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Oligomerisation of the structural proteins of Rubella virus
VIROLOGY
185, 811-819
(1991)
Oligomerisation
of the Structural
MICHAEL Department
of Molecular
Biology,
Karolinska Received
D. BARON’** Institute May
Centre 20,
Proteins
of Rubella Virus
KERSTIN FORSELL
AND
for Biotechnology,
199 1; accepted
August
Blickagdngen 23,
6, S- 14 1 52 Huddinge.
Sweden
199 1
Rubella virus contains, in addition to its RNA genome, a nucleocapsid protein (C) and two membrane proteins (E2 and El). We have studied the association of these proteins during viral assembly and when expressed from cDNA constructs. The C protein was found to dimerize very shortly after synthesis; this dimer became disulfide-linked in the virion. Formation of the dimer was independent of the presence of other RV proteins. The membrane glycoproteins formed an E2El heterodimer, a minor fraction of which was also found to be disulfide-linked in the virion. This heterodimer also formed when the two proteins were coexpressed from cloned cDNA. Formation of the heterodimer preceded the transport of E2 to the Golgi, as judged by modification of the protein by Golgi-located enzymes. In the absence of E2, the El protein was slowly converted to high molecular weight aggregates. Q fQQi Academic press, hc.
INTRODUCTION
tions in most cell lines (Abernathy et a/., 1990). The C protein, unlike that of alphaviruses, is not an autoprotease and is cleaved from the following proteins by signal peptidase (Clarke et al., 1987; Hobman and Gillam, 1989) leaving the C protein membrane-associated (Suomalainen et al., 1990). In addition, the C protein in isolated virus has been reported to be a disulfide-linked dimer (Waxham and Wolinsky, 1983); a fraction of E2 and El were also observed to be in disulfidelinked dimers in the same work. No such covalent linkages have been seen in the alphaviruses. The relative amounts of the structural proteins in the isolated virus are in some doubt, having been reported as either (5: 1:5) (El :E2:C) (Vaheri and Hovi, 1972; Waxham and Wolinsky, 1985b) or 1 :l :l (Bowden and Westaway, 1984). We have been studying the synthesis and transport of the viral structural proteins expressed from cloned cDNA (Suomalainen et al., 1990; Baron et al., submitted for publication) and have attempted to elucidate how these proteins interact during assembly of the virus in infected cells. We have found that the C protein spontaneously forms homodimers even if expressed in the absence of other RV proteins: these dimers appear to become disulfide-linked after virus budding or if exposed to an oxidizing environment. The El and E2 proteins form heterodimers; this oligomerization appears to be necessary to prevent later aggregation of El and for efficient transport of E2 and El from the ER to the Golgi.
Rubella virus (RV) is a membrane-enveloped, positive-stranded RNA virus, the only member of the genus Rubivirus in the Togavirus family. The virus contains three structural proteins (Bowden and Westaway, 1984; Ho-Terry and Cohen, 1980, 1982; Oker-Blom et a/., 1983; Toivonen et a/., 1983; Trudel et a/., 1982; Vaheri and Hovi, 1972; Waxham and Wolinsky, 1983) with molecular weights variously reported as between 33 and 38 kDa (the capsid or C protein), between 53 and 62 kDa (membrane protein El), and a broad band covering about 6 kDa in the region 43-51 kDa (membrane protein E2). The C protein forms a shell surrounding the genome (Vaheri and Hovi, 1972). This nucleocapsid is, in turn, surrounded by the membrane envelope, which contains El and E2 (Vaheri and Hovi, 1972; Waxham and Wolinsky, 1985b). The structural proteins are translated from a single, subgenomic message as a polyprotein in the order CE2-El (Oker-Blom, 1984). The subgenomic message is derived from the 3’-end of the viral genome (OkerBlom et al,, 1984); the 5’-end appears to encode a number of nonstructural proteins involved in viral replication (Dominguez et al., 1990). In these respects, RV resembles the alphaviruses (reviewed in Garoff eta/., 1982) in its replication strategy and (to some extent) in its structure. However, RV differs from the alphaviruses in several aspects of both replication and structure. Replication is much slower than in alphaviruses (Hemphill et a/., 1988) and the virus is able to form persistent infec’ To whom reprint ’ Present address: Ash Road, Pirbright,
requests Institute Woking,
should be addressed. for Animal Health Pirbright Surrey GU24 ONF. UK.
METHODS Cells and virus B-Vero and RK,, cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal
Laboratory,
811
0042.6822/91
$3.00
Copyright Q 1991 by Academic Press, Inc All rights of reproduction in any form reserved.
