Adaptation of Alphaviruses to Heparan Sulfate: Interaction of Sindbis and Semliki Forest Viruses with Liposomes Containing Lipid-Conjugated Heparin

ABSTRACT Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycosaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 (E2:1, E2:70, and E2:114) have been identified that mutate during adaptation and independently confer on the virus the ability to bind to cell surface HS (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). In this study, we used HS-adapted SIN mutants to evaluate a new model system involving target liposomes containing lipid-conjugated heparin (HepPE) as an HS receptor analog for the virus. HS-adapted SIN, but not nonadapted wild-type SIN TR339, interacted efficiently with HepPE-containing liposomes at neutral pH. Binding was competitively inhibited by soluble heparin. Despite the efficient binding of HS-adapted SIN to HepPE-containing liposomes at neutral pH, there was no fusion under these conditions. Fusion did occur, however, at low pH, consistent with cellular entry of the virus via acidic endosomes. At low pH, wild-type or HS-adapted SIN underwent fusion with liposomes with or without HepPE with similar kinetics, suggesting that interaction with the HS receptor analog at neutral pH has little influence on subsequent fusion of SIN at low pH. Finally, Semliki Forest virus (SFV), passaged frequently on BHK-21 cells, also interacted efficiently with HepPE-containing liposomes, indicating that SFV, like other alphaviruses, readily adapts to cell surface HS. In conclusion, the liposomal model system presented in this paper may serve as a novel tool for the study of receptor interactions and membrane fusion properties of HS-interacting enveloped viruses.

Download Full-text

The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

PLoS Pathogens ◽

10.1371/journal.ppat.1009803 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009803

Author(s):

Dipanwita Mitra ◽

Mohammad H. Hasan ◽

John T. Bates ◽

Michael A. Bierdeman ◽

Dallas R. Ederer ◽

...

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Virus Entry ◽

Degree Of Polymerization ◽

Host Cells ◽

Virus Infectivity ◽

Wild Type ◽

Enveloped Viruses ◽

Host Membrane ◽

Mutant Cells

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.

Download Full-text

Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.74.7.3353-3365.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3353-3365 ◽

Cited By ~ 183

Author(s):

Chi-Long Lin ◽

Che-Sheng Chung ◽

Hans G. Heine ◽

Wen Chang

Keyword(s):

Cell Surface ◽

Vaccinia Virus ◽

Heparan Sulfate ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

Download Full-text

Low-pH-Dependent Fusion of Sindbis Virus with Receptor-Free Cholesterol- and Sphingolipid-Containing Liposomes

Journal of Virology ◽

10.1128/jvi.73.10.8476-8484.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8476-8484 ◽

Cited By ~ 113

Author(s):

Jolanda M. Smit ◽

Robert Bittman ◽

Jan Wilschut

Keyword(s):

Sindbis Virus ◽

Core Protein ◽

Fusion Reaction ◽

Low Ph ◽

Viral Envelope ◽

Cell Entry ◽

Receptor Interaction ◽

Neutral Ph ◽

Virus Fusion ◽

Entry Mechanism

ABSTRACT There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.

Download Full-text

Interaction of an Outer Membrane Protein of Enterotoxigenic Escherichia coli with Cell Surface Heparan Sulfate Proteoglycans

Infection and Immunity ◽

10.1128/iai.70.3.1530-1537.2002 ◽

2002 ◽

Vol 70 (3) ◽

pp. 1530-1537 ◽

Cited By ~ 44

Author(s):

James M. Fleckenstein ◽

James T. Holland ◽

David L. Hasty

Keyword(s):

