Two distinct size classes of immature and mature subviral particles from tick-borne encephalitis virus

Abstract

Flaviviruses assemble in the endoplasmic reticulum by a mechanism that appears to be driven by lateral interactions between heterodimers of the envelope glycoproteins E and prM. Immature intracellular virus particles are then transported through the secretory pathway and converted to their mature form by cleavage of the prM protein by the cellular protease furin. Earlier studies showed that when the prM and E proteins of tick-borne encephalitis virus are expressed together in mammalian cells, they assemble into membrane-containing, icosahedrally symmetrical recombinant subviral particles (RSPs), which are smaller than whole virions but retain functional properties and undergo cleavage maturation, yielding a mature form in which the E proteins are arranged in a regular T = 1 icosahedral lattice. In this study, we generated immature subviral particles by mutation of the furin recognition site in prM. The mutation resulted in the secretion of two distinct size classes of particles that could be separated by sucrose gradient centrifugation. Electron microscopy showed that the smaller particles were approximately the same size as the previously described mature RSPs, whereas the larger particles were approximately the same size as the virus. Particles of the larger size class were also detected with a wild-type construct that allowed prM cleavage, although in this case the smaller size class was far more prevalent. Subtle differences in endoglycosidase sensitivity patterns suggested that, in contrast to the small particles, the E glycoproteins in the large subviral particles and whole virions might be in nonequivalent structural environments during intracellular transport, with a portion of them inaccessible to cellular glycan processing enzymes. These proteins thus appear to have the intrinsic ability to form alternative assembly products that could provide important clues about the role of lateral envelope protein interactions in flavivirus assembly.

FIG. 1.

Mutation of the furin cleavage site in prM. (A) Schematic diagram of the portion of the TBE virus genome included in the wild-type expression plasmid SV-PEwt (1) and the SV-P(Δ88)E deletion mutant used in this study. These constructs contained the coding region for prM and E under the control of the SV40 early promoter as well as short flanking regions from the genes encoding the capsid protein (C) and nonstructural protein 1 (NS1). The sites where the polyprotein is cleaved by host cell signalase (28) are indicated by small arrows, and the furin cleavage site in prM is indicated by a large arrow. (B) Sequence changes at the furin recognition site. The position of the N terminus of the M protein, which is normally created by furin cleavage, is indicated by an M, and the Arg 88 codon at position −2 that was deleted in SV-P(Δ88)E is indicated by an open triangle. Additional silent mutations in SV-P(Δ88)E resulting in a new XbaI site are shown in italics above the wild-type sequence.

FIG. 2.

Kinetics of E protein secretion from COS-1 cells transfected with plasmid SV-PEwt (wild-type) or SV-P(Δ88)E (mutant). The cell culture medium was replaced with fresh medium 24 h after transfection (arrow).

FIG. 3.

Rate-zonal sucrose density gradient centrifugation of clarified cell culture supernatants. (A) Cells transfected with the prM mutant. (B) Cells transfected with wild-type plasmid. The sedimentation direction is left to right.

FIG. 4.

Electron micrographs of mutant and wild-type subviral particles stained with formyl acetate and photographed at the same magnification. (A) Peak 1 (prM mutant); (B) peak 1 (wild type); (C) peak 2 (prM mutant); (D) peak 2 (wild type).

FIG. 5.

SDS-PAGE of proteins from purified virions and subviral particles. (A) Gel stained with Coomassie blue. (B) Immunoblot with polyclonal antiserum KVIII. Lanes: 1, mature TBE virus; 2, small wild-type subviral particle; 3, large wild-type subviral particle; 4, immature TBE virus; 5, small mutant subviral particle; 6, large mutant subviral particle. The positions of the bands for proteins E, prM, C (capsid protein), and M are indicated at the right. A minor band, corresponding to a previously identified SDS-resistant M dimer (43), is also indicated (*).

FIG. 6.

Detergent sensitivity of small and large subviral particles. The peak fractions from the experiment shown in Fig. 3A were analyzed again by rate-zonal sucrose density gradient centrifugation after treatment with 0.5% Triton X-100 (open circles) or no detergent treatment (filled circles). Triton X-100 (0.1%) was included in the gradients of detergent-treated samples to prevent aggregation. (A) Small immature mutant subviral particle (peak 1); (B) large immature mutant subviral particle (peak 2); (C) small mature subviral particle (RSP control). The sedimentation direction is left to right.

FIG. 7.

Antibody binding profiles of immature (A) and mature (B) subviral particles. The binding of 18 E-protein-specific MAbs with each of the different subviral particle types was compared by four-layer ELISA. Immature TBE virus produced by treating cells with 20 mM NH₄Cl was used as a control for the immature forms. The structural domain of the E protein to which each of the MAbs binds (38) is indicated at the bottom.

FIG. 8.

(A) Endoglycosidase treatment of glycoproteins from secreted immature TBE virus from infected cells treated with 20 mM NH₄Cl (lanes 1 to 4), secreted small immature subviral particles (prM mutant) (lanes 5 to 8), and secreted large immature subviral particles (prM mutant) (lanes 9 to 12). (B) Same treatment as described for panel A but using the corresponding mature forms. Samples were treated with 200 or 1,000 U of endo H_f, as indicated, or 500 U of PNGase F (F) and compared to untreated controls (U) by SDS-PAGE and immunoblotting with polyclonal antiserum KVIII. The positions of the E and prM protein bands are indicated at the right.