992). Heparan sulfate glycosaminoglycans as primary cell surface heparin. Trends Biochem. Sci. 11, 221–225. receptors for herpes simplex virus. Adv. Exp. Med. Biol. 313, 341–353. Lindahl, U., Kusche, M., Lidholt, K., and Oscarsson, L. G. (1989). Bio- Suzuki, S., Saito, H., Yamagata, T., Anno, K., Seno, N., Kawai, Y., and synthesis of heparin and heparan sulfate. Ann. NY Acad. Sci. 556, Furuhashi, T. (1968). Formation of three types of disulfated disaccha- 36–50. rides from chondroitin sulfates by chondroitinase digestion. J. Biol. Linhardt, R. J., al Hakim, A., Liu, J. A., Hoppensteadt, D., Mascellani, G., Chem. 243, 1543–1550. Bianchini, P., and Fareed, J. (1991). Structural features of dermatan Tekotte, H., Engel, M., Margolis, R. U., and Margolis, R. K. (1994). sulfates and their relationship to anticoagulant and antithrombotic Disaccharide composition of heparan sulfates: Brain, nervous tissue activities. Biochem. Pharmacol. 42, 1609–1619. storage organelles, kidney, and lung. J. Neurochem. 62, 1126–1130. Lohse, D. L., and Linhardt, R. J. (1992). Purification and characterization Toida, T., Yoshida, H., Toyoda, H., Koshiishi, I., Imanari, T., Hileman, of heparin lyases from Flavobacterium heparinum. J. Biol. Chem. 267, R. E., Fromm, J. R., and Linhardt, R. J. (1997). Structural differences 24347–24355. and the presence of unsubstituted amino groups in heparan sul- Lories, V., de Boeck, H., David, G., Cassiman, J. J., and Van Den, B. H. phates from different tissues and species. Biochem. J. 322, 499–506. (1987). Heparan sulfate proteoglycans of human lung fibroblasts: Turnbull, J. E., Fernig, D. G., Ke, Y., Wilkinson, M. C., and Gallagher, J. T. Structural heterogeneity of the core proteins of the hydrophobic (1992). Identification of the basic fibroblast growth factor binding cell-associated forms. J. Biol. Chem. 262, 854–859. sequence in fibroblast heparan sulfate. J. Biol. Chem. 267, 10337– Maccarana, M., Sakura, Y., Tawada, A., Yoshida, K., and Lindahl, U. 10341. (1996). Domain structure of heparan sulfates from bovine organs. J. Underhill, C. (1992). CD44: The hyaluronan receptor. J. Cell Sci. 103, Biol. Chem. 271, 17804–17810. 293–298. Michelacci, Y. M., and Dietrich, C. P. (1975). A comparative study van Kuppevelt, T. H., Cremers, F. P., Domen, J. G., and Kuyper, C. M. between a chondroitinase B and a chondroitinase AC from Flavobac- (1984). Staining of proteoglycans in mouse lung alveoli. II. Charac- terium heparinum: Isolation of a chondroitinase AC- susceptible terization of the Cuprolinic blue-positive, anionic sites. Histochem. J. dodecasaccharide from chondroitin sulphate B. Biochem. J. 151, 16, 671–686. 121–129. van Kuppevelt, T. H., Cremers, F. P., Domen, J. G., van Beuningen, H. M., Mondor, I., Ugolini, S., and Sattentau, Q. J. (1998). Human immunode- van den Brule, A. J., and Kuyper, C. M. (1985). Ultrastructural local- GLYCOSAMINOGLYCANS IN RSV INFECTION 275

ization and characterization of proteoglycans in human lung alveoli. Whitfield, D. M., Choay, J., and Sarkar, B. (1992). Heavy metal binding to Eur. J. Cell Biol. 36, 74–80. heparin disaccharides. I. Iduronic acid is the main binding site. Walsh, E. E., and Hruska, J. (1983). Monoclonal antibodies to respiratory Biopolymers 32, 585–596. syncytial virus proteins: Identification of the fusion protein. J. Virol. 47, WuDunn, D., and Spear, P. G. (1989). Initial interaction of herpes simplex 171–177. virus with cells is binding to heparan sulfate. J. Virol. 63, 52–58. Walter, A., and Downing, L. A. (1993). Respiratory syncytial virus inter- Wyatt, L. S., Moss, B., and Rozenblatt, S. (1995). Replication-deficient actions with host cell membranes: An enigma among the paramyxo- vaccinia virus encoding bacteriophage T7 RNA polymerase for viridae. In “Viral Fusion Mechanisms” (J. Bentz, Ed.), pp. 363–383. transient gene expression in mammalian cells. Virology 210, 202– CRC Press, Inc., Boca Raton, FL 205. Westergren-Thorsson, G., Onnervik, P. O., Fransson, L. A., and Malm- Yamagata, T., Saito, H., Habuchi, O., and Suzuki, S. (1968). Purification strom, A. (1991). Proliferation of cultured fibroblasts is inhibited by and properties of bacterial chondroitinases and chondrosulfatases. L-iduronate- containing glycosaminoglycans. J. Cell. Physiol. 147, J. Biol. Chem. 243, 1523–1535. 523–530. Zhang, L., David, G., and Esko, J. D. (1995). Repetitive Ser-Gly se- Westergren-Thorsson, G., Persson, S., Isaksson, A., Onnervik, P. O., quences enhance heparan sulfate assembly in proteoglycans. J. Biol. Malmstrom, A., and Fransson, L. A. (1993). L-iduronate-rich gly- Chem. 270, 27127–27135. cosaminoglycans inhibit growth of normal fibroblasts indepen- Zhang, L., and Esko, J. D. (1994). Amino acid determinants that drive dently of serum or added growth factors. Exp. Cell Res. 206, heparan sulfate assembly in a proteoglycan. J. Biol. Chem. 269, 93–99. 19295–19299.