Two Distinct Size Classes of Immature and Mature Subviral Particles from Tick-Borne Encephalitis Virus

Flaviviruses assemble in the endoplasmic reticulum by a mechanismthat appears to be driven by lateral interactions between heterodimersof the envelope glycoproteins E and prM. Immature intracellularvirus particles are then transported through the secretory pathwayand converted to their mature form by cleavage of the prM proteinby the cellular protease furin. Earlier studies showed thatwhen the prM and E proteins of tick-borne encephalitis virusare expressed together in mammalian cells, they assemble intomembrane-containing, icosahedrally symmetrical recombinant subviralparticles (RSPs), which are smaller than whole virions but retainfunctional properties and undergo cleavage maturation, yieldinga mature form in which the E proteins are arranged in a regularT = 1 icosahedral lattice. In this study, we generated immaturesubviral particles by mutation of the furin recognition sitein prM. The mutation resulted in the secretion of two distinctsize classes of particles that could be separated by sucrosegradient centrifugation. Electron microscopy showed that thesmaller particles were approximately the same size as the previouslydescribed mature RSPs, whereas the larger particles were approximatelythe same size as the virus. Particles of the larger size classwere also detected with a wild-type construct that allowed prMcleavage, although in this case the smaller size class was farmore prevalent. Subtle differences in endoglycosidase sensitivitypatterns suggested that, in contrast to the small particles,the E glycoproteins in the large subviral particles and wholevirions might be in nonequivalent structural environments duringintracellular transport, with a portion of them inaccessibleto cellular glycan processing enzymes. These proteins thus appearto have the intrinsic ability to form alternative assembly productsthat could provide important clues about the role of lateralenvelope protein interactions in flavivirus assembly.

INTRODUCTION

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Introduction

Flaviviruses are small enveloped viruses that are assembledintracellularly, apparently by budding into the endoplasmicreticulum (ER) (28). The entire virion, consisting of a nucleocapsid,lipid membrane, and two envelope glycoproteins, prM and E, isthen transported through the secretory pathway (32) and, afterundergoing posttranslational modifications that include thecleavage of the prM protein, is exported from the cell by exocytosis.Newly synthesized prM and E proteins rapidly associate to formheterodimers (6, 30, 46), which then further associate to organizethe envelope into a regular icosahedral structure (8, 25, 48).The idea that lateral envelope protein interactions play a dominantrole in assembly is supported by the observations that orderedmembrane-containing subviral particles can be obtained by coexpressingprM and E in the absence of other viral components (4, 9, 19-23,35, 36, 41) and that subviral particles containing only theenvelope proteins are also secreted as a by-product of naturalinfections (39).

Shortly before being released from the cell, the prM proteinsin the immature virus are cleaved by the trans-Golgi networkresident proprotein convertase furin (43), resulting in theloss of approximately the N-terminal half of this protein andleaving the small (8 kDa) C-terminal portion, the M protein,anchored in the membrane. The cleavage of prM converts the viralenvelope proteins to a metastable state (44), allowing themto undergo the further structural changes that are requiredfor membrane fusion (3, 43).

Studies with recombinant subviral particles (RSPs) from tick-borneencephalitis (TBE) virus have shown that they are assembledin the ER and transported through the secretory pathway in thesame manner as whole virus particles (31), that they undergosimilar posttranslational modifications during transport (41),and that their envelope proteins are presented in a fully functionalstate (5, 41). The molecular organization of a mature RSP fromTBE virus was previously determined by cryoelectron microscopyand icosahedral image reconstruction (8). It was demonstratedthat the prevalent species secreted from transfected COS-1 cellsexpressing prM and E is an icosahedrally symmetrical particlewith a diameter of 31.5 nm (compared to approximately 50 nmfor the whole virus). Fitting of the X-ray crystal structureof the TBE virus E protein (38) into the experimental electrondensity revealed that the E proteins in the mature RSP makea regular lattice structure consisting of 30 E homodimers ina T = 1 arrangement. The lateral interactions that stabilizethe E protein network are apparently identical at each positionof the particle. Based on this structure, a model was proposedfor the arrangement of the E proteins in the native mature TBEvirion particle by using quasiequivalent interactions to makea T = 3 icosahedral lattice with a diameter of 50 nm (8). Sincethen, it has been shown by Kuhn et al. (25) that the E proteinarrangement in another flavivirus, dengue virus, does not conformto this model but instead fits a herringbone model that is notgoverned by quasiequivalence and features different types oflateral interactions between the E proteins at different siteson the icosahedral lattice.

