Identification of residues in West Nile virus pre-membrane protein that influence viral particle secretion and virulence.

The pre-membrane protein (prM) of West Nile virus (WNV) functions as a chaperone for correct folding of the envelope (E) protein, and prevents premature fusion during virus egress. However, little is known about its role in virulence. To investigate this, we compared the amino acid sequences of prM between a highly virulent North American strain (WNV(NY99)) and a weakly virulent Australian subtype (WNV(KUN)). Five amino acid differences occur in WNV(NY99) compared with WNV(KUN) (I22V, H43Y, L72S, S105A and A156V). When expressed in mammalian cells, recombinant WNV(NY99) prM retained native antigenic structure, and was partially exported to the cell surface. In contrast, WNV(KUN) prM (in the absence of the E protein) failed to express a conserved conformational epitope and was mostly retained at the pre-Golgi stage. Substitutions in residues 22 (Ile to Val) and 72 (Leu to Ser) restored the antigenic structure and cell surface expression of WNV(KUN) prM to the same level as that of WNV(NY99), and enhanced the secretion of WNV(KUN) prME particles when expressed in the presence of E. Introduction of the prM substitutions into a WNV(KUN) infectious clone (FLSDX) enhanced the secretion of infectious particles in Vero cells, and enhanced virulence in mice. These findings highlight the role of prM in viral particle secretion and virulence, and suggest the involvement of the L72S and I22V substitutions in modulating these activities.

Expression of recombinant West Nile virus prM protein fused to an affinity tag for use as a diagnostic antigen.

Previous studies have concluded that the Flavivirus prM protein is a suitable viral antigen to distinguish serologically between infections with closely related Flaviviruses (Cardosa et al., 2002). To express the recombinant West Nile virus (WNV) prM antigen fused to a suitable affinity tag for purification, a series of prM-His-tag and prM-V5-tag fusion proteins were generated. Analysis of the prM-His-tag fusion proteins revealed that either prM epitopes were disrupted or the His-tag was not presented properly depending on the location of the His tag and the presence of the prM transmembrane domains in these constructs. This identified domains critical for proper folding of prM, and arrangements that allowed the correct presentation of the His-tag. However, the inclusion of the V5 epitope tag fused to the C terminus of prM allowed formation of the authentic antigenic structure of prM and the proper presentation of the V5 epitope. Capture of tagged recombinant WNV(NY99) prM antigen to the solid phase with anti-V5 antibody in ELISA enabled the detection of prM-specific antibodies in WNV(NY99)-immune horse serum, confirming its potential as a useful diagnostic reagent.

Mammalian expression of functional autologous red cell agglutination reagents for use in diagnostic assays.

The autologous red cell agglutination assay reagent consists of an antibody or antibody fragment of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to an antigen of interest. This bi-functional reagent causes the agglutination of the patient's erythrocytes in the presence of the antigen-specific antibodies in the patient's serum. Previously, such reagents have been produced either by chemical conjugation or recombinant expression in bacteria. These protocols required laborious processes for purification and refolding. The aim of the work reported in this article was to explore the production of the agglutination assay reagent as both a single chain Fv (scFv) antibody fragment and recombinant full-length mAb, expressed in a secreted form in commonly used mammalian cell lines. The DNA encoding the anti-erythrocyte antibodies was linked to that of a diagnostic peptide from West Nile virus, which requires glycosylation for recognition by antibodies present in the sera of infected horses. The expression vectors were designed to allow the rapid directional insertion of DNA encoding other immunogenic peptides to mediate the secretion of agglutinating scFv and full-length mAb reagents from transfected mammalian cells. Stable cell lines were produced for the expression of most, but not all of the constructs. The recombinant reagents could be used directly from the cell culture media after a simple concentration step. The results indicate that further modifications to increase the yield of recombinant protein will enable the direct use of culture supernatant in diagnostic assays without further processing.