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Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes.

Budding of enveloped viruses has been shown to be driven by interactions between a nucleocapsid and a proteolipid membrane. By contrast, we here describe the assembly of viral envelopes independent of a nucleocapsid. Membrane particles containing coronaviral envelope proteins were assembled in and released from animal cells co-expressing these proteins' genes from transfected plasmids. Of the three viral membrane proteins only two were required for particle formation, the membrane glycoprotein (M) and the small envelope protein (E). The spike (S) protein was dispensable but was incorporated when present. Importantly, the nucleocapsid protein (N) was neither required not taken into the particles when present. The E protein, recently recognized to be a structural protein, was shown to be an integral membrane protein. The envelope vesicles were found by immunogold labelling and electron microscopy to form a homogeneous population of spherical particles indistinguishable from authentic coronavirions in size (approximately 100 nm in diameter) and shape. They were less dense than virions and sedimented slightly slower than virions in sucrose velocity gradients. The nucleocapsid-independent formation of apparently bona fide viral envelopes represents a novel mode of virus assembly. Images


Introduction
Basically, viruses are mobile genetic elements equipped for swift spread from cell to cell; enveloped viruses employ a membrane to facilitate their intercellular transport. Budding at cellular membranes allows the virus to acquire its envelope and leave the cell without injury. The viral envelope protects the genome in the extracellular environment and provides a convenient device to deliver it into the target cell's cytoplasm by membrane fusion. Like intracellular transport vesicles, which also use processes of budding and fusion, the viral envelope contains proteins that mediate binding to, and fusion with, the acceptor membrane. Envelope proteins are also involved in the formation of the particle and in maintaining its integrity.
The process of viral envelope formation is not well understood. Available evidence points to a crucial role played by the viral nucleocapsid (NC). In retroviruses, it is all that is required, as the gag core particle can direct its envelopment without the need for envelope proteins (Delchambre et al., 1989;Gheysen et al., 1989;Karacostas et al., 1989). Capsids of togaviruses are not sufficient to drive their own budding. Here, the viral membrane proteins are indispensable, and virion assembly involves direct interactions between them and the NC (Suomalainen et al., 1992;Hobman et al., 1994;Lopez et al., 1994;Qiu et al., 1994;Zhao et al., 1994). Probably, the icosahedral capsid acts as a scaffold responsible for the curvature of the envelope. Viral membrane proteins are in some cases secreted in the absence of core components. Expression of the hepatitis B virus surface antigen, for instance, leads to the assembly and release of subviral particles (Patzer et al., 1986;Simon et al., 1988;Bruss and Ganem, 1991), which, however, are not simply 'empty' envelopes as they are morphologically distinct from hepatitis B virions (Dubois et al., 1980;Laub et al., 1983;Patzer et al., 1986). Subviral lipoprotein particles were also observed after synthesis of flavivirus envelope proteins (Mason et al., 1991;Konishi et al., 1992;Yamshchikov and Compans, 1993;Allison et al., 1995).
Coronaviruses are among the largest enveloped RNA viruses, measuring 80-120 nm in diameter. Their membrane, which surrounds the 30 kb genome packaged in a helical nucleocapsid, contains two major glycoproteins. The type I spike (S) glycoprotein makes up the surface projections typical for the viruses. The M glycoprotein is most abundant, largely embedded within the membrane bilayer and spans the membrane three times. The small envelope protein (E) is a third, minor structural component in infectious bronchitis virus (IBV), transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) particles (Liu and Inglis, 1991;Godet et al., 1992;Yu et al., 1994); it has only recently been identified and is not extensively characterized. Some coronaviruses contain an additional membrane protein with haemagglutinin/ esterase activity (HE; reviewed by Spaan et al., 1988).