In this study, we generated immature subviral particles by introducinga mutation in the furin recognition site of prM. The mutationresulted in the secretion of two distinct, relatively homogeneouspopulations of prM-containing particles with diameters of about30 and 55 nm. Further characterization showed that these twoparticle classes probably represent specific alternative assemblyproducts with subtle differences in glycan modification thatcould indicate that, in contrast to the small particles, theE proteins in the large subviral particles and virions are innonequivalent structural environments during intracellular transport.

MATERIALS AND METHODS

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Materials and Methods

Construction of prM cleavage mutant. Plasmid SV-PEwt (1), which contains nucleotides 388 to 2550from TBE virus strain Neudoerfl (34) (GenBank accession no.U27495) under the control of a simian virus 40 (SV40) earlypromoter, was the parent construct used for mutant constructionand for wild-type controls. To construct the prM cleavage mutantSV-P( ${Delta}$ 88)E, a restriction fragment (SapI-AgeI, nucleotides 648to 960) corresponding approximately to the prM coding regionof SV-PEwt was replaced by the corresponding fragment from plasmiddR204, an intermediate construct used previously for constructingthe full-length mutant clone pTNd/prM( ${Delta}$ 88) (7). This fragmentcontained the deletion of the codon for arginine 88 of prM (nucleotides742 to 744) as well as silent mutations at nucleotides 735,736, and 738 to produce a new XbaI site (Fig. 1). Plasmids werepropagated in Escherichia coli HB101 and purified with a Qiagenplasmid mega kit. Sequence analysis of the prM and E codingregions was performed with an automated DNA sequencing system(ABI) to confirm that only the desired mutations were present.

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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( ${Delta}$ 88)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( ${Delta}$ 88)E is indicated by an open triangle. Additional silent mutations in SV-P( ${Delta}$ 88)E resulting in a new XbaI site are shown in italics above the wild-type sequence.

Production of subviral particles and virus. COS-1 cells (ATCC CRL 1650) were grown in Dulbecco's modifiedEagle medium (Life Technologies) supplemented with 10% fetalcalf serum, 4 mM glutamine, 100 U of penicillin per ml, and100 µg of streptomycin per ml at 37°C in 5% CO₂. Cellswere transfected with purified plasmid with a Bio-Rad gene pulserapparatus and allowed to grow for another 22 to 24 h in thenormal growth medium. The medium was then replaced with serum-freebicarbonate- and HEPES-buffered Dulbecco's modified Eagle medium(Life Technologies), and incubation was continued for another24 h.

The cell supernatants were cleared by centrifugation at 10,000rpm for 30 min at 4°C in a Beckman JLA16.250 rotor, andparticles were collected by polyethylene glycol (PEG) precipitationas described previously (8). In some cases, harvesting was doneinstead by pelleting in an ultracentrifuge (Beckman 45 Ti rotor,44,000 rpm, 4°C, 120 min). The particles were then separatedby rate-zonal sucrose density gradient centrifugation (41) and,when high purity was required, further purified by equilibriumdensity centrifugation on 20 to 50% sucrose gradients as describedpreviously (41).

TBE virus strain Neudoerfl was grown in primary chicken embryocells and purified by two cycles of sucrose gradient centrifugationas described previously (15). For production of secreted immaturevirus, ammonium chloride was added to the cell culture mediumduring virus growth to a final concentration of 20 mM to suppressprM cleavage (16).

