Binding of Sindbis Virus to Cell Surface Heparan Sulfate

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.     

The amino-terminal one-third of pseudorabies virus glycoprotein gIII contains a functional attachment domain, but this domain is not required for the efficient penetration of Vero cells.

We have examined the attachment and penetration phenotypes of several glycoprotein gIII mutants of pseudorabies virus (PRV) and have identified the first one-third of gIII as a region that mediates efficient virus attachment to PK15 and Vero cells. This portion of gIII, amino acids 25 through 157 of the wild-type sequence, appeared to support attachment by binding to heparinlike molecules on cell surfaces. Virions containing the first one-third of gIII were sensitive to heparin competition and showed greatly reduced infectivity on cells treated with heparinase. PRV virions lacking the first one-third of the mature glycoprotein exhibited only residual binding to cells if challenged by vigorous washing with phosphate-buffered saline at 2 h postinfection at 4 degrees C. This residual binding was resistant to heparin competition, and strains lacking the first one-third of gIII were able to infect cells treated with heparinase as effectively as untreated cells. When we determined the penetration phenotypes for each strain, we found that gIII-mediated virus attachment was necessary for timely penetration of PK15 cells but remarkably was not required for efficient virus penetration of Vero cells. Moreover, wild-type PRV was actually prohibited from rapid penetration of Vero cells by a gIII-heparan sulfate interaction. Our results indicate that initial virus binding to heparan sulfate via glycoprotein gIII is not required for efficient PRV infection of all cell types and may in fact be detrimental in some instances.     

Bovine herpesvirus 1 glycoprotein B does not productively interact with cell surface heparan sulfate in a pseudorabies virion background.

Attachment to cell surface heparan sulfate proteoglycans is the first step in infection by several alphaherpesviruses. This interaction is primarily mediated by virion glycoprotein C (gC). In herpes simplex virus, in the absence of the nonessential gC, heparan sulfate binding is effected by glycoprotein B. In contrast, gC-negative pseudorabies virus (PrV) infects target cells via a heparan sulfate-independent mechanism, indicating that PrV virion gB does not productively interact with heparan sulfate. To assay whether a heterologous alphaherpesvirus gB protein will confer productive heparan sulfate binding on gC-negative PrV, gC was deleted from an infectious PrV recombinant, PrV-9112C2, which expresses bovine herpesvirus 1 (BHV-1) gB instead of PrV gB. Our data show that gC-negative PrV-BHV-1 gB recombinant 9112C2-delta gCbeta was not inhibited in infection by soluble heparin, in contrast to the gC-positive parental strain. Similar results were obtained when wild-type BHV-1 was compared with a gC-negative BHV-1 mutant. Moreover, infection of cells proficient or deficient in heparan sulfate biosynthesis occurred with equal efficiency by PrV-9112C2-delta gCbeta, whereas heparan sulfate-positive cells showed an approximately fivefold higher plating efficiency than heparan sulfate-negative cells with the parental gC-positive virus. In summary, our data show that in a PrV gC-negative virion background, BHV-1 gB is not able to mediate infection by productive interaction with heparan sulfate, and they indicate the same lack of heparin interaction for BHV-1 gB in gC-negative BHV-1.     

Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants

The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32 degrees C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37 degrees C and was undetectable at 39 degrees C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting a ts phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 x 10(6) and 3 x 10(3), respectively), demonstrating that this is a relatively tight mutant. In addition to the ts Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg(2+)-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep ts mutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.     

The gIII glycoprotein of pseudorabies virus is involved in two distinct steps of virus attachment.