Coronaviruses are assembled intracellularly at membranes of the intermediate compartment, between the endoplasmic reticulum (ER) and the Golgi complex (Tooze et al., 1984(Tooze et al., , 1988Klumperman et al., 1994;Krijnse Locker et al., 1994). The helical nucleocapsids which are synthesized in the cytoplasm align at these membranes (Dubois-Dalcq et al., 1982) and probably interact with the cytoplasmic domains of the viral membrane proteins. They are incorporated into viral particles by budding. The immature virions are exported from the cell along the exocytic pathway while undergoing various post-assembly maturation events including proteolytic and oligosaccharide processing.
Little is known about the molecular interactions that drive coronaviral budding and control the specificity of the process. Direct interactions have been demonstrated between the M and the S protein: when co-expressed from their cloned genes they associate into large heteromeric complexes (Opstelten et al., 1995). Surprisingly, these complexes do not localize to the intermediate (i.e. budding) compartment but rather accumulate in the Golgi complex, the site where the coronaviral M protein generally localizes when synthesized alone (Krijnse Locker et al., 1992;Klumperman et al., 1994). The S protein, which on its own is transported to the plasma membrane, is thus retained in the Golgi complex through its interaction with M. Interestingly, M also forms complexes in the absence of S. Independently synthesized M protein accumulates in the Golgi apparatus in homomultimeric, detergentinsoluble structures, presumably as part of its retention mechanism (Krijnse Locker et al., 1995).
For the study of coronavirus assembly a system is required in which the roles of and the interactions between the assembly partners can be analysed in molecular detail. Since an infectious cDNA clone is presently not feasible due to the large size of the coronaviral genome we have exploited the co-expression approach. We show here that viral envelope proteins can self-assemble into membranebound structures that are morphologically indistinguishable from coronavirions.

Results
Co-expression of selected MHV-A59 genes Expression of viral envelope protein genes has been shown in some cases to result in the production of subviral lipoprotein particles. We never observed such particles in the culture medium when expressing the coronavirus MHV-A59 M or S genes either individually or together (Opstelten et al., 1995). Minor amounts of the S1 cleavage product of S were detected but this was merely due to dissociation from its membrane-anchored partner S2 at the cell surface (see below). In the experiments to be described below we included the E protein gene and this proved to be essential for the formation of coronaviruslike particles.
Particle assembly and secretion were initially assayed by measuring the release of structural proteins into the culture medium of cells expressing MHV protein genes by the recombinant vaccinia virus bacteriophage T7 RNA polymerase system. In the experiment of Figure 1 the cells and media were harvested separately and processed for immunoprecipitation with a polyclonal anti-MHV rabbit serum mixed with anti-S monoclonal antibodies, followed by SDS-PAGE analysis. We found that neither the M protein nor the 180 kDa S protein (S/gp 180) was released into the medium when their genes were expressed individually (lanes 2 and 4). However, co-expression of the M and E gene resulted in release of the M protein into the medium (lane 6). Release  gene or the E gene were omitted (lanes 10 and 12). Appearance of the proteins in the medium did not result from cell lysis since the 150 kDa immature S protein precursor (S/gpl 50), which was still abundant in the cell lysate after 2 h of chase (lanes 3, 7, 9 and 11), was not found in the medium. Thus, co-expression of selected MHV genes leads to specific release of MHV envelope proteins. All medium samples from cells expressing the S gene contained the 90 kDa Sl cleavage product. This was therefore not a suitable marker for particle assembly and release. Rather, the M protein and the 180 kDa S protein species were used as such, since these were not released from cells transfected with non-productive combinations of genes. In later experiments we used the non-productive combination of the M and S gene as our negative control. Subsequently, we also included the gene encoding the viral nucleocapsid protein (N) in our transfections. Coexpression of N with the three envelope protein genes did not affect the release of the 180 kDa S protein nor of the M protein into the culture medium (data not shown). Some N protein was found in the medium but this was also the case and to a similar extent when its gene was expressed alone. Therefore, the N protein was not released specifically and release of envelope proteins was independent of nucleocapsid. We also tested the generality of our findings by repeating the above experiments with the analogous genes of another coronavirus, feline infectious peritonitis virus (FIPV). Again, viral membrane protein release was fully dependent on the M and E gene products (data not shown).