Escherichia Coli ◽

Epithelial Cell ◽

Epithelial Cells ◽

Cell Surface ◽

Heparan Sulfate ◽

Heparan Sulfate Proteoglycans ◽

Eukaryotic Cells ◽

Heparin Binding ◽

Wild Type ◽

E Coli

ABSTRACT We have previously shown that enterotoxigenic invasion protein A (Tia), a 25-kDa outer membrane protein encoded on an apparent pathogenicity island of enterotoxigenic Escherichia coli (ETEC) strain H10407, mediates attachment to and invasion into cultured human gastrointestinal epithelial cells. The epithelial cell receptor(s) for Tia has not been identified. Here we show that Tia interacts with cell surface heparan sulfate proteoglycans. Recombinant E. coli expressing Tia mediated invasion into wild-type epithelial cell lines but not invasion into proteoglycan-deficient cells. Furthermore, wild-type eukaryotic cells, but not proteoglycan-deficient eukaryotic cells, attached to immobilized polyhistidine-tagged recombinant Tia (rTia). Binding of epithelial cells to immobilized rTia was inhibited by exogenous heparan sulfate glycosaminoglycans but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfate. Similarly, pretreatment of eukaryotic cells with heparinase I, but not pretreatment of eukaryotic cells with chrondroitinase ABC, inhibited attachment to rTia. In addition, we also observed heparin binding to both immobilized rTia and recombinant E. coli expressing Tia. Heparin binding was inhibited by a synthetic peptide representing a surface loop of Tia, as well as by antibodies directed against this peptide. Additional studies indicated that Tia, as a prokaryotic heparin binding protein, may also interact via sulfated proteoglycan molecular bridges with a number of mammalian heparan sulfate binding proteins. These findings suggest that the binding of Tia to host epithelial cells is mediated at least in part through heparan sulfate proteoglycans and that ETEC belongs on the growing list of pathogens that utilize these ubiquitous cell surface molecules as receptors.

Download Full-text

The Cholesterol Requirement for Sindbis Virus Entry and Exit and Characterization of a Spike Protein Region Involved in Cholesterol Dependence

Journal of Virology ◽

10.1128/jvi.73.5.4272-4278.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4272-4278 ◽

Cited By ~ 119

Author(s):

Yanping E. Lu ◽

Todd Cassese ◽

Margaret Kielian

Keyword(s):

Sindbis Virus ◽

Semliki Forest Virus ◽

Virus Entry ◽

Single Point ◽

Endocytic Pathway ◽

Progeny Virus ◽

Single Point Mutation ◽

Entry And Exit ◽

Wild Type

ABSTRACT Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV,srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had thesrf-3 amino acid sequence from E1 position 224 to 235 showed increases of ∼100-fold in infection and ∼250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGMexit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.

Download Full-text

Adaptation of Sindbis Virus to BHK Cells Selects for Use of Heparan Sulfate as an Attachment Receptor

Journal of Virology ◽

10.1128/jvi.72.9.7357-7366.1998 ◽

1998 ◽

Vol 72 (9) ◽

pp. 7357-7366 ◽

Cited By ~ 266

Author(s):

William B. Klimstra ◽

Kate D. Ryman ◽

Robert E. Johnston

Keyword(s):

Cell Culture ◽

Cell Surface ◽

Heparan Sulfate ◽

Sindbis Virus ◽

Consensus Sequence ◽

Alternative Mechanism ◽

Bhk Cells ◽

Adaptive Mutations ◽

Culture Adaptation ◽

Agarose Beads

ABSTRACT Attachment of Sindbis virus to the cell surface glycosaminoglycan heparan sulfate (HS) and the selection of this phenotype by cell culture adaptation were investigated. Virus (TR339) was derived from a cDNA clone representing the consensus sequence of strain AR339 (K. L. McKnight, D. A. Simpson, S. C. Lin, T. A. Knott, J. M. Polo, D. F. Pence, D. B. Johannsen, H. W. Heidner, N. L. Davis, and R. E. Johnston, J. Virol. 70:1981–1989, 1996) and from mutant clones containing either one or two dominant cell culture adaptations in the E2 structural glycoprotein (Arg instead of Ser at E2 position 1 [designated TRSB]) or this mutation plus Arg for Ser at E2 114 [designated TRSB-R114]). The consensus virus, TR339, bound to baby hamster kidney (BHK) cells very poorly. The mutation in TRSB increased binding 10- to 50-fold, and the additional mutation in TRSB-R114 increased binding 3- to 5-fold over TRSB. The magnitude of binding was positively correlated with the degree of cell culture adaptation and with attenuation of these viruses in neonatal mice. HS was identified as the attachment receptor for the mutant viruses by the following experimental results. (i) Low concentrations of soluble heparin inhibited plaque formation on and binding of mutant viruses to BHK cells by >95%. In contrast, TR339 showed minimal inhibition at high concentrations. (ii) Binding and infectivity of TRSB-R114 was sensitive to digestion of cell surface HS with heparinase III, and TRSB was sensitive to both heparinase I and heparinase III. TR339 infectivity was only slightly affected by either digestion. (iii) Radiolabeled TRSB and TRSB-R114 attached efficiently to heparin-agarose beads in binding assays, while TR339 showed virtually no binding. (iv) Binding and infectivity of TRSB and TRSB-R114, but not TR339, were greatly reduced on Chinese hamster ovary cells deficient in HS specifically or all glycosaminoglycans. (v) High-multiplicity-of-infection passage of TR339 on BHK cell cultures resulted in rapid coselection of high-affinity binding to BHK cells and attachment to heparin-agarose beads. Sequencing of the passaged virus population revealed a mutation from Glu to Lys at E2 70, a mutation common to many laboratory strains of Sindbis virus. These results suggest that TR339, the most virulent virus tested, attaches to cells through a low-affinity, primarily HS-independent mechanism. Adaptive mutations, selected during cell culture growth of Sindbis virus, enhance binding and infectivity by allowing the virus to attach by an alternative mechanism that is dependent on the presence of cell surface HS.