Sedimentation analysis. Subviral particles were analyzed by centrifugation in 5 to 20%(wt/wt) sucrose gradients made with TAN buffer (0.05 M triethanolamine[pH 8.0], 0.1 M NaCl). For membrane solubilization experiments,samples were treated first for 1 h at room temperature withTriton X-100 (final concentration, 0.5% [wt/wt]) and the correspondingsucrose gradients were made with 0.1% Triton X-100 to preventaggregation. The gradients were then centrifuged in a BeckmanSW 40 rotor at 38,000 rpm at 4°C for 90 min and fractionatedfrom the top with a BioComp piston gradient fractionator. Theamount of E protein in the fractions was quantified by a four-layerenzyme-linked immunosorbent assay (ELISA) after heating thesamples and virus standard at 65°C for 30 min with 0.4%sodium dodecyl sulfate (SDS) (16).

Electron microscopy. Sucrose gradient fractions were first diluted approximatelythreefold in TAN buffer plus 10 mM MgCl_2. The particles werethen concentrated by ultracentrifugation in a Beckman SW40 rotorat 43,000 rpm for 3 h at 4°C and resuspension in a smallvolume of buffer. Samples were adsorbed to glow-discharged carbon-coatedcopper grids, washed with two drops of deionized water, andstained with two drops of freshly prepared 0.75% uranyl formate.Images were taken with a Tecnai 12 electron microscope (Philips,Eindhoven, The Netherlands) at 120 kV, with a magnificationof x21,000 and a defocus of 2 to 3 µm.

Endoglycosidase treatment, gel electrophoresis, and immunoblotting. Endoglycosidase H_f (endo H_f) and peptide N-glycosidase F (PNGaseF) (New England Biolabs) were used according to the manufacturer'sinstructions with the provided buffers. Protein samples wereprecipitated with deoxycholate and trichloroacetic acid andseparated by SDS-polyacrylamide gel electrophoresis (PAGE) asdescribed by Laemmli and Favre (26). For analysis of proteincomposition, gels containing 15% acrylamide were used. For analysisof glycosidase-treated E and prM proteins, gels with 10% acrylamidewere used.

Protein bands were visualized with Coomassie PhastGel Blue R(Pharmacia) or by immunoblotting onto a polyvinylidene difluoridemembrane (Bio-Rad) with a Bio-Rad semidry transfer cell as describedpreviously (41). In the latter case, a polyclonal rabbit serum(KVIII) recognizing the TBE virus structural proteins (43) wasused together with a donkey anti-rabbit immunoglobulin horseradishperoxidase conjugate (Amersham) for detection.

Analysis of monoclonal antibody binding. A four-layer ELISA (17) was used to measure the reactivity ofthe different preparations with a panel of E-protein-specificmonoclonal antibodies (MAbs) (13, 18) as described previouslyby Schalich et al. (41). The antigen (0.5 µg of E proteinper ml) was captured on the solid phase by a guinea pig anti-TBEvirus immunoglobulin and tested with a single dilution of eachMAb and a peroxidase-labeled rabbit anti-mouse immunoglobulin(Nordic) for detection.

RESULTS

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Results

Mutation of the furin cleavage site in prM. To make subviral particles that are secreted from cells whilestill in their immature state, we altered the furin recognitionsite in prM to prevent cleavage during exocytosis. In an earlierstudy (7), it was shown with an infectious clone system thatthe deletion of arginine 88, two residues before the furin cleavagesite in prM, prevented prM cleavage and eliminated the abilityof the virus to be passaged in cell culture. This deletion disruptedthe furin recognition motif R-X-R/K-R and rendered it uncleavableby furin. In the same study, it was also shown that infectivitycould be restored by the addition of exogenous trypsin, indicatingthat the mutation had caused the envelope proteins to be heldin a latent but nevertheless potentially functional state. Wetherefore introduced this same mutation into the correspondingsite of plasmid SV-PEwt, which contains the coding region forprM and E under the control of an SV40 promoter and has beenused in several previous studies for generating mature RSPs(2, 4, 5, 8, 41). The position of the deletion in the resultingplasmid construct, SV-P( ${Delta}$ 88)E, is shown in Fig. 1.