The E protein is essential for secretion of M The E gene expression construct pTM5ab used above contains the unique region of MHV-A59 mRNA 5 behind a T7 promoter. In this region the ORFs Sa and 5b are present, the latter encodes the E protein which is translated by internal initiation (Thiel and Siddel, 1994). The mRNA transcribed from our construct may allow translation of both ORFs, as has previously been shown for a similar construct by Budzilowicz and Weiss (1987). To confirm that the observed effect was due to the ORF Sb expression product alone we prepared two additional constructs. In construct pTM5b, ORF Sa was eliminated and ORF Sb was positioned immediately behind a T7 promoter. In construct pTM5a, a frameshift mutation was introduced into pTM5ab to render ORF Sb inactive. Both constructs and pTM5ab were co-transfected with the M gene expressing plasmid pTUMM, and viral proteins were assayed as before. Analysis of the cell lysates ( Figure 2) showed a protein of the expected size (9.7 kDa) in the lanes of pTM5ab and pTM5b, not of pTM5a. The protein was detected only after prolonged exposure of the gel. Since the rabbit antiserum used had been raised against highly purified virus, its ability to immunoprecipitate the E protein, though only to a low extent, indicates that E is a structural protein of MHV. However, we could not rule out the possibility that the overproduced protein was precipitated non-specifically or co-precipitated with the M protein. No potential Sa protein band was seen after immunoprecipitation. In the functional assay, elimination of ORF Sa, as in construct pTM5b, had no noticeable effect: the M protein was released into the medium as efficiently as with the complete construct. Knocking out the expression of ORF Sb as in pTM5a, however, completely abolished the release of M; its secretion from cells is therefore fully dependent on the E protein alone. Secretion of E was not detected in these experiments. The MHV E protein is an integral membrane protein In view of its apparent importance for MHV assembly the E protein was further characterized. Its primary sequence predicted a large NH2-terminal hydrophobic domain that might anchor the protein in the membrane. To demonstrate that it is an integral membrane protein we used an alkaline extraction method. Dounce homogenates were prepared of cells expressing either the M, S and E genes, the M and S genes, or the M and E genes. After removal of the nuclei by pelleting, the homogenates were treated with 100 mM Na2CO3 at pH I for 10 min on ice. This treatment disrupts membrane vesicles, releases their protein content, and strips off peripheral membrane proteins, leaving integral membrane proteins unaffected (Fujiki et al., 1982). The membrane pellet after ultracentrifugation was analysed by SDS-PAGE and found to contain all three proteins ( Figure 3A). The E protein was readily identified by its absence from the sample of cells transfected with the M and S gene only. No coronaviral proteins were detected in the supernatant fractions. Subsequently, MHVinfected cells were fractionated similarly after three consecutive 1-h periods of 35S-labelling, in order to follow the appearance of E ( Figure 3B). The protein was identified by co-electrophoresis of the recombinant expression product prepared in the same way. (A) To one of a set of two equal samples of culture medium Triton X-100 was added to a concentration of 1%. The samples were ultracentrifuged and the supernatants (s) and pellets (p) were analysed after immunoprecipitation. (B) Two other samples of culture medium were diluted 2-fold with carbonate buffer adjusted to pH 11 or to pH 7 as indicated, and ultracentrifuged. The resulting pellets were analysed directly in a 15% polyacrylamide gel. Only the sections of the gels containing the M proteins are shown.
integral membrane protein. In MHV-infected cells it is one of the specific proteins that are increasingly produced in the course of infection.
