Cell-surface heparan sulfate facilitates human immunodeficiency virus Type 1 entry into some cell lines but not primary lymphocytes

Virus Research 60 (1999) 159 – 169

Cell-surface heparan sulfate facilitates human immunodeficiency virus Type 1 entry into some cell lines but not primary lymphocytes Jamal Ibrahim a, Philip Griffin a,1, Deirdre R. Coombe b, Christopher C. Rider c, William James a,* a

Sir William Dunn School of Pathology, Uni6ersity of Oxford, South Parks Road, Oxford OX1 3RE, UK b Institute for Child Health Research, PO Box 855, West Perth, WA 6872, Australia c Department of Biochemistry, Royal Holloway Uni6ersity of London, Egham TW20 0EX, UK Received 12 August 1998; accepted 5 February 1999

Abstract Many viruses have evolved to exploit cell-surface glycosaminoglycans (GAG), particularly heparan sulfate, to facilitate their attachment and infection of host cells. Here, the case for the involvement of heparan sulfate GAG in cellular infection by human immunodeficiency virus Type 1 (HIV-1) compared with herpes simplex virus Type 1 (HSV-1) is re-examined. It is shown that HIV-1 infection is facilitated by heparan sulfate GAG in only one of three highly permissive cell lines tested, whereas HSV-1 infection is facilitated to varying extents in all three. To evaluate the physiological relevance of these findings, primary peripheral blood lymphocytes (PBL), the physiological host for HIV-1, were examined. It was found that treatment of PBL with heparitinase, to remove any traces of heparan sulfate GAG, did not alter their sensitivity to infection by either lymphocyte-tropic, X4-type strain HIV-1IIIB, nor the monocyte-tropic, R5-type strain, HIV-1Ba-L. It is concluded that heparan sulfate GAG has little physiological role in the infection of lymphocytes by HIV-1 and that evidence derived from studies on immortalized cell lines suggesting a significant role must be interpreted with caution. © 1999 Elsevier Science B.V. All rights reserved. Keywords: HIV-1 entry; Heparan sulfate; Receptor

1. Introduction * Corresponding author. Tel.: +44-1865-275-548. E-mail address: [email protected] (W. James) 1 Present address: Glaxo Wellcome Research and Development, Stevenage, SG1 2NY, UK.

Some viruses use more than one cellular receptor, each of which may be used in concert, either sequentially or independently. For example, her-

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pes simplex virus Type-1 (HSV-1) makes initial attachments to susceptible cells using cell surface glycosaminoglycans (GAG), principally heparan sulfate (WuDunn and Spear, 1989; Lycke et al., 1991; Shieh et al., 1992; Gruenheid et al., 1993; McClain and Fuller, 1994; Shieh and Spear, 1994) via its envelope glycoproteins, gC (Herold et al., 1991; Svennerholm et al., 1991; Trybala et al., 1993) and gB (Herold et al., 1994). Subsequently, the viral gD glycoprotein interacts with HVEM, a cell-surface glycoprotein of the TNF receptor family, to effect entry (Montgomery et al., 1996). Although the interactions with heparan sulfate greatly enhance the efficiency of infection they are not essential (Karger et al., 1995) and it is arguable that HVEM should be considered the primary receptor. For human immunodeficiency virus Type-1 (HIV-1), the leukocyte glycoprotein, CD4 is thought of as the ‘primary’ receptor because its role was identified at an early date (Maddon et al., 1986), its interaction with the viral glycoprotein, gp120SU is of high affinity, and it determines the species specificity and the global cellular tropism of HIV-1 in vivo. The binding energy of gp120SU –CD4 interaction is used, in part, for conformational changes that allow gp120SU to interact with a second, essential receptor molecule. This is usually CCR5 in primary, monocyte-tropic strains and CXCR4 in laboratory-adapted, T-cell line-tropic strains, giving rise to the recent nomenclature, R5 and X4 for such strains, respectively (Berger et al., 1998). The second interaction is necessary, in an unexplained way, for allowing subsequent fusion steps to occur. Nevertheless, it is possible that HIV-1 exploits additional cell-surface molecules, including adhesion molecules, to increase the efficiency of infection in vivo. Although nonessential in vitro, they could be quantitatively significant in the establishment of infection in one or more target tissues and for the development of pathology. Heparan sulfate GAGs are attractive candidates as additional or adjunct receptors for HIV-1 for a number of reasons. First, they are known to be a component of the cellular receptor not only for alpha herpes viruses (see above) but also for Dengue virus (Chen et al., 1997), respiratory