Expression and secretion of mutant subviral particles. To investigate the effect of the mutation on the efficiencyof particle assembly, transport, and secretion, COS-1 cellswere transfected either with the wild-type plasmid or with theconstruct carrying the prM mutation, and the kinetics of secretionwere monitored by quantifying the amount of E protein presentin the cell culture medium at different time points. The transfectionefficiency (approximately 50% for both the wild type and themutant) was monitored by immunofluorescence staining (data notshown). As shown in Fig. 2, transfection with each of the plasmidsresulted in a significant accumulation of extracellular E protein,but the amount secreted was somewhat less with the mutant thanwith the wild-type construct. In three separate experiments,the rate of extracellular E protein production by the mutantwas 40 to 60% of that of the wild-type control, suggesting eitherthat the mutation had caused a minor decrease in the overallrate of synthesis and particle assembly or that the mutant proteinsrequired more time to pass through the secretory pathway.

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FIG. 2. Kinetics of E protein secretion from COS-1 cells transfected with plasmid SV-PEwt (wild-type) or SV-P( ${Delta}$ 88)E (mutant). The cell culture medium was replaced with fresh medium 24 h after transfection (arrow).

Two populations of subviral particles with different sedimentation properties. In order to characterize the mutant particles, larger-scalepreparations were made. Particles were harvested from clarifiedcell culture supernatants by PEG precipitation and further purifiedby sucrose gradient centrifugation under conditions used previouslyfor mature wild-type RSPs (8, 41). Unexpectedly, the harvestedmaterial from the prM mutant was found to contain two distinctmajor populations of particles that sedimented at differentrates in rate-zonal sucrose density gradients (Fig. 3A). Peak1 migrated to the same position as the previously reported matureRSP (41), whereas peak 2 sedimented much faster, suggestinga larger particle size. The ratio of the amount of E proteinin peak 1 to that in peak 2 was approximately 1.5 to 1 and variedonly slightly when samples were analyzed at different time pointsbetween 42 and 66 h posttransfection or when they were harvestedby ultracentrifugation instead of PEG precipitation (data notshown).

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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.

Similar analyses were performed to see whether cells transfectedwith the wild-type construct also secrete a faster-sedimentingparticle form. As shown in Fig. 3B, the pattern of distributionin supernatants from wild-type-transfected cells was quite differentfrom that of the mutant, and as expected, peak 1, which wasknown from earlier studies to correspond to the previously describedmature RSP (8, 41), was by far the dominant form. Nevertheless,a second, minor population of particles with a sedimentationrate similar to that of the mutant peak 2 could also be consistentlydetected when sufficient amounts of starting material were analyzed(Fig. 3B).

Two different particle sizes shown by electron microscopy. Particles from sucrose gradient peaks 1 and 2 were concentrated,stained with uranyl formate, and analyzed by electron microscopy.As shown in Fig. 4A, the predominant species in peak 1 fromthe mutant preparation was a spherical particle with a diameterof about 30 nm (n = 74, mean = 28.2 nm, standard deviation [SD]= 2.69 nm). This corresponds in size and shape to the maturewild-type RSP (n = 108, mean = 30.23 nm, SD = 2.35 nm) (Fig.4B), which has been shown previously to have a T = 1 icosahedralarrangement of 30 E protein homodimers on its surface (8). Inaddition, the negatively stained images revealed a number ofmisshapen particles that probably represent damaged RSPs. Peak2 from the prM mutant also contained mostly spherical particles(Fig. 4C), although these appeared somewhat more heterogeneousin shape than the peak 1 material. In contrast to peak 1, however,the most prevalent form in this population had a diameter ofabout 55 nm (n = 170, mean = 55.4 nm, SD = 3.22 nm), which correspondsapproximately to the diameter of the whole virion. In addition,a few of the smaller 30-nm-diameter particles were visible inthese preparations (Fig. 4C), probably due a slight overlapbetween the peaks or particle aggregation. The faster-sedimentingpeak (peak 2) from the wild-type preparations also consistedprimarily of round, approximately 55-nm-diameter particles (Fig.4D). These two major size classes thus appear to be distinctassembly products.

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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).