The released envelope proteins are membrane associated Detergent treatment of the structures released from the co-transfected cells gave us a first indication that the proteins were contained in membranes. Culture medium from cells co-expressing the M and E genes was analysed by ultracentrifugation directly or after treatment with the detergent Triton X-100. The pellets were solubilized and processed for RIPA in parallel with their respective supematants. In the untreated controls, the M protein was present in the pellet and virtually absent in the supernatant (Figure 4). After treatment of the culture medium with Triton X-100, however, the M protein remained largely in the supernatant; only a small fraction was pelleted. The alkaline extraction method was then applied to the culture medium. Medium samples were incubated for 10 min on ice in 100 mM Na2CO3 at pH 11 or, as a control, at pH 7. After centrifugation the pellets were solubilized in sample buffer and analysed directly by SDS-PAGE. The high pH treatment did not affect the M protein's appearance in the pellet (Figure 4), indicating that it is an integral part of membranes. These data support the notion that the envelope proteins secreted from the cells are constituents of membranous structures, presumably in the form of particles.

Density of particles in sucrose gradients
The particle density was analysed by flotation in sucrose gradients. To this end we dissolved crystalline sucrose in the culture supernatant to a final concentration of 60% (w/w). Subsequently, 1.5 ml of this solution was overlayed with 0.9 ml each of 50, 40, 30 and 20% (w/w) sucrose solutions. The gradients were centrifuged and five fractions of 1 ml were collected. This fractionation scheme allowed us to collect each interphase in one fraction. Fractions were diluted 2-fold to allow pelleting of particles by ultracentrifugation. The selected vector combinations for transfection were M+ S +E, M+E and M+S, the latter serving as a negative control. As a positive control we used culture medium of MHV-infected cells treated in the same manner. The MHV particles were found at the highest density, in fraction 3 which contains the 40/ 50% interphase ( Figure 5A). A polypeptide of~10 kDa, presumably representing the E protein (indicated with an arrowhead), co-purified with MHV virions. The particles derived from M+S+E-expressing cells always floated to a slightly higher position in these gradients and appeared in fractions 3 and 4 corresponding with the 40/50% and 30/40% interphases, respectively. Here, a weak band of the E protein could be discerned in fraction 4 after overexposing the gel. Particles devoid of the S protein produced by M+E-expressing cells, on the other hand, floated consistently to lower density than particles containing S; they were found mainly in fraction 4, which contains the 30/40% interphase ( Figure SB). No viral proteins were seen floating when the S and M genes were co-expressed. In fraction 5 of the sample of MHV-infected cells ( Figure SA) there appears to be only M protein present. These particles might be very similar to the particles produced by M+E-expressing cells.
The data allow a comparison of the yields of viral particles released from transfected and MHV-infected cells. Taking the radioactivity in the M protein as a measure we estimated that the amounts of particles produced by cells expressing M+S+E and M+E were~20% and 10% that of virions secreted from infected cells, respectively. The yields of particles varied between 5% and 50% relative to MHV among different experiments, probably due to variations in transfection efficiency. Frequently, more particles were released when the S protein was co-expressed than in its absence. Apparently, S proteininduced fusion of neighbouring cells containing incomplete sets of genes increased productivity by increasing the availability of individual components to the assembly system.

Analysis of particles by electron microscopy
In order to visualize the particles that were apparently assembled when coronaviral envelope proteins were cosynthesized in cells, preparations of extracellular material purified by pelleting through a 30% (w/w) sucrose cushion were analysed by electron microscopy (EM). Immunogold labelling was employed for particle identification using the polyclonal anti-MHV serum and a protein-A-gold conjugate. Labelled particles were observed in material from cells expressing the M+E or M+S +E genes ( Figure  6). The particles were spherical and had the same appearance as MHV particles analysed in parallel. They also had the same dimensions. While the mean diameter of MHV virions was 108.9 ± 13.4 nm (n = 30), that of the M+S+E particles was 102.6 ± 15.2 nm (n = 30) and that of M+E particles 102.6 ± 9.8 nm (n = 30). Material derived from cells expressing the M+S genes showed no particles. Occasional gold labelling was seen here, probably due to the presence of some S1 material released into the medium (data not shown). No labelling of particles occurred with control sera. Sedimentation analysis of particles MHV and the virus-like particles (VLPs) were compared by velocity centrifugation in 5-30% (w/w) sucrose gradients. Fractions were collected and analysed as described above. Consistently, MHV particles sedimented slightly After centrifugation, five fractions were collected from which the particles were analysed in a 15% polyacrylamide gel. Vector combinations used are indicated above the panels and the positions of the viral proteins are given at the right. Fractions I and 5 are bottom and top fraction, respectively. ahead of M+ S + E VLPs which in turn sedimented slightly faster than M+E particles (Figure 7). The sedimentation profiles were similar with respect to peak width for all types of particles, indicating that the particles were of comparable homogeneity. Sedimentation analysis of VLPs produced by cells expressing M+E+ S +N did not reveal N protein co-purifying with the particles containing S and M, indicating that it was not physically associated with the envelopes.