812
BARON AND FORSELL
calf serum, 10 mll/l HEPES (AI-2-hydroxyethylpiperazine-N’-2-ethane sulfonic acid), 100 units liter-’ penicillin, 100 pg liter-’ streptomycin. RV infection was as described (Suomalainen et a/., 1990). The Therien strain of RV was used throughout; the cloning of the structural region of this strain has been described (Suomalainen et a/., 1990; Takkinen et a/., 1988; Vidgren et a/., 1987). The construction of recombinant vaccinia virus expressing the complete structural protein polyprotein (wRVS4), E2(wE2), El(wEl), or the E2El part of the polyprotein (wE2El) is described elsewhere (Baron et al., submitted for publication). The same techniques were used to construct the recombinant wC17, which contains a cDNA clone containing the first 1500 bases of the structural protein coding sequence (Suomalainen et a/., 1990). All media were from GIBCO; all cell culture plasticware were from Costar or NUNC (Denmark). Radiolabeling
of RV and RV-infected
cells
Radiolabeled virus was prepared as previously described (Suomalainen et al., 1990). RV-infected cells were labeled 24 hr postinfection; for studies on infected cells, 35-mm dishes of cells were routinely starved of methionine by incubating for 30 min in methionine-free MEM containing 10 mM HEPES and 0.2% bovine serum albumin. This medium was then replaced by 0.5 ml of the same medium containing 50 &i [35S]methionine (SJl515, Amersham, UK) and incubated (unless otherwise indicated) for 15 min (pulse). The labeling medium was removed and replaced with 2 ml of complete medium for the indicated time (chase time). Labeled cells were washed once with cold PBS (15 mM NaPO,, pH 7.4, 150 mM NaCI) and then solubilized in 250 ~1 of Tris lysis buffer (50 mll/l Tris-Cl, pH 7.4, 150 mM NaCI, 2 mM EDTA, 1% (v/v) Nonidet P-40 (NP-40) 20 pg ml-’ phenylmethylsulfonyl fluoride (PMSF)) or in the same volume of phosphate lysis buffer (50 mM NaPO,, pH 7.4, 0.4 1\/1 NaCI, 5 rnM EDTA, 10 mM ATP, 1% NP-40, 20 pg ml-’ PMSF). Where indicated, the lysis buffer contained 10 mM iodoacetamide (IAA). Lysates were spun for 2 hr at 17,000 rpm in a Beckman JAI 8.1 rotor, and the supernatants used for immunoprecipitation (Wahlberg et a/., 1989). Samples were analyzed by polyacrylamide gel electrophoresis after denaturation in sodium dodecyl sulphate (SDS-PAGE) as previously described (Baron and Garoff, 1990). Quantification of immunoprecipitated proteins was performed by excising gel slices and determining the [35S] content by scintillation counting (Wahlberg et al., 1989).
Antibodies The antibodies used in these studies were: polyclonal rabbit anti-RV(the gift of R. Pettersson, Ludwig Institute for Cancer Research, Stockholm, Sweden); polyclonal rabbit anti-E2ct, a polyclonal serum that recognizes the last 12 amino acids of E2 (Baron eT al., submitted for publication); monoclonal anti-C-l, antiE2-1, anti-El -10, 17, 19 and 23 (the generous gift of J. Wolinsky, University of Texas Health Science Center, Houston, TX). Labeling
of vaccinia-infected
cells
Confluent or near confluent monolayers of RK,, cells were infected with recombinant or wild-type vaccinia virus at a multiplicity of infection of -5. The cells were labeled as for RV-infected cells but 5 hr postinfection and using a 30-min pulse. Labeled cells were lysed and RV proteins immunoprecipitated as normal. Sucrose velocity gradients Samples (100-300 ~1) were layered onto 1 1.5-ml 5-20% linear gradients of sucrose in 50 mM Tris-Cl, pH 7.5, 100 mM NaCI, 2 mlM EDTA, 0.1% NP-40 and centrifuged for 28-30 hr at 40,000 rpm in a Beckman SW41 rotor. Fractions (50 drops) were collected from the bottom of the tube and labeled proteins immunoprecipitated with various antibodies. RESULTS Virion C protein
is a disulfide-linked
dimer
It has been reported (Waxham and Wolinsky, 1983) that there are disulfide linkages between the structural proteins in the RV virion. However, in those studies no attempt was made to prevent the formation of disulfide bonds between free cysteine residues during solubilization and immunoprecipitation. In order to confirm this observation, therefore, we first pretreated the virus with iodoacetamide and then solubilized it in the presence of the same reagent. The viral proteins were precipitated with the respective monospecific antibodies and the resultant proteins analyzed by SDS-PAGE after denaturing in the presence or absence of dithiothreitol (Fig. 1). It is clear from these studies that the C protein in the virus is present as a disulfide-linked dimer, since it is found entirely in this form (C,) despite the presence of a high concentration of IAA during lysis (Fig. 1, lane 4). The membrane glycoprotein El, but not E2, showed increased mobility in the gel if disulfide bond reduction was omitted, as has previously been reported (Waxham and Wolinsky, 1983); this difference
OLIGOMERISATION +DR 123456
-DTT
FIG. 1. Disulfide bond linkages in RV. [35S]Methionine-labeled RV was solubilized in the presence of 10 mM IAA and the proteins were immunoprecipitated by using monoclonal antibodies anti-C-l (lanes 1 and 4) or anti-El -17 (lanes 2 and 5) or a polyclonal antibody to the carboxy-terminal of E2 (anti-E2ct) (lanes 3 and 6). Half of each im,munoprecipitate was denatured in SDS in the presence (lanes l-3) and half in the absence (lanes 4-6) of 50 mM dithiothreitol (DTT). The samples were analyzed by SDS-PAGE and fluorography. The two panels are from different parts of the same gel, exposed identically.