Biochemical and physical characteristics of subviral particles. The four types of subviral particles were further purified andanalyzed by SDS-PAGE (Fig. 5). Mature TBE virus (Fig. 5, lane1) and immature TBE virus produced by treating infected cellswith ammonium chloride (16) (Fig. 5, lane 4) were used as controls.In the Coomassie blue-stained gel shown in Fig. 5A, it can beseen that both the small (30-nm diameter) (Fig. 5, lane 5) andlarge (55-nm diameter) (Fig. 5, lane 6) particles produced byusing the prM mutant construct contained E and uncleaved prMproteins, with no detectable M. In contrast, most of the prMprotein had been cleaved in both the small (Fig. 5, lane 2)and large (Fig. 5, lane 3) particles produced by using the wild-typeconstruct. These results were confirmed by immunoblotting, asshown in Fig. 5B, and the data together demonstrate that thedeletion of arginine 88 in the furin recognition site of prMwas indeed sufficient to abolish prM cleavage in COS-1 cells.Thus, both sizes of particles containing the mutation representimmature forms. Consistent with their immature state (43) andin contrast to both sizes of wild-type subviral particles andmature virus, they also lacked the ability to hemagglutinategoose erythrocytes at pH 6.4 (data not shown).

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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 (*).

To get a first indication of whether both particle size classescontain a lipid membrane, samples from the peak fractions shownin Fig. 3A were treated with 0.5% Triton X-100 for 1 h and centrifugedin 5 to 20% sucrose gradients containing 0.1% Triton X-100.Untreated samples in gradients without detergent were used ascontrols. As shown in Fig. 6, both the small particles frompeak 1 and the large particles from peak 2 were dispersed bythe detergent treatment, causing the E protein peak to be shiftedto the top of the gradient. This is consistent with the presenceof a lipid membrane in both particle size classes. It is alsosignificant that there was no change in the sedimentation behaviorof the untreated controls between the first and second sedimentationanalyses (compare Fig. 3 and 6), confirming that each peak representsa relatively stable and defined subpopulation.

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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.

To assess the conformational state of the envelope glycoproteinsin each of the particle types, we tested their reactivity ina four-layer ELISA, with a panel of 18 E-specific MAbs (13,18), which has been used in several earlier studies for theanalysis of the conformation of protein E. These MAbs have beenshown to bind equally well to whole virions and RSPs (14, 41),but several of them react differently with the immature prM-containingforms, yielding different reactivity profiles in the ELISA (7,16, 41). The data shown in Fig. 7 reveal that both populationsof mutant, prM-containing, immature RSPs are very similar intheir reactivity patterns to immature whole virions producedby suppressing prM cleavage by treating cells with ammoniumchloride. This suggests that the E proteins in both the smalland large immature subviral particles are in the same conformationas found on immature virions. The mature small and large wild-typeparticles yielded profiles that were very similar to each otherbut distinctly different from those of the immature forms (Fig.7, compare panels A and B). The most pronounced differencesbetween the mature and immature forms were observed with MAbsrecognizing domain II (especially MAbs A3, A4, A5, and IE3),as has been observed previously (7, 16, 41). From these results,it therefore appears that the antigenic structure and, thus,the envelope protein conformation are independent of particlesize.

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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.

Differences in glycan processing. The prM and E proteins of TBE virus each contain a single N-linkedoligosaccharide moiety (30, 47). It has been shown previouslythat, in secreted mature 30-nm-diameter RSPs, all of the N-linkedglycans attached to Asn 154 in the E protein are processed toan endo H-resistant form, whereas in secreted mature TBE virusparticles produced in primary chicken embryo cells or COS-1cells, a substantial portion consistently remains sensitiveto endo H cleavage (41), indicating that only a subset of theE proteins possesses a fully processed glycan. It is not known,however, whether this difference is attributable to differencesin accessibility to the processing enzymes or to other factorsaffecting the cellular machinery during infection. Therefore,to investigate potential differences in the degree of glycanprocessing, we treated all four subviral particle types as wellas mature and immature virus controls with endo H_f, an enzymethat cleaves immature high-mannose-type glycans, and PNGaseF, which removes both complex and high-mannose sugar chains(33).