Discussion
Co-expression of coronaviral envelope protein genes in cells leads to the assembly and secretion of particles morphologically indistinguishable from authentic coronavirions. Only the M and E proteins are required to drive their formation; the S protein and the N protein (and, by consequence, the nucleocapsid) are dispensable. Earlier we have shown that the MHV M protein alone can associate into large homomeric complexes, presumably by lateral interactions between M molecules in the plane of the membrane (Krijnse Locker et al., 1995). Extensive EM work with M proteins from various coronaviruses never gave any indication that the protein produced on its own would form particles or induce curvature in the intracellular membranes in which it appears (Machamer et al., 1990;Krijnse Locker et al., 1992;Klumperman et al., 1994). This implies that the E protein is pivotal in inducing this curvature and that coronaviruses acquire their particular shape and dimension by the concerted action of just these two membrane proteins.
Through our observations we have identified a novel budding principle for enveloped viruses: nucleocapsidindependent assembly of bona fide virion envelopes. In the two other well-studied models the nucleocapsid plays a central role (Figure 8). In type D retroviruses the gag core particle is the sole requirement: It is able to bud in the absence of envelope proteins (see Hunter, 1994).
Specific interactions between the nucleocapsid and viral membrane proteins are the driving force in the alphavirus model (Garoff et al., 1994;Strauss and Strauss, 1994). Here, enveloped particles are formed neither by the nucleocapsid alone nor by the membrane proteins alone (Suomalainen et al., 1992). However, nucleocapsidindependent formation of enveloped particles has been demonstrated before. Subviral particles were, for instance, produced by the synthesis of the hepatitis B or Japanese encephalitis virus envelope proteins (Simon et al., 1988;Bruss and Ganem, 1991;Konishi et al., 1992). Heterogeneous enveloped particles were also released from cells producing the VSV G protein (Rolls et al., 1994). Apparently, these were generated by vesiculation at the plasma membrane rather than by a specific assembly process. We can only speculate why coronaviruses have the capacity to assemble uniform envelopes from their membrane proteins: the helical nature of the (probably random-coil) nucleocapsid and the lack of a classical matrix protein may necessitate an intrinsic morphogenetic potential of the envelope proteins.
While the M protein has long been recognized as being essential for coronavirus assembly (for a review, see , the crucial role of the E protein came as a surprise. Only recently has E been identified as a virion protein, having been overlooked due to its small size and low abundance. It was estimated to occur in IBV and TGEV at a rate of about 100 and 20 molecules per particle, respectively (Liu and Inglis, 1991;Godet et al., 1992). For MHV our preliminary estimates were some 5-10 molecules per virion using M as a reference, with 726 molecules per virion as calculated for human coronavirus (HCV) OC43 (Hogue and Brian, 1986). The stoichiometry of M and E in VLPs appeared similar although the low particle yields did not allow accurate measurements. This low abundance suggests that the E protein has no genuine structural function in the virion envelope in that it occupies frequent, regular positions within the lattice built by  M molecules. Rather, it may have a morphogenetic function by taking infrequent but strategic positions within the lattice to generate the required membrane curvature. Alternatively, its role might merely be 'catalytic', serving for instance to close the 'neck' of the viral particle as it pinches off from the membrane in the terminal phase of budding.
