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

1999 ◽  
Vol 73 (8) ◽  
pp. 6299-6306 ◽  
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.

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

1998 ◽  
Vol 72 (9) ◽  
pp. 7357-7366 ◽  
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.

scholarly journals Isolation of an adhesion-mediating protein from chick neural retina adherons.

1985 ◽  
Vol 101 (3) ◽  
pp. 1071-1077 ◽  
Author(s):  
D Schubert ◽  
M LaCorbiere
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Cultured Cells ◽  
Cell Types ◽  
Neural Retina ◽  
Cell Surface Receptor ◽  
Isolation And Characterization ◽  
Surface Receptor ◽  
Adhesive Interactions

Adherons are high molecular weight glycoprotein complexes which are released into the growth medium of cultured cells. They mediate the adhesive interactions of many cell types, including those of embryonic chick neural retina. The cell surface receptor for chick neural retina adherons has been purified, and shown to be a heparan sulfate proteoglycan (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 100:56-63). This paper describes the isolation and characterization of a protein in neural retina adherons which interacts specifically with the cell surface receptor. The 20,000-mol-wt protein, called retinal purpurin (RP), stimulates neural retina cell-substratum adhesion and prolongs the survival of neural retina cells in culture. The RP protein interacts with heparin and heparan sulfate, but not with other glycosaminoglycans. Monovalent antibodies against RP inhibit RP-cell adhesion as well as adheron-cell interactions. The RP protein is found in neural retina, but not in other tissues such as brain and muscle. These data suggest that RP plays a role in both the survival and adhesive interactions of neural retina cells.

scholarly journals Characterization of a Chinese Hamster Ovary Cell Line Developed by Retroviral Insertional Mutagenesis That Is Resistant to Sindbis Virus Infection

1999 ◽  
Vol 73 (6) ◽  
pp. 4919-4924 ◽  
Author(s):  
Jia-Tsrong Jan ◽  
Andrew P. Byrnes ◽  
Diane E. Griffin
Keyword(s):  
Heparan Sulfate ◽  
Cell Line ◽  
Virus Replication ◽  
Chinese Hamster Ovary ◽  
Sindbis Virus ◽  
Insertional Mutagenesis ◽  
Cultured Cells ◽  
Chinese Hamster ◽  
Ovary Cell

ABSTRACT The alphavirus Sindbis virus (SV) has a wide host range and infects many types of cultured cells in vitro. The outcome of infection is dependent on the strain of virus used for infection and the properties of the cells infected. To identify cellular determinants of susceptibility to SV infection we mutagenized Chinese hamster ovary (CHO) cells by retroviral insertion with a vector containing the neomycin resistance gene that allowed selection for integration into transcriptionally active genes. Cells were then selected for survival after infection with SV. The most resistant cell line (CHO-18.4m) exhibited delayed virus replication and virus-induced cell death, had a single retroviral insertion, and was defective in SV binding to the cell surface. Further analysis revealed that CHO-18.4m cells were deficient in the expression of the sulfated glycosaminoglycans heparan sulfate and chondroitin sulfate. This further confirms the importance of heparan sulfate as an attachment molecule for SV in vitro and demonstrates the usefulness of this technique for identifying cellular genes that are important for virus replication.

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

2002 ◽  
Vol 76 (20) ◽  
pp. 10128-10137 ◽  
Author(s):  
Jolanda M. Smit ◽  
Barry-Lee Waarts ◽  
Koji Kimata ◽  
William B. Klimstra ◽  
Robert Bittman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Model System ◽  
Low Ph ◽  
Wild Type ◽  
Enveloped Viruses ◽  
Neutral Ph ◽  
Receptor Interactions

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.

scholarly journals Attenuation of Sindbis virus variants incorporating uncleaved PE2 glycoprotein is correlated with attachment to cell-surface heparan sulfate

Virology ◽  
2004 ◽  
Vol 322 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Kate D Ryman ◽  
William B Klimstra ◽  
Robert E Johnston
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Sindbis Virus

scholarly journals Heparan Sulfate Binding Can Contribute to the Neurovirulence of Neuroadapted and Nonneuroadapted Sindbis Viruses