Figure 8A shows an immunoblot of a gel on which different glycosidase-treatedsamples were analyzed by electrophoresis. In this experiment,immature virus and the small and large immature subviral particleswere treated with two different concentrations of endo H_f aswell as with PNGase F. Treatment with PNGase F resulted in completedeglycosylation of E and prM for all particle types, causinga shift in the electrophoretic mobility of both proteins. However,with endo H_f, the E protein deglycosylation pattern was differentfor the small subviral particles than for the large subviralparticles and whole immature virions. As expected from earlierwork (41), nearly all of the glycans linked to the E proteinsof the small immature subviral particles were endo H_f resistant,indicating that most of these were of the complex type. Thelarge immature subviral particles, in contrast, displayed apattern more similar to that of the immature virus, with anapparently larger proportion of the total E protein still inan endo H_f-sensitive form. An analysis of the E proteins ofthe corresponding mature forms yielded essentially identicalresults (Fig. 8B), as was expected because all of the sugarmodifications occur while the particles are still in an immaturestate and are therefore independent of the subsequent prM cleavagestep. The prM proteins, on the other hand, were in the fullyprocessed endo H_f-resistant form, regardless of which immatureparticle type was analyzed (Fig. 8A). These data reveal differencesin glycan processing patterns between the small and large particlesthat are possibly attributable to local differences in the accessibilityof sugar chains to the processing enzymes of the secretory pathway.These data are consistent with a nonequivalent arrangement ofE proteins in the large immature subviral particles and immaturevirions during intracellular transport.

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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.

DISCUSSION

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Discussion

It is an interesting feature of flaviviruses that their envelopeproteins appear to play a dominant role in the assembly process.Their ability to form capsidless subviral particles suggeststhat lateral interactions between the heterodimers of prM andE in the ER membrane may be sufficient to induce budding intothe lumen of this organelle, although it has not been ruledout that cellular components could also play at least a chaperone-likerole in this process. Furthermore, the apparent scarcity ofpreformed core particles in the cytoplasm of infected cells(28) would suggest that an envelope protein lattice in the ERmight even guide capsid assembly by ordering C protein capsomersat the budding site. A similar proposal has also been made inthe case of the alphaviruses based on the properties of capsidmutants (10), but alphaviruses, in contrast to flaviviruses,bud at the plasma membrane and apparently do not make subviralparticles (11). However, secreted subviral particles producedby expression of envelope proteins alone have been reportedfor hepatitis B virus (27) and the coronavirus mouse hepatitisvirus (45), but also in these cases, the exact role of the envelopeproteins in the budding mechanism is still unknown.

In this study, we found that coexpression of proteins E andprM leads to the secretion of two distinct classes of subviralparticles, the first of which corresponds in size to the previouslydescribed RSPs (8, 41) and the second of which has approximatelythe same outer dimensions as a whole virus particle. Since bothof these forms are assembled in the same cells from the samebuilding blocks in the absence of capsid or other viral proteins,it appears that the ability to make particles of two specificsizes is a property that is encoded in the envelope proteinsthemselves. The intrinsic ability of viral structural proteinsto form more than one type and size of closed icosahedral shellhas been demonstrated previously with capsid proteins from severaldifferent viruses (24, 29, 42, 49). Although it is not yet knownwhether the immature subviral particles described in this studyhave icosahedral symmetry, the fact that they form two definedand stable subpopulations strongly suggests that the numberof subunits per particle is not random and is most likely governedby specific lateral interactions that would favor an icosahedralarrangement.

Although the production and characterization of flavivirus subviralparticles has been reported in a number of previous studies(4, 9, 19-23, 35-37, 40, 41), secreted particles of the largersize class had not yet been reported. In studies with wild-typeRSPs, the larger particles were probably overlooked becauseof their low abundance relative to the small particles as wellas the lack of sensitivity of certain assays. It is also possiblethat the ability to secrete the larger particles is a propertythat is not shared by all flaviviruses.

The successful use of mutations in the furin recognition sitein prM to produce stable CHO cell lines secreting immature subviralparticles has been reported recently for the Japanese encephalitisvirus (21) and dengue virus type 2 (20) envelope proteins, butin each of these studies only one size class was reported. Theseauthors showed that making prM uncleavable by furin improvedthe stability of the cell lines by eliminating the ability ofthe secreted particles to induce syncytium formation. For TBEvirus, on the other hand, it has been shown recently that aCHO cell line constitutively producing wild-type RSPs couldbe established without mutating the prM cleavage site or suppressingfusogenic activity (12).

