Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. To determine the step in virus replication during which apoptosis is triggered, we used UV-inactivated SV, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for SV infection of many cells. In initial experiments, UV-inactivated neuroadapted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure to acidic pH, apoptosis was induced in CHO cells with or without heparan sulfate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus strain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH(4)Cl or bafilomycin A1. These studies indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.     

Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans

The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV.     

Heparan sulfates of mouse cells. Analysis of parent and transformed 3T3 cell lines.

Heparan sulfate from the surface of a variety of mouse cells at different cell densities was examined by ion-exchange chromatography. The results of this analysis show that: (1) The heparan sulfate from new isolates of Swiss 3T3 cells transformed by SV40 virus (a DNA tumor virus) elutes from DEAE-cellulose at a lower ionic strength than that from the parent cell type. This finding confirms our earlier observation with an established SV40-transformed cell line (Underhill and Keller, '75) and eliminates the possibility that this change is caused by extended passage in culture. (2) For both parent and transformed 3T3 cells, the heparan sulfates from low and high density cultures were the same as judged by chromatography on DEAE-cellulose. This result demonstrates that the transformation-dependent change which we have observed is independent of cell density. (3) The heparan sulfate from Balb/c 3T3 cells transformed with Kirsten murine sarcoma virus (an RNA tumor virus) elutes from DEAE-cellulose prior to that from parent Balb/c 3T3 cells. This result extends the transformation dependent change in heparan sulfate to the Balb/c 3T3 cell line and to cells transformed with an RNA virus.     

A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

Simian virus 40-based replication of catalytically inactive human immunodeficiency virus type 1 integrase mutants in nonpermissive T cells and monocyte-derived macrophages

Integrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into the nef position of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies.     

Simian virus 40 small-t antigen stimulates viral DNA replication in permissive monkey cells.

The simian virus 40 (SV40) large-T antigen is essential for SV40 DNA replication and for late viral gene expression, but the role of the SV40 small-t antigen in these processes is still unclear. We have previously demonstrated that small t inhibits SV40 DNA replication in vitro. In this study, we investigated the effect of small t on SV40 replication in cultured cells. CV1 monkey cell infection experiments indicated that mutant viruses that lack small t replicate less efficiently than the wild-type virus. We next microinjected CV1 cells with SV40 DNA with and without purified small-t protein and analyzed viral DNA replication efficiency by Southern blotting. Replication of either wild-type SV40 or small-t deletion mutant DNA was increased three- to fivefold in cells coinjected with purified small t. Thus, in contrast to our in vitro observation, small t stimulated viral DNA replication in vivo. This result suggests that small t has cellular effects that are not detectable in a reconstituted in vitro replication system. We also found that small t stimulated progression of permissive monkey cells--but not of nonpermissive rodent cells--from G0-G1 to the S phase of the cell cycle, possibly leading to an optimal intracellular environment for viral replication.     

Cathepsin X binds to cell surface heparan sulfate proteoglycans

Glycosaminoglycans have been shown to be important regulators of activity of several papain-like cathepsins. Binding of glycosaminoglycans to cathepsins thus directly affects catalytic activity, stability or the rate of autocatalytic activation of cathepsins. The interaction between cathepsin X and heparin has been revealed by affinity chromatography using heparin-Sepharose. Conformational changes were observed to accompany heparin-cathepsin X interaction by far UV-circular dichroism at both acidic (4.5) and neutral (7.4) pH. These conformational changes promoted a 4-fold increase in the dissociation constant of the enzyme-substrate interaction and increased 2.6-fold the kcat value also. The interaction between cathepsin X and heparin or heparan sulfate is specific since dermatan sulfate, chondroitin sulfate, and hyaluronic acid had no effect on the cathepsin X activity. Using flow cytometry cathepsin X was shown to bind cell surface heparan sulfate proteoglycans in wild-type CHO cells but not in CHO-745 cells, which are deficient in glycosaminoglycan synthesis. Moreover, fluorescently labeled cathepsin X was shown by confocal microscopy to be endocytosed by wild-type CHO cells, but not by CHO-745 cells. These results demonstrate the existence of an endocytosis mechanism of cathepsin X by the CHO cells dependent on heparan sulfate proteoglycans present at the cell surface, thus strongly suggesting that heparan sulfate proteoglycans can regulate the cellular trafficking and the enzymatic activity of cathepsin X.     

Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.     

Membrane-Associated Heparan Sulfate Proteoglycan Is a Receptor for Adeno-Associated Virus Type 2 Virions

The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, we demonstrate that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrated dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduced AAV attachment and infectivity. Finally, mutant cell lines that do not produce heparan sulfate proteoglycans were significantly impaired for both AAV binding and infection. This is the first report that proteoglycan has a role in cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector.     

