In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.Dengue virus (DENV) is an enveloped, positive-strand RNA virus belonging to the family Flaviviridae, which also includes tick-borne encephalitis virus, yellow fever virus, and West Nile virus. Flavivirus virions contain three structural proteins: the C (capsid) protein, the M (membrane) protein, and the E (envelope) protein (12,26). Multiple copies of the C protein associate with the viral RNA to form the nucleocapsid (26). The nucleocapsid is surrounded by a lipid bilayer in which the M and E glycoproteins are inserted. In the infected cell, the M protein is produced as a precursor protein called prM, which is believed to function as a chaperone during the folding and assembly of the E protein (2, 27). The E glycoproteins are assembled as homodimers on the surface of mature virions and mediate the infectious entry of flaviviruses into cells (14, 21). The crystal structure of the major external part of the E glycoprotein has been solved and reveals that the protein contains three distinct domains: domain I is the structurally central domain, domain II is the dimerization domain and contains the fusion peptide, and domain III has an immunoglobulin-like fold and mediates receptor binding (1,6,7,21,39).The initial step in the viral life cycle of DENV is attachment of the virus to a cellular receptor. DENV has been proposed to bind to the glycosaminoglycan heparan sulfate, ...

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

Human lactoferrin is a component of the non-specific immune system with distinct antiviral properties. We used alphaviruses, adapted to interaction with heparan sulfate (HS), as a tool to investigate the mechanism of lactoferrin's antiviral activity. Lactoferrin inhibited infection of BHK-21 cells by HS-adapted, but not by non-adapted, Sindbis virus (SIN) or Semliki Forest virus (SFV). Lactoferrin also inhibited binding of radiolabeled HS-adapted viruses to BHK-21 cells or liposomes containing lipid-conjugated heparin as a receptor analog. On the other hand, low-pH-induced fusion of the viruses with liposomes, which occurs independently of virus-receptor interaction, was unaffected. Studies involving preincubation of virus or cells with lactoferrin suggested that the protein does not bind to the virus, but rather blocks HS-moieties on the cell surface. Charge-modified human serum albumin, with a net positive charge, had a similar antiviral effect against HS-adapted SIN and SFV, suggesting that the antiviral activity of lactoferrin is related to its positive charge. It is concluded that human lactoferrin inhibits viral infection by interfering with virus-receptor interaction rather than by affecting subsequent steps in the viral cell entry or replication processes.

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