in mobility is probably due to the presence of intrachain disulfide bonds in El. In addition, a fraction of El and E2 was found as the reported disulfide-linked dimers (d) (Fig. 1, lanes 5 and 6). Although these have been described as El-El and El-E2 dimers (Waxham and Wolinsky, 1983) they were both precipitated in equal proportions by either anti-El or anti-E2; this suggests that these dimers may be part of larger complexes containing both El and E2, and therefore precipitated by either antibody. It is clear from Fig. 1 that El and E2 are coprecipitated by either anti-El or anti-E2 even when the proteins are not covalently linked. This coprecipitation has been reported (although not discussed) before with some anti-El monoclonals (Waxham and Wolinsky, 1983, 1985a,b); it is abolished by denaturing El and E2 before immunoprecipitation (Waxham and Wolinsky, 1985a; our own unpublished observations). Anti-E2 monoclonals failed to coprecipitate noncovalently linked El and precipitated virion E2 poorly; denaturation of virion proteins in SDS resulted in improved precipitation of E2 by monoclonal anti-E22 (not shown). These data suggest that the epitope seen by this monoclonal is at least partly concealed in the heterodimer. Nevertheless, it must be a strong epitope since all of the anti-E2 monoclonals we tested showed the same phenotype. The RV C protein forms noncovalent
homodimers
The C protein is cytoplasmic, so disulfide links cannot form until the protein moves out of the cytoplasmic compartment; this will happen when the virus buds, whether that budding occurs at the plasma membrane or at an intracellular membrane. We therefore performed pulse-chase studies on RV-infected B-Vero
OF RV PROTEINS
813
cells to determine the time after synthesis when disulfide-linked C protein could be seen. We were surprised to find that, when cells were solubilized in our normal Tris-based lysis buffer, C protein appeared entirely as a disulfide-linked dimer even after only the 15-min labeling period (e.g. Fig. 2a). However, when 10 mM IAA was included in the lysis buffer, no disulfide-linked dimer was seen (Fig. 2a). The formation of disulfidelinked C protein in the cell therefore appeared to be artifactual, apparently promoted by oxidizing conditions in the lysis buffer. Those conditions were specific to the Tris-based lysis buffer; when we tried a phosphate-based lysis buffer also in use in our lab, no covalently linked dimer was seen whether or not IM was present during lysis (not shown). We therefore used the phosphate-based lysis buffer in all other experiments, except when we specifically wanted to use the formation of the disulfide bond as an assay for dimerization. The efficiency of disulfide bond formation between C protein monomers was such that it was clear that the C protein in the cytoplasm must already be in a noncovalent dimer or other oligomer. (Note that the concentration of IAA used was sufficient to suppress entirely the formation of disulfide bonds between the proteins in the dimer, and we are therefore confident that this concentration was sufficient to inhibit spontaneous disulfide bond formation during lysis of the isolated virus.) When RV-infected cells were pulse-labeled and chased for up to 22 hr (Fig. 2b), it became possible to observe the formation of MA-resistant covalent dimers (C,); these were detectable at 2 hr chase and clearly visible at 4 hr chase. At this time, labeled C protein was detected in medium virus. Quantification showed that the rate of release of C protein into the medium was roughly the same as the rate of loss of C, from the cell between 8 and 22 hr, suggesting that the C, in the cell is a precursor to C protein in released virus, which was, as we had shown above, all in the C, state. The labeled C protein in the cell remained predominantly in the noncovalently linked form, although it is not clear at this stage whether this is all free C protein or includes intracellular virus, since we have no measure of the rate at which virion C protein becomes disulfide-linked after budding. The amount of labeled C protein released into the medium after 22 hr was only a small fraction of the total labeled during the pulse. We also consistently found an apparently disulfidelinked form of C protein (marked C*, Fig. 2b) at all time points. This form migrated more slowly on SDS-PAGE than the viral form of the C protein dimer and was never seen in the released virion; unlike C,, it disappeared from the cells at about the same rate as noncovalently linked C protein (fllP = 8 hr). The state of the C protein