syncitial virus (Krusat and Streckert, 1997) and type O strains of foot and mouth disease virus (Jackson et al., 1995). Second, their natural functions include the entrapment and presentation of chemokines to chemokine receptors on leukocytes (Webb et al., 1993; Witt and Lander, 1994). Third, their interactions are dominated by ionic forces that could increase the rate of association between virus and host cells above the diffusion limit by electrostatic steering. Finally, experimentally added heparin and other polyanions have long been known to block the infectivity of HIV-1 in vitro (Ito et al., 1987; Callahan et al., 1991) and this might, in principle, result from competition with a heparan sulfate GAG component of the cell surface receptor, for the virus. In previous reports, it was shown that gp120SU binds to heparan sulfate GAG and that removal of heparan sulfate GAG from the surface of H9 lymphoblastoid and HeLa-CD4 cell lines reduced its susceptibility to infection by tissue-culture adapted strains of HIV-1 (Patel et al., 1993; Roderiquez et al., 1995; Mondor et al., 1998). In addition, using a cell-binding assay, Ohshiro et al. showed that heparan sulfate GAGs played a quantitatively significant role in attachment of virions to some cell lines, if not to PBL (Ohshiro et al., 1996). It might, therefore, be reasonable to conclude that heparan sulfate GAG forms part of the cellular receptor complex for HIV-1. However, in some of these studies the techniques used did not allow one to distinguish the effects of sulfated-GAG removal on entry per se from those on later events in virus replication. This is particularly possible given the discovery that the paracrine trans-activating activity of Tat is mediated through heparan sulfate GAG (Chang et al., 1997). Further, the use of laboratory-adapted, X4 strains of HIV-1 and lymphoblastoid or CD4transfected cell lines in each of these is unrepresentative of the physiological situation. Accordingly, this question has been re-examined, using a wider range of host cells, including primary human lymphocytes, and both an X4 and an R5 strain of HIV-1. It was found that H9 cells are atypical in their possession of abundant heparan sulfate GAGs and that heparan sulfate GAGs do not form a quantitatively significant

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component of the HIV-1 receptor complex in other susceptible cells, particularly primary lymphocytes.

2. Methods

2.1. Viruses and cells HIV-1IIIB (Popovic et al., 1984) and HIV-1Ba-L (Gartner et al., 1986) were obtained from the NIH AIDS Research and Reference Program. HIV1IIIB was propagated by acute infection in C8166 cells and HIV-1Ba-L in peripheral blood mononuclear cells (PBMC). Stocks of virus were rendered sulfate-free by the transfer of cells to sulfate-free medium 1 day before harvesting. HSV-1 (strain HFEM) was a kind gift of Professor A.C. Minson, University of Cambridge and was propagated in HeLa cells (Gey et al., 1952), which were obtained from Flow Laboratories. HeLa CD4 cells (Simon et al., 1993) were from laboratory stocks. C8166 cells (Salahuddin et al., 1983) were obtained from Professor R.A. Weiss, Chester Beatty Laboratories, London. H9 cells (Popovic et al., 1984) sub-line of the Hut 78 lymphoma, were obtained through the NIH AIDS Reagent Program. Peripheral blood lymphocytes (PBL) and PBMC were isolated as previously described (Collin et al., 1994). C8166 cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin (complete RPMI). HeLa and HeLa CD4 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin (complete DMEM). PBMC were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, 5% autologous human serum and 10% PBL-conditioned medium. PBL were cultured in RPMI 1640 supplemented with 10% FBS, 10% interleukin (IL)-2, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin. The depletion of GAG sulfation was by culture of cells for 6 days in sulfate-free RPMI 1640 medium (GIBCO) supplemented with 20 mM

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NaClO4, 10% dialysed FBS, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, with trypsinization every 3 days to remove residual proteoglycans. Removal of heparan sulfate was by treatment of cells with heparitinase (heparinase III; Sigma, St. Louis, MO; 1 U/ml in DMEM) for 2 h at 37°C.