Characterization of an in vitro model of alphavirus infection of immature and mature neurons

Terminally differentiated, mature neurons are essential cells that are not easily regenerated. Neurotropic viruses, such as Sindbis virus (SV), cause encephalomyelitis through their ability to replicate in neurons. SV causes the death of immature neurons, while mature neurons can often survive infection. The lack of a reproducible and convenient neuronal cell culture system has hindered a detailed study of the differences in levels of virus replication between immature and mature neurons and the molecular events involved in virus clearance from mature neurons. We have characterized SV replication in immortalized CSM14.1 rat neuronal cells that can be differentiated into neurons. During differentiation, CSM14.1 cells ceased dividing, developed neuronal morphology, and expressed neuron-specific cell markers. SV infection of undifferentiated CSM14.1 cells was efficient and resulted in high levels of virus replication and cell death. SV infection of differentiated CSM14.1 cells was less efficient and resulted in the production of 10- to 100-fold less virus and cell survival. In undifferentiated cells, SV induced a rapid shutdown of cellular protein synthesis and pE2 was efficiently processed to E2 (ratio of E2 to pE2, 2.14). In differentiated cells, the SV-induced shutdown of cellular protein synthesis was transient and pE2 was the primary form of E2 in cells (ratio of E2 to pE2, 0.0426). We conclude that age-dependent restriction of virus replication is an intrinsic property of maturing neurons and that the CSM14.1 cell line is a convenient model system for investigating the interactions of alphaviruses with neurons at various stages of differentiation.     

Initial interaction of herpes simplex virus with cells is binding to heparan sulfate.

We have shown that cell surface heparan sulfate serves as the initial receptor for both serotypes of herpes simplex virus (HSV). We found that virions could bind to heparin, a related glycosaminoglycan, and that heparin blocked virus adsorption. Agents known to bind to cell surface heparan sulfate blocked viral adsorption and infection. Enzymatic digestion of cell surface heparan sulfate but not of dermatan sulfate or chondroitin sulfate concomitantly reduced the binding of virus to the cells and rendered the cells resistant to infection. Although cell surface heparan sulfate was required for infection by HSV types 1 and 2, the two serotypes may bind to heparan sulfate with different affinities or may recognize different structural features of heparan sulfate. Consistent with their broad host ranges, the two HSV serotypes use as primary receptors ubiquitous cell surface components known to participate in interactions with the extracellular matrix and with other cell surfaces.     

Cell Surface Proteoglycans Are Necessary for A27L Protein-Mediated Cell Fusion: Identification of the N-Terminal Region of A27L Protein as the Glycosaminoglycan-Binding Domain

We previously showed that vaccinia virus infection of BSC40 cells was blocked by soluble heparin, suggesting that cell surface heparan sulfate mediates vaccinia virus binding (C.-S. Chung, J.-C. Hsiao, Y.-S. Chang, and W. Chang, J. Virol. 72:1577–1585, 1998). In this study, we extended our previous work and demonstrated that soluble A27L protein bound to heparan sulfate on cells and interfered with vaccinia virus infection at a postbinding step. In addition, we investigated the structure of A27L protein that provides for its binding to heparan sulfate on cells. A mutant of A27L protein, named D-A27L, devoid of a cluster of 12 amino acids rich in basic residues, was constructed. In contrast to the soluble A27L protein, purified D-A27L protein was inactive in all of our assays, including binding to heparin in vitro, binding to heparan sulfate on cells, and the ability to block virus infection. These data demonstrated that the N-terminal region acts as a glycosaminoglycan (GAG)-binding domain critical for A27L protein binding to cells. Previously A27L protein was thought to be involved in fusion of virus-infected cells induced by acid treatment. When we investigated whether cell surface GAGs also participate in A27L-dependent fusion, our results indicated that soluble A27L protein blocked cell fusion, whereas D-A27L protein did not. Taken together, the results therefore demonstrated that A27L-mediated cell fusion is triggered by its interaction with cell surface GAGs through the N-terminal domain.     

Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate

The conservation of positively charged residues in the N terminus of the hepatitis C virus (HCV) envelope glycoprotein E2 suggests an interaction of the viral envelope with cell surface glycosaminoglycans. Using recombinant envelope glycoprotein E2 and virus-like particles as ligands for cellular binding, we demonstrate that cell surface heparan sulfate proteoglycans (HSPG) play an important role in mediating HCV envelope-target cell interaction. Heparin and liver-derived highly sulfated heparan sulfate but not other soluble glycosaminoglycans inhibited cellular binding and entry of virus-like particles in a dose-dependent manner. Degradation of cell surface heparan sulfate by pretreatment with heparinases resulted in a marked reduction of viral envelope protein binding. Surface plasmon resonance analysis demonstrated a high affinity interaction (KD 5.2 x 10-9 m) of E2 with heparin, a structural homologue of highly sulfated heparan sulfate. Deletion of E2 hypervariable region-1 reduced E2-heparin interaction suggesting that positively charged residues in the N-terminal E2 region play an important role in mediating E2-HSPG binding. In conclusion, our results demonstrate for the first time that cellular binding of HCV envelope requires E2-HSPG interaction. Docking of E2 to cellular HSPG may be the initial step in the interaction between HCV and the cell surface resulting in receptor-mediated entry and initiation of infection.     