Download Full-text

The Furin Protease Cleavage Recognition Sequence of Sindbis Virus PE2 Can Mediate Virion Attachment to Cell Surface Heparan Sulfate

Journal of Virology ◽

10.1128/jvi.73.8.6299-6306.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 6299-6306 ◽

Cited By ~ 55

Author(s):

William B. Klimstra ◽

Hans W. Heidner ◽

Robert E. Johnston

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Sindbis Virus ◽

Cultured Cells ◽

Consensus Sequence ◽

Recognition Sequence ◽

Furin Cleavage ◽

Virus Particles ◽

Agarose Beads ◽

Protease Cleavage

ABSTRACT Cell culture-adapted Sindbis virus strains attach to heparan sulfate (HS) receptors during infection of cultured cells (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357–7366, 1998). At least three E2 glycoprotein mutations (E2 Arg 1, E2 Lys 70, and E2 Arg 114) can independently confer HS attachment in the background of the consensus sequence Sindbis virus (TR339). In the studies reported here, we have investigated the mechanism by which the E2 Arg 1 mutation confers HS-dependent binding. Substitution of Arg for Ser at E2 1 resulted in a significant reduction in the efficiency of PE2 cleavage, yielding virus particles containing a mixture of PE2 and mature E2. Presence of PE2 was associated with an increase in HS-dependent attachment to cells and efficient attachment to heparin-agarose beads, presumably because the furin recognition site for PE2 cleavage also represents a candidate HS binding sequence. A comparison of mutants with partially or completely inhibited PE2 cleavage demonstrated that efficiency of cell binding was correlated with the amount of PE2 in virus particles. Viruses rendered cleavage defective due to deletions of portions or all of the furin cleavage sequence attached very poorly to cells, indicating that an intact furin cleavage sequence was specifically required for PE2-mediated attachment to cells. In contrast, a virus containing a partial deletion was capable of efficient binding to heparin-agarose beads, suggesting different requirements for heparin bead and cell surface HS binding. Furthermore, virus produced in C6/36 mosquito cells, which cleave PE2 more efficiently than BHK cells, exhibited a reduction in cell attachment efficiency correlated with reduced content of PE2 in particles. Taken together, these results strongly argue that the XBXBBX (B, basic; X, hydrophobic) furin protease recognition sequence of PE2 can mediate the binding of PE2-containing Sindbis viruses to HS. This sequence is very similar to an XBBXBX heparin-HS interaction consensus sequence. The attachment of furin protease cleavage sequences to HS may have relevance to other viruses whose attachment proteins are cleaved during maturation at positively charged recognition sequences.

Download Full-text

Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells.

The Journal of Cell Biology ◽

10.1083/jcb.133.2.247 ◽

1996 ◽

Vol 133 (2) ◽

pp. 247-256 ◽

Cited By ~ 165

Author(s):

T Yoshimori ◽

P Keller ◽

M G Roth ◽

K Simons

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Cho Cells ◽

Semliki Forest Virus ◽

Mdck Cells ◽

Wild Type ◽

Vitro Assay ◽

Delivery Routes ◽

The One

The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.

Download Full-text