2.2. Infecti6ity assay by limiting dilution for HIV-1 and HSV-1 This was done essentially as described (Simon et al., 1993). Viruses and cells were in sulfate-free medium at all stages in experiments studying the effects of desulfation of GAGs on infectivity. Infection of primary blood lymphocytes was carried out at a multiplicity of infection of 0.01 for 16 h in medium containing 20 mM sodium chlorate. For HIV-1, polymerase chain reaction (PCR) amplification of LTR-containing reverse transcripts was used to determine infection at 6 days post-infection. For HSV-1, endpoints were determined by visual inspection of wells for cytopathic effects. First and second order statistics of infectious titer were determined using the ID50 program of John Spouge (Wu et al., 1996).

2.3. COCAL(HIV-1) pseudotype assay This was as previously described (Gregory et al., 1993, 1994; Simon et al., 1993) with the exception that the media used were sulfate-free. Briefly, HeLa CD4 cells were grown in normal medium and in sulfate-free medium containing 20 mM sodium chlorate for 6 days before challenge. These were mixed in sulfate-free medium with COCAL(HIV-1) pseudotype virions previously incubated with anti-COC (COCAL) neutralizing antiserum or with anti-COC serum plus pooled human Ig against HIV-1. After 4 h, the monolayers were overlain with medium containing 1.5% carboxymethyl cellulose (CMC), incubated for 2 days at 37°C and stained with naphthalene black as described by Porterfield (de Madrid and Porterfield, 1969). The pseudotype titer is defined as the difference in plaque forming units (PFU)/ ml in the presence of anti-COC serum and the PFU/ml in the presence of anti-COC and antiHIV-1 serum.

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2.4. Titration of HSV-1 infecti6ity in HeLa CD4 cells by plaque assay Briefly, virus and trypsin-dispersed cells were mixed in sulfate-free medium and seeded into 24-well plates. After 4 h, cells were washed once with phosphate-buffered saline (PBS) and overlain with CMC and sulfate-containing growth medium supplemented with pooled human immunoglobulin (Sigma) at 200 mg/ml, which provides antiHSV-1 antibodies that prevent secondary plaque formation. Plaques were stained with naphthalene black 72 h following addition of virus.

2.5. Semi-quantitati6e PCR assay of HIV-1 pro6irus This assay measures the amount of proviral LTR formed by reverse transcription in cells 20 h after high multiplicity challenge with HIV-1 and was largely as previously described (Collin et al., 1994). Briefly, triplicates of 100.5-fold serial dilutions of test sample in control cell lysate were subjected to PCR using U3 + and U5 − primers and the abundance of the LTR-specific PCR product was estimated from its fluorescence output following staining of the agarose gel with ethidium bromide. The image of the gel was captured using an Appligene Imager and analysed using NIH Image. The intensity of product in mock-infected samples spiked with 100.5-fold serial dilutions containing known copy numbers of the proviral clone, HXB2, was used to interpolate copy number from signal intensity in test samples.

0–1 M LiCl in acetate buffer on an automated, low pressure chromatography system (Pharmacia). The concentration of Li + was determined by measuring conductivity and the mobility of GAG standards was determined by the 3-phenyl phenol assay for uronic acids (Blumenkrantz and AsboeHansen, 1973).

2.6.1. FACS analysis of heparitinase III-treated and non-treated cells HeLa CD4 cells at 70% density were either treated or not treated in the culture flask with heparitinase III at 1 U/ml in complete DMEM for 2 h at 37°C, then subjected to either direct or indirect staining approaches. For direct staining, after harvesting with cell scrapers, 106 cells were directly stained with FITC-conjugated mouse monoclonal anti-heparan sulfate IgM clone 10E4 (Seikagaku). For indirect-staining, cells still attached in culture flasks were incubated with crude ascites mouse monoclonal anti-heparan sulfate IgM 615 (Ianelli et al., 1998), After detachment they were stained with goat anti-mouse IgMFITC (The Binding Site, Birmingham). Non-heparitinase-treated, unstained cells were used as negative control for the direct staining while mouse anti-rat IgM was used as the negative control primary antibody for the indirect staining procedure.