The entry of herpesviruses into cells involves two distinct stages: attachment or adsorption to the cell surface followed by internalization. The virus envelope glycoproteins have been implicated in both stages. Pseudorabies virus attaches to cells by an early interaction that involves the viral glycoprotein gIII and a cellular heparinlike substance. We examined the role of gIII in the attachment process by analysis of a set of viruses carrying defined gIII mutations. The initial attachment of gIII mutants with an internal deletion of 134 amino acids (PrV2) to MDBK cells was indistinguishable from that of wild-type virus. The adsorption of these mutants was, however, much more sensitive than that of wild-type virus to competing heparin. Furthermore, while attachment of wild-type virus to MDBK cells led to a rapid loss of sensitivity to heparin, this was not the case with PrV2, which could be displaced from the cell surface by heparin after it had attached to the cells. We conclude that glycoprotein gIII is involved in two distinct steps of virus attachment and that the second of these steps but not the first is defective in PrV2.     

Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity.

The purpose of this study was to identify the herpes simplex virus glycoprotein(s) that mediates the adsorption of virions to cells. Because heparan sulfate moieties of cell surface proteoglycans serve as the receptors for herpes simplex virus adsorption, we tested whether any of the viral glycoproteins could bind to heparin-Sepharose in affinity chromatography experiments. Two glycoproteins, gB and gC, bound to heparin-Sepharose and could be eluted with soluble heparin. In order to determine whether virions devoid of gC or gB were impaired for adsorption, we quantitated the binding of wild-type and mutant virions to cells. We found that at equivalent input concentrations of purified virions, significantly fewer gC-negative virions bound to cells than did wild-type or gB-negative virions. In addition, the gC-negative virions that bound to cells showed a significant delay in penetration compared with wild-type virus. The impairments in adsorption and penetration of the gC-negative virions can account for their reduced PFU/particle ratios, which were found to be about 5 to 10% that of wild-type virions, depending on the host cell. Although gC is dispensable for replication of herpes simplex virus in cell culture, it clearly facilitates virion adsorption and enhances infectivity by about a factor of 10.     

Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle.

Isolates of foot-and-mouth disease virus (FMDV) exist as complex mixtures of variants. Two different serotype O1 Campos preparations that we examined contained two variants with distinct plaque morphologies on BHK cells: a small, clear-plaque virus that replicates in BHK and CHO cells, and a large, turbid-plaque virus that only grows in BHK cells. cDNAs encoding the capsids of these two variants were inserted into a genome-length FMDV type A12 infectious cDNA and used to produce chimeric viruses that exhibited the phenotype of the original variants. Analyses of these viruses, and hybrids created by exchanging portions of the capsid gene, identified codon 56 in VP3 (3056) as the critical determinant of both cell tropism and plaque phenotype. Specifically, the CHO growth/clear-plaque phenotype is dependent on the presence of the highly charged Arg residue at 3056, and viruses with this phenotype and genotype were selected during propagation in tissue culture. The genetically engineered Arg 3056 virus was highly attenuated in bovines, but viruses recovered from animals inoculated with high doses of this virus had lost the ability to grow in CHO cells and contained either an uncharged residue at 3056 or a negatively charged Glu substituted for a Lys at a spatially and antigenically related position on VP2 (2134). Comparison of these animal-derived viruses to other natural and engineered viruses demonstrated that positively charged residues are required at both 2134 and 3056 for binding to heparin. Taken together, these results indicate that in vitro cultivation of FMDV type O selects viruses that bind to heparin and that viruses with the heparin-binding phenotype are attenuated in the natural host.     

A heterologous heparin-binding domain can promote functional attachment of a pseudorabies virus gC mutant to cell surfaces.

The efficient attachment of pseudorabies virus to cultured cells is dependent on an electrostatic interaction between negatively charged cell surface heparan sulfate and the viral envelope glycoprotein gC. Deletion of the first one-third of gC severely impairs virus attachment, but the mutant virions are still capable of entering cells and establishing an infection via a gC-independent pathway. This region of gC contains three clusters of positively charged amino acids that exactly or nearly conform to proposed consensus motifs for heparin-binding domains (HBDs), and the loss of one or more of these potential HBDs may be responsible for the observed attachment defect. To more directly show the involvement of HBDs in pseudorabies virus attachment to cells, we replaced the first one-third of gC with a single, biochemically defined HBD from apolipoprotein B-100. On the basis of the results of attachment, penetration, and heparin competition assays, the heterologous HBD mediated heparan sulfate-dependent virus attachment, but not to fully wild-type levels. Although the intermediate phenotype is not understood, the apolipoprotein B-100 HBD may represent the smallest defined amino acid sequence that promotes functional herpesvirus attachment to cultured cells.     

