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).Alphaviruses, such as Ross River virus (RR), Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus (VEE), are enveloped positive-strand RNA viruses belonging to the family Togaviridae. The viral genome consists of a single-stranded RNA molecule, which is complexed with 240 copies of the capsid protein (50). The nucleocapsid is surrounded by a lipid bilayer in which the spike proteins are inserted. A single virion contains 80 hetero-oligomeric spikes, each spike consisting of a trimer of E2/E1 heterodimers. The E1 and E2 glycoproteins mediate the infectious entry of alphaviruses into cells. The E2 glycoprotein is primarily involved in the interaction of the virus particle with an attachment receptor on the cell surface (7, 28, 49), whereas E1 is required for the subsequent fusion process (19, 53).The spike proteins of RNA viruses are capable of rapid adaptation to their growth environment. Recently, it has been shown that viruses from different families interact with glycosaminoglycans (GAGs), in most cases heparan sulfate (HS), as a cell culture adaptation. Virus families or genera that exhibit such GAG adaptation include alphaviruses (2, 21, 28), flaviviruses (33), pestiviruses (25), picornaviruses (16, 43), and retroviruses (38, 41). GAGs are highly sulfated polymers of disaccharide repeats and hence are negatively charged. They are ubiquitously expressed on cell surfaces but vary with respect to their composition and quantity in different tissues and cell types (3, 52).Positively charged amino acid substitutions that are responsible for interaction with HS have been identified in the viral spike protein E2 of SIN, RR, and VEE (2,21,28). For SIN, three loci in E2 (E2:1, E2:70, and E2:114) appeared to mutate during adaptation of the virus to baby hamster kidney (BHK-21) cells, each mutation independently conferring on the virus the ability to bind to cell surface HS (28). The sequence XBX BBBX or XBBXBX (where X is any residue and B is a basic residue) is a linear binding motif that allows proteins to attach to HS (9). The positive-charge mutation at E2:1 results in the formation (although in the opposite orientation) of a linear HS interaction sequence. The HS-binding motifs are not present in the E2:70 and E2:114 regions, which suggests that these viruses interact with HS in a conformation-dependent manner. This phenomenon is known to occur in foot-and-mouth disease virus type O, structural studies of which have revealed that heparin makes contact with all three major capsid proteins, VP1, VP2, and VP3 (18). Despite the efficient interaction of the selected mutants of SIN, VEE, and RR with HS, the

Reversible Acid-Induced Inactivation of the Membrane Fusion Protein of Semliki Forest Virus

“…In addition, these studies have revealed a striking dependence of SFV fusion on the presence of both cholesterol (Chol) and sphingolipids in the target membrane (3,5,15,16,24,25,36,37,39,40). Studies conducted with another prototype alphavirus, Sindbis virus, have led to similar conclusions (30,31,32,34).…”

“…In addition, these studies have revealed a striking dependence of SFV fusion on the presence of both cholesterol (Chol) and sphingolipids in the target membrane (3,5,15,16,24,25,36,37,39,40). Studies conducted with another prototype alphavirus, Sindbis virus, have led to similar conclusions (30,31,32,34).Membrane fusion of alphaviruses is mediated by the E1 component of the heterodimeric E1/E2 envelope glycoprotein (6, 37). Recent X-ray crystallographic analyses of the structure of the alphavirus membrane fusion protein (19) have revealed that it has striking similarities with the structure of membrane fusion protein E of flaviviruses (22, 27) and major differences with the structure of the fusion protein hemagglutinin (HA) of influenza virus (29, 41) and other HA-related viral membrane fusion proteins.…”

Oncolytic Capacity of Attenuated Replicative Semliki Forest Virus in Human Melanoma Xenografts in Severe Combined Immunodeficient Mice

“…Correspondingly, small interfering RNA-mediated down-regulation of the high-affinity laminin receptor was shown to proportionally reduce the infectivity of Sindbis virus in ovarian cancer cells (28). On the other hand, alphaviruses can use several cell surface receptors for infection, including the high-affinity laminin receptor (29), heparan sulfate (30), and cell surface C-type lectins (31), which suggests that alphaviruses are not likely to be completely blocked at the level of entry and that the tropism of these viruses is largely determined by intracellular factors, which influence the viral replication machinery. Such factors include the double-stranded RNA-dependent protein kinase (PKR), RNase L, Mx class proteins, and components of the IFN-a/h-activated pathway (32).…”

Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems

“…Many viruses, including most laboratory strains of SINV, use the abundantly expressed negatively charged glycosaminoglycan heparan sulfate (HS) for initial interaction with host cells (6,11,14,30,48,57). To assess the effects of deletion of individual glycosylation sites on SINV interaction with heparin, the NaCl concentration required to elute each of the viruses from a heparin-Sepharose column was determined ( Fig.…”