3. Results

3.1. Inhibition of GAG sulfation by growth in chlorate-containing medium

2.6. GAG-labeling and analysis Cultured cells were washed in PBS and resuspended at 106 cells/ml in growth medium supplemented with [3H]glucosamine (100 mCi/ml) overnight. After washing in complete PBS, GAGs were extracted by treatment of 107 cells in 1 ml of PBS containing pronase and trypsin (each 1 mg/ ml) and EDTA (1 mM) for 1.5 h at 37°C. Samples were transferred to sodium acetate buffer (50 mM, pH 7.4) using PD10 columns (Pharmacia) and analysed by ion-exchange chromatography on DEAE-Sepharose using a linear gradient of

Chlorate is a competitive inhibitor of the incorporation of sulfate into preformed GAG chains and can effectively eliminate the sulfation of a range of GAGs when added to the low-sulfate growth medium of cells (Greve et al., 1988; Humphries et al., 1989; Keller et al., 1989). In a series of preliminary experiments, it was established that the cell lines, C8166, H9 and HeLaCD4 could tolerate low-sulfate medium supplemented with 20 mM sodium chlorate for up to 12 days without a significant reduction in cell viability or replication rate. To check that this

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treatment was effective at preventing GAG sulfation, cultures of each cell line were grown in control and low-sulfate, chlorate-supplemented medium for 6 days. Then the GAGs were metabolically labeled by supplementing the medium with [3H]glucosamine (Yanagishita et al., 1989). The GAGs were extracted and separated according to charge density by ion-exchange chromatography (as described in Section 2). The results show that the clearly discernable peak corresponding to highly-sulfated heparan sulfate fraction in control H9 cells is abolished by growth in chlorate-containing medium (see Fig. 1). However, the levels of expression of highly-sulfated GAGs on C8166 and HeLa CD4 cells were too low to be reliably detected using this method (results not shown). To provide a sensitive biological control for inhibition of GAG sulfation, herpes simplex virus Type-1 (HSV-1), was used because it is known to depend upon cell-surface heparan sulfate for highefficiency infection (WuDunn and Spear, 1989; Lycke et al., 1991; Shieh et al., 1992; Spear et al., 1992; Yura et al., 1992). Following growth for 6 days in chlorate-supplemented low-sulfate medium, the susceptibility of each cell line to HSV-1 was tested. The results show that there

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was approximately 3-fold reduction in infectivity in HeLa CD4 cells (statistically significant, if modest) and approximately 30-fold reduction in infectivity in C8166 and H9 cells following chlorate treatment (see Fig. 2). This compares with the 6–7-fold reduction in HSV-1 infectivity reported in heparan sulfate deficient variants of L cells (Gruenheid et al., 1993). The variation in sensitivity of the control cell lines and their differing susceptibility to chlorate treatment probably reflects differences in the amount of sulfated proteoglycans present on these cells and is reminiscent of the differences in HSV-1 entry kinetics described for Vero and Hep-2 cells.

3.2. HIV-1 infection is facilitated by sulfated GAGs in some cell lines but not others C8166, H9 and HeLaCD4 cells that had been depleted of sulfated heparan sulfate GAG by chlorate-treatment, as determined by the studies above, were tested in parallel for their sensitivity to infection by the strain, HIV-1IIIB, a prototype of the ‘X4’ strains (Berger et al., 1998). Three assays were used to register early infection events. The first assay used pseudotype viruses comprising the core of the Vesiculovirus, Cocal (COC)

Fig. 1. Analysis of glycosaminoglycans from control and chlorate-treated H9 cells. Cells grown in control medium or in sulfate-free medium supplemented with 20 mM sodium chlorate were metabolically labeled using [3H]glucosamine. The intact cells were treated with proteases to release cell-surface glycosaminoglycans (GAGs) and other protein-linked oligosaccharides and this fraction was analysed by ion-exchange chromatography on DEAE-Sepharose, developed using a 0 – 1 M linear gradient of lithium chloride. The dashed line represents the concentration of lithium chloride estimated from measurements of the conductivity of column fractions. The position of fractions from parallel runs that contained unlabeled, control hyaluronic acid (Sigma, St. Louis, MO) or heparan sulfate (Sigma) is indicated with hatched and stippled boxes, respectively. The radioactivity in fractions from the control cell extract or chlorate-treated cell extract is indicated with filled and open circles, respectively, and the change in activity of the heparan sulfate peak is indicated with a double-headed arrow.