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.     

Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses.

We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.     

The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells

Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.     

Herpes simplex virus type 2 glycoprotein G is targeted by the sulfated oligo- and polysaccharide inhibitors of virus attachment to cells

Variants of herpes simplex virus type 2 (HSV-2) generated by virus passage in GMK-AH1 cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to PI-88 in their initial infection of cells and/or their cell-to-cell spread. The major alteration detected in all variants resistant to PI-88 in the initial infection of cells was a frameshift mutation(s) in the glycoprotein G (gG) gene that resulted in the lack of protein expression. Molecular transfer of the altered gG gene into the wild-type background confirmed that the gG-deficient recombinants were resistant to PI-88. In addition to PI-88, all gG-deficient variants of HSV-2 were resistant to the sulfated polysaccharide heparin. The gG-deficient virions were capable of attaching to cells, and this activity was relatively resistant to PI-88. In addition to having a drug-resistant phenotype, the gG-deficient variants were inefficiently released from infected cells. Purified gG bound to heparin and showed the cell-binding activity which was inhibited by PI-88. Many PI-88 variants produced syncytia in cultured cells and contained alterations in gB, including the syncytium-inducing L792P amino acid substitution. Although this phenotype can enhance the lateral spread of HSV in cells, it conferred no virus resistance to PI-88. Some PI-88 variants also contained occasional alterations in gC, gD, gE, gK, and UL24. In conclusion, we found that glycoprotein gG, a mucin-like component of the HSV-2 envelope, was targeted by sulfated oligo- and polysaccharides. This is a novel finding that suggests the involvement of HSV-2 gG in interactions with sulfated polysaccharides, including cell surface glycosaminoglycans.     

Large-plaque mutants of Sindbis virus show reduced binding to heparan sulfate, heightened viremia, and slower clearance from the circulation

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.     

An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells

Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.     

Temperature-sensitive mutants of vesicular stomatitis virus are conditionally defective particles that interfere with and are rescued by wild-type virus.

Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to complementation groups I, II and IV inhibited the replication of wild-type vesicular stomatitis virus when mixed infections were carried out in BHK21 cells at 32, 37, and 39.5 C. The group IV mutant (ts G 41) was most effective in this regard; wild-type virus yields were inhibited almost 1,000-fold in mixed infections with this mutant at 32 C. In the case of group I and II mutants, inhibition of wild-type virus replication at 37 and 39.5 C was accompanied by an enhancement (up to 15,000-fold) of the yields of the coinfecting ts mutant. The yields of the group IV mutant (ts G 41) were not enhanced by mixed infections with wild-type virus at any temperature, although this mutant inhibited wild-type virus replication at all temperatures. The dominance of the replication of ts mutants at 37 C provides a rationale for the selection and maintenance of ts virus in persistently infected cells.     

Selection of COS cell mutants defective in the biosynthesis of heparan sulfate proteoglycan.