In this study, the mutation of the furin cleavage site in prMresulted in a substantial shift in the particle size ratio infavor of the larger size class. The reason for this phenomenonis not clear. It is possible that the structure of the matureenvelope is relatively unstable in the absence of the viralcore and that the presence of prM lends a degree of additionalstability that favors an accumulation of the larger form. Alternatively,it is possible that the prM mutation causes a change in prMor E that is slightly unfavorable for the formation or releaseof the smaller particle type. COS-1 cells transfected with SV-PEwt(the same wild-type plasmid used in this study) appear to containtwo different sizes of RSP-like particles in the ER and othercompartments of the secretory pathway, including post-Golgivesicles (31). These intracellular particles are approximatelyof the same sizes as the extracellular particles reported here,with the larger size accounting for about 25% of the total.In the present study, we found that with the prM mutant, theratio of the amount of E protein in peak 1 to that in peak 2was about 1.5 to 1. If we assume, based on the available structuralstudies with TBE virus RSPs (8) and dengue virus (25) that thelarge particles contain three times as many E protein moleculesas the small ones (180 versus 60 monomers), the calculated particlenumber ratio for the prM mutant would be about 4.5 to 1, whichis fairly consistent with what has been observed in cell sectionsby electron microscopy (31). In the case of the mature wild-typeparticles, however, we observed much higher small-to-large particleratios in cell supernatants. The results of both studies togethersuggest that the large wild-type particles are probably efficientlysynthesized and transported through the secretory pathway butthat preventing prM cleavage somehow alters their relative rateof release from the cell or their ability to accumulate in thecell culture medium due to enhanced stability.

The large subviral particles and whole virions, in contrastto the small subviral particles, were found to contain a relativelylarge proportion of E proteins with sugar chains that had notbeen converted to the complex form during intracellular transport.Furthermore, because the two subviral particle types comparedin this study came from the same cells, it can be concludedthat these differences were not caused by any effects on theprocessing enzymes or cell machinery but instead were probablydue to differences in the structural environment of the sugarchains themselves, although subtle differences in intracellulartransport pathways have not yet been ruled out. The endo H_fdigestion patterns therefore suggest that the E proteins inthe virions and large subviral particles are in nonequivalentenvironments at the time that the particle is transported throughthe Golgi, consistent with the observed nonequivalence of theE protein contacts in mature dengue virus (25) and spike asymmetryin immature dengue and yellow fever viruses (48). The glycosylationpattern is a reflection of the state of the immature particlebefore transport to the trans-Golgi network, where prM cleavageand virus maturation presumably occur, and not of the E proteinarrangement in the mature state, whose final geometry couldbe the result of further rearrangements.

Although medium-resolution structures for both mature and immaturedengue virus have recently been determined by cryoelectron microscopy(25, 48), the way in which the envelope proteins rearrange tothe mature configuration upon prM cleavage remains enigmatic.This is because each of these forms has a complex geometry whoserelationship to its counterpart is not obvious (48). It is likelythat the two classes of subviral particles described here havespatially different packing arrangements but nevertheless undergosimilar rearrangements upon prM cleavage. Their use in comparativestructural studies might therefore simplify the process of identifyingthe underlying mechanisms that are important for flavivirusmaturation.

ACKNOWLEDGMENTS

We thank Angela Dohnal for excellent technical assistance throughoutthe course of this work. We are also grateful to Walter Holzerfor help with virus production, to Elisabeth Krach for helpwith secretion time course experiments, and to Yifan Cheng forhelp with electron microscopy.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Virology, Kinderspitalgasse 15, A-1095 Vienna, Austria. Phone: 43-1-40490, ext. 79505. Fax: 43-1-40490-9795. E-mail: steven.allison{at}univie.ac.at.

Present address: Department of Biochemistry and Cell Biology,Rice University, Houston, Texas.

REFERENCES

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