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Fig. 2. The dependence of efficient herpes simplex virus Type 1 (HSV-1) infection of cell lines on sulfated glycosaminoglycans (GAGS). The infectivity of a stock of HSV-1 for control cells (solid bars) and chlorate-treated cells (hatched bars) was determined. (a) Infectivity in HeLa CD4 cells was determined by plaque assay. Error bars represent the S.D. of estimated from three independent assays. (b) Infectivity in H9 and C8166 cells by limiting dilution assay and the mean and S.D. (N = 8) were determined by the method of Spouge (Wu et al., 1996).

enclosed in a functional HIV-1 envelope. This assay is suitable for adherent, HIV-1-susceptible cells, such as HeLa CD4. These pseudotype virions enter cells in the same manner as parental HIV-1 and can be neutralized by antibodies to HIV-1 gp120. After entry, however, the Cocal virus genome is uncoated, leading to the production of Cocal virus plaques after 2 days. Fig. 3a shows the mean results of titrations of three separate COC(HIV-1) pseudotype stocks in control and chlorate-treated cells that were tested in parallel for HSV-1 infectability (see Fig. 2). For each stock, the ratio between the titer of pseudotype in control and sulfate-depleted cells was close to unity suggesting that the HIV-1 envelope-dependent entry of COC(HIV-1) pseudotype viruses into HeLa CD4 cells is sulfate-independent. The second assay is a determination of infectivity by limiting dilution. This depends on replication of single infectious units to a modest detectable level (10 – 100 copy numbers, in this case) and the predictable distribution of these units in replicated limiting dilutions according to the Poisson equation. Fig. 3b shows the results of infectivity determinations of a stock of HIV-1 in control and chlorate-treated H9 and C8166 cells that were used in parallel for the studies on

HSV-1 (see Fig. 2). The results show that the inhibition of GAG sulfation reduces the apparent infectivity of HIV-1 for H9 cells but not for C8166 or HeLa CD4 cells, suggesting that sulfated proteoglycans facilitate infection in one cell line but not the others. To confirm the insensitivity of HIV-1 entry into C8166 cells to GAG sulfation, the copy number of HIV-1 provirus in cells 20 h after high multiplicity infection was quantified. The primers used detect reverse transcripts containing a complete LTR, i.e. after first strand exchange. Under the conditions routinely used, no PCR product is seen in CD4-negative cells or susceptible cells treated with AZT or the CD4 mAb, Q425, or with virus treated with polyclonal anti-HIV-1Ig (Collin et al., 1991, 1994; Simon et al., 1993, 1994). Only assays in which test signal lay in the linear portion of the standard curve were considered. The results (see Fig. 3c) show the proviral copy number found in triplicate serial dilutions of control and chlorate-treated C8166 cell lysates 20 h after challenge with HIV-1IIIB. Although the assay has substantial error variation, it is clear that entry and initial stages of reverse transcription in these cells are not dependent to any large degree on the sulfation of GAG.

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3.3. Enzymatic remo6al of heparan GAGs affects HIV-1 infection in a similar way to the inhibition of GAG sulfation The chlorate method, used above, ensures that GAGs (including heparan, chondroitin, and dermatan) are not sulfated but leaves the sugar backbones otherwise intact. In order to test whether the principal GAG involved in HIV-1 infection in H9 cells was heparan sulfate, rather than another GAG, heparitinase treatment was used. In contrast to chlorate treatment, this method does not affect chondroitin sulfate but removes the oligosaccharide backbone of heparan sulfates and not just the sulfate groups. In order to confirm that the treatment removed heparan sulphate from cells, we stained HeLa CD4 cells with the heparan sulfate-specific IgM antibodies, clone 10E4 (Seikagaku) and 615 (Ianelli et al., 1998) and analysed using FACS® (see Section 2). Both indicated that the treatment removed the majority of heparan sulphate from the cell surface under the conditions used (results not shown). The results of challenge experiments in these cells confirmed those obtained above. Namely,

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heparitinase treatment reduced the susceptibility of both C8166 and H9 cells to HSV-1 by more than 10-fold but had little effect on the susceptibility of HeLaCD4 cells to HSV-1 (see Fig. 4A). However, while heparitinase treatment reduced the susceptibility of H9 cells to HIV-1 by over 10-fold, the susceptibility of C8166 and HeLa CD4 cells remained unchanged (see Fig. 4B).

