Biophysical Characterization and Vector-Specific Antagonist Activity of Domain III of the Tick-Borne Flavivirus Envelope Protein

Abstract

The molecular determinants responsible for flavivirus host cell binding and tissue tropism are largely unknown, although domain III of the envelope protein has been implicated in these functions. We examined the solution properties and antagonist activity of Langat virus domain III. Our results suggest that domain III adopts a stably folded structure that can mediate binding of tick-borne flaviviruses but not mosquito-borne flaviviruses to their target cells. Three clusters of phylogenetically conserved residues are identified that may be responsible for the vector-specific antagonist activity of domain III.Flaviviruses (family Flaviviridae, genus Flavivirus) are organized into distinct vector-specific classes and serocomplex-specific subgroups (6), although little is known of the molecular determinants that dictate vector-specific pathogenesis, host cell specificity, and tissue tropism. Both mosquito-borne and tick-borne flaviviruses are responsible for epidemics throughout the developing world and pose serious public health threats in developed countries (7, 9, 10). Vaccines are only available to help control infection by yellow fever, Japanese encephalitis, Central European tick-borne encephalitis (TBE), and louping ill flaviviruses (1,2,16,17). No antiviral therapy is available to treat any flavivirus infection.Flavivirus infection requires attachment and entry into a target cell, mediated by binding of the viral envelope (E) proteins to cell surface receptors. The host cell receptor (or receptors) and the region (or regions) of the E protein responsible for flavivirus attachment are unknown, although heparan sulfate has been suggested as one factor mediating the interaction between dengue 2 virus and its target cells (3). It is unknown whether different cell surface receptors and/or envelope-receptor interactions are responsible for flavivirus pathogenesis, host range, and tissue tropism.The structure of the E protein ectodomain from TBE virus has been determined (13). This ectodomain forms a homodimer, with each dimer subunit organized into three domains, designated I, II, and III. Comparisons of E proteins from wild-type viruses with those of attenuated or escape mutant viruses have identified a number of residues in domain III that may be responsible for receptor recognition (4,8,(12)(13)(14). We examined the solution properties of domain III of the E protein from Langat virus (a tick-borne flavivirus) and the ability of this domain to function as an antagonist for virus infectivity. We demonstrate that recombinant domain III adopts a highly stable folded structure in solution and shows reduced infectivity in tick-borne flaviviruses but not mosquitoborne flaviviruses. This suggests that interactions between the envelope protein and its host cell receptor may be vector specific, although it is likely that other factors also influence fla-* Corresponding author. Mailing address:

Complete genome characterization of Rocio virus (Flavivirus: Flaviviridae), a Brazilian flavivirus isolated from a fatal case of encephalitis during an epidemic in São Paulo state

“…Interestingly, in the E protein, the tripeptide (TGP) of ROCV within domain III was found to be different from those observed in all other JEV members (RGX, where X is D, E or T). As the RGD motif is speculated to be involved in the virus adsorption to host cells for JEV and MVEV, two important JEV group flaviruses (Bhardwaj et al, 2001;Lee & Lobigs, 2000) and as it was also found in ALFV and USUV, further experimental studies for ROCV are needed to investigate the role of the TGP tripeptide in the mechanisms that involve the viruscell interaction.…”

“…Interestingly, in the E protein, the tripeptide (TGP) of ROCV within domain III was found to be different from those observed in all other JEV members (RGX, where X is D, E or T). As the RGD motif is speculated to be involved in the virus adsorption to host cells for JEV and MVEV, two important JEV group flaviruses (Bhardwaj et al, 2001;Lee & Lobigs, 2000) and as it was also found in ALFV and USUV, further experimental studies for ROCV are needed to investigate the role of the TGP tripeptide in the mechanisms that involve the viruscell interaction.The functional roles of the 39-NCR of flaviviruses have been recently studied and the roles in the genomic cyclization between the 59-and 39-NCRs, viral replication and translation were elucidated. Moreover, the putative changes in the highly conserved regions related to important biological functions of the flaviviruses (survival and/or pathogenicity) were recognized (Bryant et al, 2005;Khromykh et al, 2001).…”

Immunization of Flavivirus West Nile Recombinant Envelope Domain III Protein Induced Specific Immune Response and Protection against West Nile Virus Infection

“…Importantly, a majority of the neutralizing epitopes have been mapped to the domain III region of the flavivirus E protein (8 -11). Furthermore, the WNV E DIII protein can be expressed as a recombinant protein that independently folds as an individual functional fragment and is capable of eliciting neutralizing Abs against WNV (12). Hence, the potential of WNV E DIII protein to function as an effective recombinant subunit vaccine against WNV is assessed and is presented in this study.…”

Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

“…DII contains a fusion loop at its distal end that is indispensable for virus-cell membrane fusion. The Ig-like C-terminal domain DIII undergoes a major, pH-triggered positional rearrangement essential for fusion, and may also be involved in receptor binding (2,(6)(7)(8)(9)(10)(11)(12). During cell entry of flaviviruses, low endosomal pH triggers a proposed E protein rearrangement cascade, including the dissociation of E dimers and the outward rotation of DII during the repositioning of E monomers into fusion-active trimers (6,9,13).…”