2007 ◽  
Vol 81 (7) ◽  
pp. 3563-3573 ◽  
Author(s):  
Kate D. Ryman ◽  
Christina L. Gardner ◽  
Crystal W. Burke ◽  
Kathryn C. Meier ◽  
Joseph M. Thompson ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Sindbis Virus ◽  
Cell Attachment ◽  
High Titer ◽  
Consensus Sequence ◽  
Cell Types ◽  
Viral Disease ◽  
Site Mutation ◽  
Adult Mice ◽  
Host Tissues

ABSTRACT Cell culture-adapted laboratory strains of Sindbis virus (SB) exhibit efficient initial attachment to cell surface heparan sulfate (HS) receptors. In contrast, non-cell-adapted strains, such as the SB consensus sequence virus TR339, interact weakly with HS and cell surfaces. Regardless of their HS binding phenotype, most SB strains do not cause fatal disease in adult mice, whether inoculated subcutaneously (s.c.) or intracranially (i.c.). However, laboratory strains of SB can be rendered neurovirulent for adult mice by introduction of a glutamine (Gln)-to-histidine (His) mutation at position 55 of the E2 envelope glycoprotein. In the current work, we have determined that E2 His 55-containing viruses require a second-site mutation (Glu to Lys) at E2 position 70 that confers efficient HS binding in order to exhibit virulence for adult mice and that virulence is correlated with very high infectivity for many cell types. Furthermore, introduction of E2 Lys 70 or certain other HS-binding mutations alone also increased morbidity and/or mortality over that of TR339 for older mice inoculated i.c. However, all viruses containing single HS-binding mutations were attenuated in s.c. inoculated suckling mice in comparison with TR339. These results suggest that HS binding may attenuate viral disease that is dependent on high-titer viremia; however, efficient cell attachment through HS binding can increase virulence, presumably through enhancing the replication of SB within specific host tissues such as the brain.

scholarly journals Large-Plaque Mutants of Sindbis Virus Show Reduced Binding to Heparan Sulfate, Heightened Viremia, and Slower Clearance from the Circulation

2000 ◽  
Vol 74 (2) ◽  
pp. 644-651 ◽  
Author(s):  
Andrew P. Byrnes ◽  
Diane E. Griffin
Keyword(s):  
Heparan Sulfate ◽  
Sindbis Virus ◽  
Cultured Cells ◽  
Single Amino Acid ◽  
Heparin Binding ◽  
Large Plaque ◽  
Recombinant Viruses ◽  
Binding Domains ◽  
Charged Amino Acids

ABSTRACT Laboratory strains of Sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. During infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. Sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the E2 glycoprotein. Recombinant viruses were constructed with these changes as single amino acid substitutions in a strain Toto 1101 background. All exhibited decreased binding to heparan sulfate and had larger plaques than Toto 1101. When injected subcutaneously into neonatal mice, large-plaque viruses produced higher-titer viremia and often caused higher mortality. Because circulating heparin-binding proteins are known to be rapidly sequestered by tissue heparan sulfate, we measured the kinetics of viral clearance following intravenous injection. Much of the parental small-plaque Toto 1101 strain of Sindbis virus was cleared from the circulation by the liver within minutes, in contrast to recombinant large-plaque viruses, which had longer circulating half-lives. These findings indicate that a decreased ability to bind to heparan sulfate allows more efficient viral production in vivo, which may in turn lead to increased mortality. Because Sindbis virus is only one of a growing number of viruses from many families which have been shown to bind to heparan sulfate, these results may be generally applicable to the pathogenesis of such viruses.

scholarly journals Passage of Classical Swine Fever Virus in Cultured Swine Kidney Cells Selects Virus Variants That Bind to Heparan Sulfate due to a Single Amino Acid Change in Envelope Protein Erns