3.4. The infection of primary lymphocytes by either monocyte-tropic or lymphocyte-tropic strains of HIV-1 is not substantially facilitated by heparan sulfate GAG Experiments, described above, had shown that sulfated heparan sulfate GAG was able to facilitate HIV-1 infection in H9 cells but not C8166 or HeLa CD4 cells. This prompted us to ask whether heparan sulfate GAGs have a role in the infection of the physiologically-relevant cell in vivo, the primary lymphocyte. As it has been shown that both inhibition of GAG sulfation by chlorate or removal of heparan sulfate GAG by heparitinase gave largely equivalent results and because prolonged growth of primary lymphocytes in chlo-

Fig. 3. The effect of glycosaminoglycan (GAG) desulfation on the efficiency of human immunodeficiency virus Type 1 (HIV-1) infection in cell lines. (a) The infectivity of Cocal(HIV-1) pseudotypes for control (solid bars) and chlorate-treated (hatched bars) HeLa T4 cells was determined by plaque assay. (b) The infectivity of a stock of HIV-1IIIB for control (solid bars) and chlorate-treated (hatched bars) H9 cells and C8166 cells was determined by limiting dilution assay in which the presence of threshold virus replication was determined by polymerase chain reaction (PCR) analysis (Collin et al., 1994). The mean and S.D. (N =8) were determined by the method of Spouge (Wu et al., 1996). (c) The efficiency of a single cycle of entry into control (solid bars) and chlorate-treated (hatched bars) C8166 cells and reverse transcription was estimated from a semi-quantitative PCR analysis of newly formed proviral sequence as previously described (Collin and Gordon, 1994).

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Fig. 4. The effect of heparitinase digestion on the efficiency of human immunodeficiency virus Type 1 (HIV-1) infection in cell lines. The infectivity of herpes simplex virus Type 1 (HSV-1) (A) and HIV-1IIIB (B) was determined in control cells (solid bars), and in cells that had been previously treated with heparitinase (hatched bars). Titrations were done using eight replicates at 100.5-fold dilution intervals with infection being determined by visual inspection for cytopathic effects (for HISV-1; A) or by polymerase chain reaction (PCR) (for HIV-1; B; as described). Determination of infectious titer ( 9S.D.) was calculated using the method of Spouge (Wu et al., 1996).

rate-supplemented, low-sulfate medium proved not to be possible, the heparitinase digestion approach was used. The infectivity assays were repeated using both the X4 strain, HIV-1IIIB and the R5 strain, HIV-1Ba-L. The results (see Fig. 5) show that removal of heparan sulfate from primary lymphocytes by heparitinase reduces their susceptibility to the X4 strain, IIIB, by a barely significant 2.5-fold and does not significantly alter their susceptibility to the R5 strain of HIV-1, Ba-L.

This conclusion, however, is in conflict with the view that attachment and infection of cells by HIV absolutely depend upon gp120–heparan sulfate interactions. Mondor et al. studied a clone of

4. Discussion It has previously been demonstrated that the binding of HIV-1 to, and efficiency of infection in, particular cell lines is facilitated by the presence of cell-surface heparan sulfate GAGs (Patel et al., 1993; Roderiquez et al., 1995; Ohshiro et al., 1996; Mondor et al., 1998). This issue has been re-examined, using a wider range of cell types, including primary lymphocytes, a range of assays of HIV-1 entry and both biochemical and virological controls of GAG sulfation. The results show that, while heparan sulfate facilitates the infection of some cell lines by HIV-1, it is not a significant factor in the infection of others, particularly primary lymphocytes.

