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 (WNVNY99) and a weakly virulent Australian subtype (WNVKUN). Five amino acid differences occur in WNVNY99 compared with WNVKUN (I22V, H43Y, L72S, S105A and A156V). When expressed in mammalian cells, recombinant WNVNY99 prM retained native antigenic structure, and was partially exported to the cell surface. In contrast, WNVKUN 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 WNVKUN prM to the same level as that of WNVNY99, and enhanced the secretion of WNVKUN prME particles when expressed in the presence of E. Introduction of the prM substitutions into a WNVKUN 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.
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