2000 ◽  
Vol 74 (20) ◽  
pp. 9553-9561 ◽  
Author(s):  
M. M. Hulst ◽  
H. G. P. van Gennip ◽  
R. J. M. Moormann
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Kidney Cells ◽  
Virus Particles ◽  
Heparinase I ◽  
Initial Binding ◽  
Swine Kidney

ABSTRACT Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein Ernsand E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV Ernspurified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of Erns with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the Erns genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of Erns (amino acid 476 in the polyprotein of CSFV). Replacement of the Erns gene of an infectious DNA copy of C1.1.1 with the Erns genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an Erns receptor.

scholarly journals Binding of Sindbis Virus to Cell Surface Heparan Sulfate

1998 ◽  
Vol 72 (9) ◽  
pp. 7349-7356 ◽  
Author(s):  
Andrew P. Byrnes ◽  
Diane E. Griffin
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Neutralizing Antibodies ◽  
Sindbis Virus ◽  
Cell Types ◽  
Surface Binding ◽  
Virus Binding ◽  
Surface Receptors ◽  
Tissue Tropisms ◽  
High Degree

ABSTRACT Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cell surface receptors which allow infection of so many different species and cell types are still incompletely characterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate in the binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and this interaction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experiments showed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However, Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate, indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross River virus, was found to be independent of heparan sulfate.

scholarly journals Human Herpesvirus 8 Envelope Glycoprotein K8.1A Interaction with the Target Cells Involves Heparan Sulfate

2001 ◽  
Vol 75 (16) ◽  
pp. 7517-7527 ◽  
Author(s):  
Fu-Zhang Wang ◽  
Shaw M. Akula ◽  
Naranatt P. Pramod ◽  
Ling Zeng ◽  
Bala Chandran
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Target Cells ◽  
Dependent Manner ◽  
Unique Region ◽  
Herpesvirus 8 ◽  
Agarose Beads ◽  
Virion Envelope

ABSTRACT Human herpesvirus-8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus K8.1 gene encodes for two immunogenic glycoproteins, gpK8.1A and gpK8.1B, originating from spliced messages. The 228-amino-acid (aa) gpK8.1A is the predominant form associated with the virion envelope, consisting of a 167-aa region identical to gpK8.1B and a 61-aa unique region (L. Zhu, V. Puri, and B. Chandran, Virology 262:237–249, 1999). HHV-8 has a broad in vivo and in vitro cellular tropism, and our studies showed that this may be in part due to HHV-8's interaction with the ubiquitous host cell surface molecule, heparan sulfate (HS). Since HHV-8 K8.1 gene is positionally colinear to the Epstein-Barr virus (EBV) gene encoding the gp350/gp220 protein involved in EBV binding to the target cells, gpK8.1A's ability to interact with the target cells was examined. The gpK8.1A without the transmembrane and carboxyl domains (ΔTMgpK8.1A) was expressed in a baculovirus system and purified. Radiolabeled purified ΔTMgpK8.1A protein bound to the target cells, which was blocked by unlabeled ΔTMgpK8.1A. Unlabeled ΔTMgpK8.1A blocked the binding of [3H]thymidine-labeled purified HHV-8 to the target cells. Binding of radiolabeled ΔTMgpK8.1A to the target cells was inhibited in a dose-dependent manner by soluble heparin, a glycosaminoglycan (GAG) closely related to HS, but not by other GAGs such as chondroitin sulfate A and C, N-acetyl heparin and de-N-sulfated heparin. Cell surface absorbed ΔTMgpK8.1A was displaced by soluble heparin. Radiolabeled ΔTMgpK8.1A also bound to HS expressing Chinese hamster ovary (CHO-K1) cells, and binding to mutant CHO cell lines deficient in HS was significantly reduced. The ΔTMgpK8.1A specifically bound to heparin-agarose beads, which was inhibited by HS and heparin, but not by other GAGs. Virion envelope-associated gpK8.1A was specifically precipitated by heparin-agarose beads. These findings suggest that gpK8.1A interaction with target cells involves cell surface HS-like moieties, and HHV-8 interaction with HS could be in part mediated by virion envelope-associated gpK8.1A.

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