Simian Virus 40-Host Cell Interaction During Lytic Infection

Both exponentially growing and serum-arrested subcloned CV-1 cell cultures were infected with simian virus 40 (SV40). By 24 h after infection 96% of the nuclei of these permissive cells contained SV40 T-antigen. Analysis of the average DNA content per cell at various times after infection indicated that by 24 h most of the cells contained amounts of DNA similar to those normally found in G(2) cells. Analysis of cell cycle distributions indicated that a G(2) DNA complement was maintained by over 90% of the cells in the infected populations 24 to 48 h postinfection. Cells continued to synthesize SV40 DNA during the first 50 h after infection, and cytopathic effect was first observed 60 h after inoculation. After infection the number of mitotic cells that could be recovered by selective detachment decreased precipitously and was drastically reduced by 24 h. A study of the kinetics of decline in the number of mitotic cells suggests that this decline is related to an event during the cell cycle at or near the G(1)-S-phase border upon which commencement of SV40 DNA replication apparently depends. It was concluded that after SV40 infection, stationary cells are induced to cycle, and cycling cells complete one round of cellular DNA synthesis but do not divide. Although the infected cells continue to synthesize viral DNA, they do not appear able to reinitiate cellular DNA replication units. These results imply that the abundance of T-antigen (produced independently of cell cycle phase) in the presence of the enzymes required for continued DNA synthesis is not sufficient for reinitiation of cellular DNA synthesis.     

Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway.

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to herpes simplex virus (HSV) infection. These cells, termed sog9, were derived from mutant parental gro2C cells, which are themselves defective in heparan sulfate biosynthesis and 90% resistant to HSV type 1 (HSV-1) infection compared with control L cells (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro, J. Virol. 67:93-100, 1993). In this report, we show that sog9 cells exhibit a 3-order-of-magnitude reduction in susceptibility to HSV-1 compared with control L cells. In steady-state labeling experiments, sog9 cells accumulated almost no [35S]sulfate-labeled or [6-3H]glucosamine-labeled glycosaminoglycans, suggesting that the initiation of glycosaminoglycan assembly was specifically reduced in these cells. Despite these defects, sog9 cells were fully susceptible to vesicular stomatitis virus (VSV) and permissive for both VSV and HSV replication, assembly, and egress. HSV plaques formed in the sog9 monolayers in proportion to the amount of input virus, suggesting the block to infection was in the virus entry pathway. More importantly, HSV-1 infection of sog9 cells was not significantly reduced by soluble heparan sulfate, indicating that infection was glycosaminoglycan independent. Infection was inhibited by soluble gD-1, however, which suggests that glycoprotein gD plays a role in the infection of this cell line. The block to sog9 cell infection by HSV-1 could be eliminated by adding soluble dextran sulfate to the inoculum, which may act by stabilizing the virus at the sog9 cell surface. Thus, sog9 cells provide direct genetic evidence for a proteoglycan-independent entry pathway for HSV-1, and results with these cells suggest that HSV-1 is a useful reagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.     

Herpes simplex virus type 1 and pseudorabies virus bind to a common saturable receptor on Vero cells that is not heparan sulfate.

Herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) infect different natural hosts but are very similar in structure, replicative cycle, and entry into cultured cells. We determined whether HSV-1 and PRV use the same cellular components during entry into Vero cells, which are highly susceptible to each virus but are not from native hosts for either. UV-inactivated virions of either HSV-1 or PRV could saturate cell surfaces to block infection of challenge HSV-1 or PRV. In the presence of saturating levels for infection of either virus, radiolabeled virus bound well and in a heparin-sensitive manner. This result shows that heparan sulfate proteoglycans on Vero cells are not the limiting cellular component. To identify the virus component required for blocking, we used an HSV-1 null mutant virus lacking gB, gD, or gH as blocking virus. Virions lacking gB were able to block infection of challenge virus to the same level as did virus containing gB. In contrast, virions lacking gD lost all and most of the ability to block infection of HSV-1 and PRV, respectively. HSV-1 lacking gH and PRV lacking gp50 also were less competent in blocking infection of challenge virus. We conclude that HSV-1 and PRV bind to a common receptor for infection of Vero cells. Although both viruses bind a heparin-like cell component on many cells, including Vero cells, they also attach to a different and limited cell surface component that is bound at least by HSV-1 gD and possibly gH and to some degree by PRV gp50 but not gB. These results clearly demonstrate binding of both HSV-1 and PRV to a common cell receptor that is not heparan sulfate and demonstrate that several types of attachment occur for both viruses during infectious entry.     