Too little is known about the E protein to understand its function. The polypeptide of 80-110 residues is hydrophobic, particularly in its NHW-terminal domain, though a classical cleaved signal sequence is not predicted. Consistently, it was shown by immunofluorescence to be associated with the cell surface and with internal membranes (Abraham et al., 1990;Smith et al., 1990;Godet et al., 1992;Yu et al., 1994); we demonstrate here that in MHV the E protein is membrane-anchored. Its membrane structure and topology can only be guessed. Antibodies to the C-terminal domain resulted in cell surface fluorescence (Godet et al., 1992) and neutralized infectivity in the presence of complement (Yu et al., 1994); these observations suggest that the C-terminal region is translocated into the lumen of the ER during biogenesis. The MHV-A59 E protein is acylated (Yu et al., 1994), probably at sites within the conserved cysteine cluster close to the middle of the molecule.
Small, often acylated proteins occur in the membranes of many enveloped RNA viruses. Examples are the influenza virus M2 and the alphavirus 6K proteins. Like E, these are synthesized in ample amounts in infected cells but are incorporated into virions in only minor amounts. The 6K protein was suggested to facilitate the budding stage (Liljestrom et al., 1991;Loewy et al., 1995). The E protein has been speculated to function as an ion channel-like influenza virus M2 (Liu and Inglis, 1991), but this activity has not yet been demonstrated. Of the several small proteins induced by lentiviruses the HIV-1 vpu protein bears resemblance to E in that it enhances virion maturation and release; however, vpu has not been detected in extracellular virions and is not essential for virus replication (see Subramanian and Cohen, 1994). An intriguing protein is p6Gag, a peptide derived from the C-terminus of the Gag precursor of primate lentiviruses. It has been implicated in a late step in budding (Gottlinger et al., 1991;Huang et al., 1995). The equivalent of this latter function was recently also identified in an unrelated retrovirus, the avian Rous sarcoma virus which lacks a similar cleavage product but has instead an assembly domain located between the matrix and capsid sequences (Parent et al., 1995). Defects in this domain lead to the accumulation of Gag proteins at the plasma membrane due to the impairment of viral particle release (Wills et al., 1994).
An intriguing feature of coronaviruses is their flexibility in accommodating various numbers of different envelope proteins. As we show here, VLPs can be assembled without S, the spike protein which mediates binding of virions to and fusion with the host cell membrane. This observation is consistent with earlier findings showing that spikeless, hence non-infectious, virions are secreted when infected cells are treated with tunicamycin (Holmes et al., 1981;Rottier et al., 1981a). Inhibition of Nglycosylation induces the S protein to aggregate in the ER (our unpublished data) thereby blocking its incorporation into viral particles. Tunicamycin similarly affects HE: when HCV OC43 was grown in the presence of the inhibitor, virions were produced which now lacked both S and HE (Mounir and Talbot, 1992). Another illustration of the flexibility of the coronavirus envelope was the incorporation of HE protein into a genetically HE-deficient strain, as shown by Liao et al. (1995). Expression of the MHV JHM(2) HE gene in MHV-A59-infected cells, yielded A59 particles containing the heterologous protein.
We have recently shown that incorporation of S into virions is mediated by its specific interaction with M (Opstelten et al., 1994(Opstelten et al., , 1995. S associates with M into large, detergent-insoluble complexes when they are coexpressed from their cloned genes and in infected cells. Since the M protein alone can assemble into higher-order complexes (Krijnse Locker et al., 1995), we speculate that specific positions are available in such an M protein matrix where S protein trimers can be accommodated. Whether the HE protein can occupy similar positions or is incorporated into virions through a different mechanism remains to be seen. The specificity of the interactions between the viral envelope proteins may function not only to include kin components into viral particles, it probably also serves to exclude host membrane proteins from them.
