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

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.

Murine encephalitis caused by HCoV-OC43, a human coronavirus with broad species specificity, is partly immune-mediated

“…While little is known about the changes required for efficient replication of HCoV-OC43 in the murine CNS, it is likely that this process includes mutations that affect binding to host cell sugar moieties. Infection with the prototypic alphavirus, Sindbis virus (SINV), or with a picornavirus, Theiler's murine encephalomyelitis virus (TMEV), is initiated by binding to a polyanionic polysaccharide, heparin sulfate (Byrnes and Griffin, 1998;Klimstra et al, 1998;Reddi and Lipton, 2002). SINV with enhanced binding to heparin sulfate is selected after passage in tissue culture cells.…”

Virus Entry: Open Sesame

“…Moreover, the presence of specific glycosphingolipids in the target cell membrane can enhance CD4/coreceptor-dependent fusion (Puri et al, 1998). Viruses, in particular those that undergo rapid mutation, are also known to be able to switch receptors (Byrnes and Griffin, 2000;Klimstra et al, 1998) or adapt to use alternative receptors when the primary receptor is absent (Vlasak et al, 2005). This is one of the risks posed by the current avian influenza epidemic: the avian hemagglutinin glycoprotein may, through mutations, evolve to interact more potently with glycoconjugates on human cells.…”

Interactions Between Virus Proteins and Host Cell Membranes During the Viral Life Cycle

“…Interactions with heparan sulfate proteoglycans have been documented for herpesviruses (Spear and Longnecker, 2003), papillomaviruses (Giroglou et al, 2001), paramyxovirus 3 (Bose and Banerjee, 2002), pestiviruses (Hulst et al, 2000), flaviviruses (Chen et al, 1997;Mandl et al, 2001), Sindbis virus (Byrnes and GriYn, 1998;Klimstra et al, 1998), adenoviruses (Dechecchi et al, 2000), and adeno-associated viruses (Opie et al, 2003;Summerford and Samulski, 1998). In most cases, these interactions are thought to be nonspecific and probably based on electrostatic attractions between the highly charged sulfated proteoglycans and arginine residues in viral particles (Chen et al, 1997;Hulst et al, 2000;Klimstra et al, 1998;Mandl et al, 2001;Opie et al, 2003). They provide initial docking sites, which concentrate viruses in the vicinity of the cell surface and facilitate interactions with specific receptors.…”

“…They provide initial docking sites, which concentrate viruses in the vicinity of the cell surface and facilitate interactions with specific receptors. For some viruses, the ability to interact with heparan sulfate is an acquired adaptation to in vitro culture (Hulst et al, 2000;Klimstra et al, 1998;Mandl et al, 2001). In addition to heparan sulfate proteoglycans, viruses may also interact with other types of carbohydrates.…”