Dengue virus induces expression of CXC chemokine ligand 10/IFN-gamma-inducible protein 10, which competitively inhibits viral binding to cell surface heparan sulfate

Dengue virus is an arthropod-borne flavivirus that causes a mild febrile illness, dengue fever, or a potentially fatal syndrome, dengue hemorrhagic fever/dengue shock syndrome. Chemokines primarily orchestrate leukocyte recruitment to the areas of viral infection, which makes them critical mediators of immune and inflammatory responses. In the present study, we investigated the induction and function of chemokines in mice early after infection with dengue virus in vivo. We found that CXCL10/IFN-gamma-inducible protein 10 (IP-10) expression was rapidly and transiently induced in liver following infection. The expressed CXCL10/IP-10 likely mediates the recruitment of activated NK cells, given that anti-CXCL10/IP-10-treated mice showed diminished NK cell infiltration and reduced hepatic expression of effector molecules in activated NK cells after dengue virus infection. Of particular interest, we found that CXCL10/IP-10 also was able to inhibit viral binding to target cells in vitro. Further investigation revealed that various CXCL10/IP-10 mutants, in which the residues that mediate the interaction between the chemokine and heparan sulfate were substituted, failed to exert the inhibitory effect on dengue binding, which suggests that CXCL10/IP-10 competes with dengue virus for binding to heparan sulfate on the cell surface. Moreover, subsequent plaque assays showed that this inhibition of dengue binding blocked viral uptake and replication. The inhibitory effect of CXCL10/IP-10 on the binding of dengue virus to cells may represent a novel contribution of this chemokine to the host defense against viral infection.     

Heparan sulfates from Swiss mouse 3T3 and SV3T3 cells: O-sulfate difference

A difference in the extent of sulfation between the heparan sulfate isolated from Swiss 3T3 mouse cells and that from Swiss 3T3 cells transformed by the DNA virus SV40 has been reported previously. This variance is manifested by different chromatographic and electrophoretic properties. Heparan sulfates from the two cell types were treated with nitrous acid under conditions that gave selective deaminative cleavage of glucosaminyl residues with sulfated amino groups in order to define the nature of the difference in sulfation further. The O-sulfate containing fragments from the heparan sulfates were compared by gel filtration and ion-exchange chromatography. The results showed that the 3T3 heparan sulfate contains 8% more O-sulfate than does the SV3T3 heparan sulfate. Analysis of uronic acids revealed that both types of heparan sulfates contain 45% L-iduronic acid and 55% D-glucuronic acid. These and other observations indicate that the primary difference in sulfation between the 3T3 and SV3T3 heparan sulfates lies in the extent of O-sulfation.     

The heparan sulfates of Swiss mouse 3T3 cells. The effect of transformation

Three major pools of heparan sulfate have been isolated from cultures of Swiss mouse 3T3 and SV40-transformed 3T3 cells: cell-surface, medium, and intracellular heparan sulfates. The cell-surface heparan sulfate is a high molecular weight proteogylcan which is partially degraded by pronase. Before pronase treatment, it has a peak molecular weight (as estimated by gel filtration) of appox. 7.2 · 10⁵ in contrast to only 2.4 · 10⁵ after pronase treatment. The medium heparan sulfate appears to be similar in structure to the cell-surface heparan sulfate, since they coelute on Bio-Gel A-15m and DEAE-cellulose, and are both proteoglycans. In contrast, the intracellular heparan sulfate has a low molecular weight (6.0 · 10³) and has little if any attached protein. Both the medium and intracellular heparan sulfate exhibit the transformation-associated change in structure reported earlier for cell-surface heparan sulfate (Underhill, C.B. and Keller, J.M. (1975) Biochem. Biophys. Res. Commun. 63, 448–454). This transformation-associated change, detected by DEAE-cellulose chromatography is not the result of changes in either molecular weight or protein core. Cellulose acetate electrophoresis of the cell-surface heparan sulfate at pH 1 suggests that the transformation-associated change in structure is due to a difference in sulfate content. Both types of heparan sulfate are produced in mixed cultures ot 3T3 and SV3T3 cells, indicating that neither serum factors in the culture medium nor secreted cell products are responsible for the transformation-associated change in heparan sulfate structure. The presented date are discussed with respect to the postulated role of heparan sulfate in cell social behavior.