The picture of coronaviral envelope formation thus emerging is one in which membrane proteins (viral and cellular) are sampled for fit into the M protein lattice. The specificity of the molecular interactions acts as a quality control system to warrant the formation of defined twodimensional assemblies containing the full complement of viral membrane proteins and excluding cellular ones. The involvement of the E protein in these structures is decisive in inducing the membrane curvature and causing budding. As the nucleocapsid is not required, it needs to entertain specific interactions with the envelope proteins in order to become sequestered into particles. As a prediction of this view, nucleocapsid-less particles might also be produced during coronavirus infection. Such particles have indeed been described for IBV. They had a typical coronavirus morphology and a lower density than virions (Macnaughton and Davies, 1980).

Expression vectors
Expression constructs used for transfection were pTUMS, with a BamHIfragment containing the MHV S gene (Vennema et al., 1990) and pTUMM, with a XhoI-fragment containing the MHV M gene (Rottier and Rose, 1987), both cloned in pTUG3 (Vennema et al., 1991), which provides the bacteriophage T7 transcription regulatory sequences. The MHV N gene was cloned as a StYI-PstI fragment from pRG68 (Bredenbeek et al., 1987) downstream of the T7 promoter in pBluescript SK-(Stratagene) to yield pBMN. Construct pTM5ab contains the MHV gene 5 ORFs 5a and Sb, the latter encoding the E protein. It was prepared by ligating a T7 promoter/MHV leader fragment, similar to a synthetic fragment described previously (van der Most et al., 1991). to the insert of clone pRG68, which contains a nearly full-length copy of mRNA 5, including most of the leader sequence. The tandemly duplicated leader sequences were eliminated by limited T4 DNA polymerase digestion in the absence of nucleotides, followed by re-annealing of complementary single-stranded ends and transformation. Correct repair in the bacterial host was confirmed by sequence analysis. The sequences downstream of ORF Sb were deleted by restriction enzyme digestion and re-ligation. A derivative of pTMSab was constructed by digestion with the restriction enzyme BstEII, which has a single recognition site in ORF Sb. The overhanging ends were filled in with Klenow DNA polymerase and re-ligated, resulting in the insertion of five nucleotides in ORF Sb. In this construct, designated pTMSa, ORF Sb is inactivated by a frameshift. Finally, pTMSb was constructed by cloning a Taiql-EagI restriction fragment from pRG68 containing ORF Sb into Accland EcigI-digested pBluescript SKand re-cloning into pTUG3.

Metabolic labelling and immunoprecipitation
Metabolic labelling was performed in methionine-free MEM without FCS with 50 pCi/ml 35S ill litro cell labelling mix (Amersham) for I h.
Pulse labelling was preceded by a starvation period of 30 min in methionine-free MEM. During chase periods the cells were incubated in DMEM containing 10% FCS and four times the normal amount of methionine. Cells were lysed in 20 mM Tris-HCI, pH 7.5, 1 mM EDTA, 100 mM NaCI (TES) containing 1% Triton X-100 and 2 mM phenylmethylsulfonyl fluoride. Lysates were kept on ice for 15 min and cleared by centrifugation for 15 min at 10(000 g and 4°C. Medium samples were cleared likewise by centrifugation at room temperature and subsequently used for radioimmunoprecipitation (RIP) or gradient analysis. For RIP. the medium was brought to x TES and 1'k Triton X-100 by adding a concentrated stock solution. followed by a further dilution with 2 volumes TES containince 0.1I% Triton X-l00.  (Fleming et oil.. 1983). The latter were added to more efficiently precipitate the S protein. RIP was carried out overnight at 4°C. The immune coniplexes were bound to Inimunoprecipitin (Gibco Laboratories) for I h at 4°C and subsequently pelleted by centrifugation. Pellets were washed three times in 10 mM Tris-HCI. pH 7.4. 0.15 M NaCI. O.1c SDS. 1%k sodium deoxycholate. I1% Nonidet P-40 (RIPA buffer) for the cell lysates and in TES-O.I%ck Triton X-100 for the medium samples. The final pellets were suspended in sample buffer and heated at 95°C for 3 min before analysis by SDS-PAGE according to Laemmli (1970).