Fig. 5. The independence of human immunodeficiency virus Type 1 (HIV-1) infection of Peripheral blood lymphocytes (PBL) on cell-surface heparan sulfate glycosaminoglycan (GAG). Stocks of the X4 strain, HIV-1IIIB, and the R5 strain, HIV-1Ba-L, were titrated on control PBLs (solid bars) and heparitinase-treated PBL (hatched bars). Titrations were done using eight replicates at 100.5-fold dilution intervals with infection being determined by polymerase chain reaction (PCR) (for HIV-1; as described). Determination of infectious titer (9S.D.) was calculated using the method of Spouge (Wu et al., 1996).

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HeLa-CD4 cells in which virion attachment to cells was independent of CD4 (Mondor et al., 1998). This contrasts with previous studies on other cell lines in which binding was demonstrated to be largely dependent on CD4 (Roderiquez et al., 1995; Ugolini et al., 1997). Further, Mondor et al. reported 100% loss of susceptibility to HIV infection following heparinase-treatment —far more profound than has been reported by others. It might be that the facilitation of infection by heparan sulfate GAG in the H9 cells used here or in the HeLa CD4 cells of (Mondor et al., 1998) is the consequence of an unphysiologically high level of heparan sulfate GAG in these cells combined with alterations in the kinetics of post-attachment events. Clearly, therefore, the quantitative contribution of heparan sulfate GAG to attachment and infection is highly dependent on the cell line being studied and ranges from negligible (C8166 cells) to substantial (H9 cells). Consequently, it is all the more important to consider the physiological host cell for HIV-1, the primary CD4+ lymphocyte. Virion attachment per se was not significantly affected by heparan sulfate GAG removal (Ohshiro et al., 1996) and here it is shown that infection itself is not significantly heparan sulfate GAG-dependent, either by R5 or X4 strains of HIV-1. This is an important demonstration because virion attachment need not necessarily lead to cellular infection if, for example, a majority of virus-heparan sulfate GAG complexes were noninfectious. One of the observations remains paradoxical. Why should infection of C8166 cells by HSV-1 but not by HIV-1 be affected by heparan sulfate GAG removal when the infection of H9 cells by both viruses is largely heparan sulfate GAG-dependent? It is believed that this can be explained by considering the process by which each virus binds productively to its primary receptor as follows. One envisages two parallel processes: the direct binding of free virions to the receptor on the one hand and the indirect binding to the receptor via an additional step involving a heparan sulfate GAG-complex on the other. The two sequential steps in the indirect route have been documented for HSV-1 and their kinetics are

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shown to depend on multiple cellular and viral parameters (McClain and Fuller, 1994). The relative contribution of the direct and indirect routes of entry, at a given concentration of virus and density of primary receptor, will be determined by the relative size of the rate constants for the two pathways and the density of heparan sulfate GAG on the cell surface. If the forward rate constants for the direct route are much greater than those for the GAG-dependent route, then the efficiency of infection will only be reduced by heparan sulfate GAG depletion in cells, such as H9, that initially carried very high levels of heparan sulfate GAG. Conversely, if the forward rate constants of the direct route are much lower than those of the GAG-dependent route, infection will be significantly reduced by heparan sulfate GAG depletion even on cells, such as C8166, that initially carried only low levels of heparan sulfate GAG. These considerations could equally explain the variation in HSV-1 reliance on heparan sulphate between cell lines noted above. Having concluded that GAGs are not a major component of the cell-surface receptor for HIV-1 on lymphocytes studied in vitro, could they still have a role in vivo? It has been shown that the inhibition of HIV-1 infection by the CC chemokine, RANTES, depends on the presence of heparan sulfate GAG (Oravecz et al., 1997). The envelope glycoprotein, gp120SU, shares with most chemokines two significant properties: it can bind to GAG and interacts with seven-pass transmembrane receptors. The interactions with GAG, though somewhat promiscuous, have a real degree of specificity, both for the GAG motif and the binding site in the protein (Witt and Lander, 1994; Koopmann and Krangel, 1997; Stringer and Gallagher, 1997). Thus, although the data show that GAG-independent events dominate the infection process of HIV-1 in its physiological host cell, the primary lymphocyte, it would not be surprising if gp120– GAG interactions were adaptively significant in vivo. For example, the possibility that heparan sulfate GAG directly facilitates infection of monocyte-derived cells or, on the surface of endothelial or dendritic cells, indirectly allows more efficient infection of lymphocytes in vivo has not been excluded.

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