BARON
814
AND
FORSELL Cell lysate
b chase
a chase(min)
+Dll RV
0
60
60
RV
0
60
2
0
4
Medium
8
22
C’ --c
-DTT 0
(h)
0
RV
2
4
8
22
4-c2 - DTT
60
-c2 +DlT -c FIG. 2. Drmerization of C protein. (a) Thirty-five-millimeter plates of RV-infected B-Vero cells were labeled as described under Methods and chased for the indicated times. The cells were solubilized in Tris lysis buffer with or without 10 mM IAA and the lysate was extracted with anti-c-l, The immunoprecipitated proteins were analyzed by SDS-PAGE after denaturing in the presence or absence of 50 mM DTT. (b) Sixty-millimeter plates of RV-infected cells were labeled with [35S]methionine and chased for the indicated trmes. after which the cells were solubilized in the presence of 10 mM WA. At 2, 4. 8. and 22 hr of chase the medium was also collected, and virus was pelleted by centrifugation and solubilized. C protein was immunoprecipitated from the cell lysates and analyzed by SDS-PAGE after denaturing in the presence or absence of 50 mM DTT. The positions of the monomeric C protein, the C, and C* dimers, and the 70.kDa protein (P) are shown. RV, labeled virus marker. Note that the Medium part of the gel was exposed to X-ray film for four times as long as the cell lysate part
in C*, and its importance in the assembly of the virus, is unclear. It is possible that it is an intermediate in the formation of the virion, but equally possible that it is a dead-end, nonproductive form of C protein, possibly attached to some other, unlabeled protein. The initial time point (0 hr chase) always contained a high molecular weight protein (marked P; Fig. 2b) of apparent molecular weight 70 kDa. This might be incompletely processed precursor polyprotein or a cell protein coprecipitating with the C protein. Preliminary experiments suggest that it is not one of the hsp70 family, since the recovery of the protein is not affected by the presence or absence of Mg/ATP in the lysis buffer or the enzyme apyrase; recovery of P is enhanced by the presence of IAA in the lysis buffer (not shown). In order to determine whether the appearance of disulfide-linked C protein was a function of virion formation or of the C protein alone, we used recombinant vaccinia virus expressing either the whole structural polyprotein (wRVS4) or the capsid plus the first half of E2 (wC17; the E2 segment was included to ensure an as near normal as possible interaction of the C protein with the ER membrane). These viruses were used to infect RK,, cells, a common host for RV that tolerated vaccinia infection better than B-Vero cells did. The infected cells were labeled, chased for 0 or 4 hr, and analyzed for the presence of oligomeric C protein by lysis in Tris lysis buffer with and without IF% As can be seen in Fig. 3, lysis in the absence of IAA allowed essentially all the C protein to become disulfide-linked, whereas the presence of IAA in the lysis buffer prevented the formation of covalent bonds between C protein monomers. In the infected cell, IA/-resistant dimers were visible after 2-4 hr chase (Fig. 2). When the C protein was
expressed from cDNA, however, no such covalent dimer was seen, even after 4 hr chase. The dimerization of the C protein, therefore, appears to occur spontaneously, with or without virion formation, and in the presence or absence of the rest of the structural proteins; the formation of IAA-resistant dimers, on the other hand, appears to occur only during productive infection with RV. Sucrose velocity gradient analysis of the C protein expressed from wRVS4 (Fig. 4) was used to determine the size of the native oligomer. Labeled, wRVS4-infected cells were lysed in the presence or absence of IAA; C protein migrated in the same position on the gradient (fractions 12/l 3) whether or not disulfide bonds were allowed to form, indicating that the olivvc17
IAA Chase
-73 +-+-+-+-
(h)
+DlT
-DlT
FIG. 3. Dimerization of C protein expressed from recombinant vaccinia virus. RK,, cells were infected with wC17 or wRVS4 and labeled with [?]methionine as described under Methods. After 0 or 4 hr of chase, cells were solubilized in the presence or absence of 10 mM IA4 and C protein was immunoprecipitated. The precipitated protein was analyzed by SDS-PAGE after denaturing in the presence or absence of 50 mM DlT. RV, labeled virus marker.