Alkaline extraction of cellular membranes Cells were lvsed in a 10-fold diluted TES buffer by 20 strokes in a tight-fitting Douilce homogenizer on ice. Nuclei were pelleted by centrifugation at 1000 g for 10 min. The supernatant was diluted 2-fold by adding an equal volume of 200 niM Na,CO adjusted to pH I1. and incubated for 10 min on ice. Membranes were pelleted in a Beckman TLAIOO rotor (Beckman Instruments. Inc.. Fullerton, CA. USA) at 65 000 r.p.m. for 30 min at 4°C. The pellet was dissolved in sample buffer and analysed by SDS-PAGE.
Flotation analysis Samples of cell culture medium were made up to 60%k sucrose (w/w) with crystalline sucrose and 1.5 nil was transferred into a centrifuge tube and overlayed with 0.9 nil each of 5f). 40. 30 and 20%7 (w/w) sucrose solutions in TES. The gradients were centrifuged for 72 h at 50 000 r.p.m. in a Beckman SW5( rotor and fractionated into five fractions. each of ml. This fractionation scheme allowed each interphase to be collected in one fraction. Fraction was 60%k sucrose, fraction 2 contained the 50-60%7 interphase. fraction 3 the 40-50%) interphase. fraction 4 the 30-40% interphase. and fraction 5 the 20-30%)9 interphase. Fractions were diluted 2-fold with TES to allow, pelleting of particles by ultracentrifugation for 30 min at 4°C in a Beckman TLA100.2 rotor at 65 000 r.p.m. Pellets were dissolved in sample buffer and analysed by SDS-PAGE.

Electron microscopy
Culture media of cells were collected at 8 h post-infection and spun for 10 min at 10 000 g to pellet cell debris. The cleared medium was then layered on a sucrose gradient consistinc of I ml 30% (w/w) sucrose. I ml 20%(7 (wAl) sucrose/ I gk alutaraldehyde and I ml I 1% (w/) sucrose in TES. pH 6.5. The gradients were centrifuged for 3 h at 30 000 r.p.m. in a SW50.1 rotor at 4°C. After the run the pellet was suspended in TES. pH 6.5. A formvar-carbon-coated copper grid *was placed on top of 5 pl of the suspension for 30 min. Subsequently. the grids were rinsed three times with PBS containing 50 mM glycine (PBG) and treated for 30 nmin with 5%k bovine serum albumin (BSA. fraction 5: Sigma) and I1c acetylated BSA (BSA-C: Aurion) in PBG. Samples were incubated for I h with the rabbit polyclonal antiserum 134 to MHV-A59 diluted 200-fold in PBG containing 0.5%'-BSA and 0.1% BSA-C (PBG+). After three washes with PBG-. samples were incubated with protein A-5 nm aold diluted to an OD520 of 0.1 in PBG- (Slot and Geuze, 1985) for 30 min. rinsed three times with PBG+ and twice with vvater. Finally. samples were negratively stained by placing the grids for 30 s in 2%7c potassium phosphotungstate solution. pH 6.8. The grids were viewed and photographed in a Philips CMI(0 electron microscope at 100 kV. Velocity gradient sedimentation Cells infected with MHV and cells expressincg combinations of MHV envelope protein genes were labelled for 4 h with 133 PCi/ml 35S il l .itr-( cell labelling mix (Amersham). Culture media were collected and cleared by centrifugation at 4°C for 15 min at 10(000 g. Particles present in medium samples were sedimented into eradients of 5-30%c (wv/w) sucrose in TES. pH 6.5. by centrifugation for I h at 40 000 r.p.m. in a Beckman SW41 rotor at 4°C. Fractions of 0.5 ml were collected froni the bottom of the tubes. Particles were pelleted from each fraction bv ultracentrifu2ation for 30 min at 4°C in a Beckman TLA 100.2 rotor at 65 000 r.p.m. aind analysed directly by SDS-PAGE.