Structure of immature West Nile virus

Abstract

The structure of immature West Nile virus particles, propagated in the presence of ammonium chloride to block virus maturation in the low-pH environment of the trans-Golgi network, was determined by cryo-electron microscopy (cryo-EM). The structure of these particles was similar to that of immature West Nile virus particles found as a minor component of mature virus samples (naturally occurring immature particles [NOIPs]). The structures of mature infectious flaviviruses are radically different from those of the immature particles. The similarity of the ammonium chloride-treated particles and NOIPs suggests either that the NOIPs have not undergone any conformational change during maturation or that the conformational change is reversible. Comparison with the cryo-EM reconstruction of immature dengue virus established the locations of the N-linked glycosylation sites of these viruses, verifying the interpretation of the reconstructions of the immature flaviviruses.

FIG. 1.

Diagram showing the structural rearrangement required for immature particles to become mature particles. The Cα backbones of the three independent E molecules per icosahedral asymmetric unit are colored green, red, and blue. The three domains in each E molecule are labeled I, II, and III. (Reprinted from the EMBO Journal [19] with permission of the publisher.)

FIG. 2.

Cryo-EM micrographs, density maps, and difference density maps showing the positions of carbohydrate sites. (A) Cryo-EM micrograph of West Nile virus ACIPs. Immature particles were produced from cells treated with ammonium chloride. Confluent Vero cells were infected with West Nile virus at a multiplicity of infection of 1.0 in the presence of 2% fetal calf serum. At 22 h after infection, the cell culture medium was exchanged with medium containing 20 mM NH₄Cl. This first overlay was discarded at 23 h after infection and replaced with fresh NH₄Cl-containing medium. The cell culture supernatant was harvested 48 h after infection, and immature virus was purified as described previously for mature particles (8). Cryo-EM micrographs showed that the ammonium chloride-treated preparation consisted almost exclusively of immature particles (ACIPs). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of these ACIPs showed that the majority of the prM protein was uncleaved. (B) Cryo-EM micrograph of West Nile virus particles produced without NH₄Cl. Black arrows point to NOIPs, whereas the white arrow points to mature particles. Scale bars in both panels A and B represent 1,000 Å. Mature West Nile virus (NY99) was propagated and purified as described elsewhere (8), except that the virus was harvested 30 h after infection and concentrated by polyethylene glycol precipitation. The analysis of these particles by SDS-PAGE showed the presence of E, C, and prM proteins, as well as M proteins. Cryo-EM showed that up to one in five particles in the resulting preparations was an NOIP. (C) Surface representation of NOIPs. Icosahedral 5-, 3-, and 2-fold axes are labeled. In one spike, prM protein is colored gray and E protein is colored green. The scale bar represents 100 Å. (D) Central cross-section showing the multilayer organization of West Nile virus NOIPs. Icosahedral symmetry operators are indicated with black lines. (E) Stereo view of a spike showing the difference density between West Nile virus and dengue virus ACIPs. The Cα backbone of the E molecules is shown in green, red, and blue. Positive and negative peaks are colored gray and yellow, respectively. The icosahedral 5-, 3-, and 2-fold axes are labeled. The N-linked glycosylation sites (Asn-15 in the prM protein of West Nile virus [prM-15], Asn-67 in the E protein of dengue virus [E-67], and Asn-69 in the prM protein of dengue virus [prM-69]) associated with the difference densities are labeled. (F) Stereo view showing the same difference map as panel E but from a side view. The three difference density peaks associated with one prM-E heterodimer are outlined with a black rectangle.