A simple procedure using human basic fibroblast growth factor (FGF) was utilized for the selection of COS cell mutants with defects in the biosynthesis or expression of heparan sulfate proteoglycan (HSPG). Our approach was based on the strong binding affinity exhibited by COS cells to human basic FGF that had been adsorbed to plastic dishes. Cell binding to basic FGF could be inhibited by heparin and heparan sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, or hyaluronic acid, suggesting that the cell binding involved an interaction between basic FGF and cell surface heparin-like molecules. COS cells were treated with ethyl methanesulfonate and four stable mutants were subsequently isolated that did not bind strongly to basic FGF adsorbed to plastic. These mutants cell lines (CM-2, CM-8, CM-9, and CM-15) exhibited significantly reduced 35SO4 incorporation into HS (40-70% depending on the cellular pool analyzed). In one of these cell lines, CM-15, the incorporation of [6-3H]glucosamine into HS was unaltered, suggesting that the extent of oligosaccharide polymerization was equivalent to that observed for the wild-type cells. Structural analysis revealed that N-sulfated glucosamine residues were present much less frequently in HS derived from these cells as compared with that derived from wild-type cells. Furthermore, CM-15 was found to be three-fold deficient in HS N-sulfotransferase activity, but contained wild-type levels of HS O-sulfotransferase activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses.

We describe a sensitive, internally controlled method for comparing the genetic adaptability and relative fitness of virus populations in constant or changing host environments. Certain monoclonal antibody-resistant mutants of vesicular stomatitis virus can compete equally during serial passages in mixtures with the parental wild-type clone from which they were derived. These genetically marked "surrogate wild-type" neutral mutants, when mixed with wild-type virus, allow reliable measurement of changes in virus fitness and of virus adaptation to different host environments. Quantitative fitness vector plots demonstrate graphically that even clones of an RNA virus are composed of complex variant populations (quasispecies). Variants of greater fitness (competitive replication ability) were selected within very few passages of virus clones in new host cells or animals. Even clones which were well adapted to BHK21 cells gained further fitness during repeated passages in BHK21 cells.     

Soluble forms of herpes simplex virus glycoprotein D bind to a limited number of cell surface receptors and inhibit virus entry into cells.

Herpes simplex virus type 1 (HSV-1) and HSV-2 plaque production was inhibited by treating cells with soluble forms of HSV-1 glycoprotein D (gD-1t) and HSV-2 glycoprotein D (gD-2t). Both glycoproteins inhibited entry of HSV-1 and HSV-2 without affecting virus adsorption. In contrast, a soluble form of HSV-2 glycoprotein B had no effect on virus entry into cells. Specific binding of gD-1t and gD-2t to cells was saturable, and approximately 4 x 10(5) to 5 x 10(5) molecules bound per cell. Binding of gD-1t was markedly reduced by treating cells with certain proteases but was unaffected when cell surface heparan sulfate glycosaminoglycans were enzymatically removed or when the binding was carried out in the presence of heparin. Together, these results suggest that gD binds to a limited set of cell surface receptors which may be proteins and that these interactions are essential for subsequent virus entry into cells. However, binding of gD to its receptors is not required for the initial adsorption of virus to the cell surface, which involves more numerous sites (probably including heparan sulfate) than those which mediate gD binding.     

Aura virus structure suggests that the T=4 organization is a fundamental property of viral structural proteins

Aura and Sindbis viruses are closely related alphaviruses. Unlike other alphaviruses, Aura virus efficiently encapsidates both genomic RNA (11.8 kb) and subgenomic RNA (4.2 kb) to form virus particles. Previous studies on negatively stained Aura virus particles predicted that there were two major size classes with potential T=3 and T=4 capsid structures. We have used cryoelectron microscopy and three-dimensional image reconstruction techniques to examine the native morphology of different classes of Aura virus particles produced in BHK cells. Purified particles separated into two components in a sucrose gradient. Reconstructions of particles in the top and bottom components were computed to resolutions of 17 and 21 A, respectively, and compared with reconstructions of Sindbis virus and Ross River virus particles. Aura virus particles of both top and bottom components have similar, T=4 structures that resemble those of other alphaviruses. The morphology of Aura virus glycoprotein spikes closely resembles that of Sindbis virus spikes and is detectably different from that of Ross River virus spikes. Thus, some aspects of the surface structure of members of the Sindbis virus lineage have been conserved, but other aspects have diverged from the Semliki Forest/Ross River virus lineage.