Solution Structure and Antibody Binding Studies of the Envelope Protein Domain III from the New York Strain of West Nile Virus

Abstract

The solution structure of domain III from the New York West Nile virus strain 385-99 (WN-rED3) has been determined by NMR methods. The West Nile domain III structure is a ␤-barrel structure formed from seven antiparallel ␤-strands in two ␤-sheets. One anti-parallel ␤-sheet consists of ␤-strands ␤1 In 2002, the mosquito-borne West Nile virus (WNV) 1 (family Flaviviridae, genus Flavivirus) was responsible for the largest outbreak of arthropod-borne encephalitis recorded in the Western hemisphere. In that year, 4156 human infections and 284 deaths were reported in the United States (1). In 2003, the WNV epidemic continued with large numbers of human and animal disease across North America, and it was detected for the first time in Mexico and Central America (e.g. Refs. 2-4). The objective of this study was to solve the solution structure for the putative envelope (E) protein, the receptor-binding domain of WNV with the long term goal of using these findings for the development of structure-based vaccines or antiviral agents. Currently, there are no approved vaccines for WNV or therapeutic treatments for West Nile encephalitis.The flaviviruses are small, enveloped, positive-sense RNA viruses that are transmitted primarily by either mosquitoes or ticks. The translation of the single open reading frame within the viral genome, followed by co-and posttranslational cleavage, results in ten viral proteins, three structural proteins (core, membrane, and E) and seven non-structural proteins. The non-structural proteins are involved in viral replication and pathogenesis, whereas the three structural proteins are assembled into the mature virus particle.The E protein is the major surface protein of the flavivirus virions. The E protein is also the primary immunogen and plays a central role in virus attachment and entry to cells via membrane fusion. The x-ray crystallographic structures of the E protein ectodomains of both the tick-borne central European encephalitis virus (5) and the mosquito-borne dengue-2 virus (6) have been solved. Both proteins contain three distinct structural domains (domains I, II, and III) that correspond to previously characterized antigenic domains (7). Domain III (D3) of the E protein was initially proposed as the likely receptorbinding domain of the flaviviruses because of its structural characteristics. These include an IgC-like fold and a four-amino-acid loop that contains an RGD integrin-binding motif in several of the mosquito-borne flaviviruses (5). More recent studies have shown that D3 is directly associated with binding of dengue-2 virus (DEN2V) (8), WNV, and the tick-borne Langat virus 2 to cells. Domain 3 of WNV (WN-rED3) has also been shown to contain epitopes recognized by virus-neutralizing monoclonal antibodies (9). X-ray crystallography (5, 6) and cryo-electron microscopy studies (6, 10, 11) of the mosquitoborne WNV, DEN2V, and yellow fever virus have found that the E protein is arranged in dimeric form on the virus surface. Located at the 5-and 3-fold axes of symmetry, D3 projects...

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

“…Domain III of the WNV E protein has been documented recently to serve as the domain of the E protein that binds to cellular receptor integrin ␣ V ␤ 3 (6). Neutralizing Abs against WNV have been mapped to epitopes localized on the WNV E DIII (7)(8)(9)(10)(11). Therapeutic trials in mice with humanized anti-WNV E DIII Ab have also been shown to be highly effective in preventing WNV infection (9,10), suggesting that the recombinant WNV E DIII protein is an attractive vaccine candidate.…”

“…Crystallography data on the ectodomain of the flavivirus E protein revealed three distinct domains (DI, DII, and DIII) (3). The high antagonistic effect of recombinant WNV E DIII protein in blocking WNV infection in both mammalian and mosquito cells strongly suggested that WNV E DIII protein functions as the receptor-binding domain (4) and is responsible for the recognition and attachment to the cellular receptor (5)(6)(7). Importantly, a majority of the neutralizing epitopes have been mapped to the domain III region of the flavivirus E protein (8 -11).…”

“…Direct ELISA for quantifying Ag-specific IgG and the IgG subclasses were conducted using plates coated with recombinant WNV E DIII protein. WNV E DIII is located at the surface of the virus particles based on the structural analysis of the E protein and represents a significant portion of the neutralizing Ab response (7,8,10,15). Microtiter plates were coated with recombinant WNV E DIII protein overnight at 4°C at 100 ng/well in coating buffer (0.015 M Na 2 CO 3 , 0.03 M NaHCO 3 , and 0.003 M NaN 3 (pH 9.6)).…”

Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus

“…Neutralizing epitope of dengue virus ED3 domains from DENV2, DENV3, DENV4, JEV and WNV, solved by nuclear magnetic resonance or X-ray crystallography, are similar (Kanai et al, 2006;Modis et al, 2003Modis et al, , 2005Nybakken et al, 2005;Volk et al, 2004Volk et al, , 2007Wu et al, 2003). Among the nine residues that belonged to the energetic epitope of mAb4E11 in ED3.DEN1-H6, six are different in YFV and seven in JEV and WNV (Table 3).…”

West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody

“…Indeed, the E glycoprotein is the principal antigen that elicits neutralizing antibodies against flaviviruses. Many of the neutralizing epitopes are located in DIII of E and in close proximity to the conserved fusion loop in DII (20)(21)(22)(23)(24)(25)(26)(27)(28). E16 is a strongly neutralizing mAb against WNV that recognizes four polypeptide loops at the tip of DIII and inhibits infection primarily subsequent to cell attachment.…”