OLIGOMERISATION 2
4
6
8
10
12
14
16
18
-C
-c2
FIG. 4. Sucrose velocity gradient analysis of C protein oligomer. WRVSCinfected RK,, cells were labeled for 2 hr and lysed in the presence or absence of 10 mM IAA. The cells were solubilized and then spun at 16,000 rpm for 10 min to remove the nuclei, and the lysates analyzed on 5-20s linear sucrose gradients as described under Methods. C protein was immunoprecipitated from each fraction and the immunoprecipitates were analyzed on SDS-PAGE after denaturation in the absence of DTT. Sedimentation is from right to left.
gomer is stable; the C protein migrated only slightly slower than the 4.5 S Semliki Forest virus spike protein heterodimer (Simons et a/., 1973; Wahlberg et al., 1989) which was used as a convenient marker, and migrated at fraction 1 1 in these gradients. From these data we conclude that the C protein exists as a dimer and that this dimer forms spontaneously both in infected cells and when the C protein is expressed from cDNA.
OF
RV PROTEINS
815
CX-El-17, 1 or 2 hr chase); indeed, immunoextraction with a monoclonal anti-E2 which only recognized monomeric E2 (E2-1) did not precipitate either El or E2, (Fig. 5a), suggesting that it is the E2 in the heterooligomer that is processed. Extended chase times (Fig. 5b) showed that essentially all the initial form of E2 (E2J was converted to E2, after 4 hr. As with the C protein, only a small fraction of labeled El or E2 was released into the medium as virus, even after prolonged chase. The size and formation of the heterooligomer were further studied by using sucrose gradients. RV-infected cells were pulse-labeled and chased for 0, 1, or 2 hr, and the cells solubilized in lysis buffer. These lysates were analyzed on isokinetic gradients as for the C protein. Gradient fractions were immunoextracted with anti-E2 or anti-El antibodies and the immunoprecipitated proteins analyzed by SDS-PAGE. As can be seen in Fig. 6a, E2 and El were found in two adjacent regions. In one of these (peak at fraction 12) the two proteins coprecipitated, indicating the presence of the heterooligomer. By comparison with the sedimentation of the SFV heterodimer and the RV C protein homodimer it is clear that El and E2 are forming a heterodimer. About half of either protein was found to sedia
a-El-1
7
wE2ct
a-E2-1
E2 and El form a heterodimer Our studies on the virion membrane proteins had shown that El and E2 could be coprecipitated by antibodies to either protein (Fig. l), suggesting that El and E2 form a noncovalent oligomer of some kind. However, this complex could be the result of interactions in the virion envelope, and we therefore sought to determine at what stage oligomers formed in the infected cell. In pulse-chase studies we found that E2 and El were coprecipitated by either anti-El or anti-E2 antibodies after 30 min chase (Fig. 5a). We consistently found that El-17 (used here) and other anti-El monoclonal and polyclonal antibodies precipitated El from RV-infected cells poorly at early times after synthesis, suggesting that the epitopes recognized in the mature protein are formed some time after completion of its synthesis; since these antibodies did precipitate El in the absence of E2 (see below), the epitope(s) did not require the formation of the dimer itself. Surprisingly, since previous reports have suggested that E2 in infected cells does not show size heterogeneity (Bowden and Westaway, 1984) E2 showed a time-dependent conversion into the broad band typical of the virion E2 (E2,) (Fig. 5a); this was especially noticeable for the E2 that coprecipitated with El (Fig. 5a,
+E2i
b
Hours of chase
lysate
medium
024822M24822 -El 1
=v
--c
Hours of chase
lysate
medium
024822M24822
E2iW
FIG. 5. Oligomerization of E2 and El, (a) Thirty-five-millimeter plates of RV-infected B-Vero cells were pulse-labeled and chased for the indicated time. Lysates were divided into three aliquots and immunoextracted with monoclonals a-El -17 or a-E2-1, or polyclonal a-E2ct, as indicated. The immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography. (b) The experiment was performed essentially as in Fig. 2b, except that the lysate or medium was divided into two and immunoextracted with a-El-17 (top) or cY-E2ct (bottom). M, labeled virus marker.
BARON
816 a
1
3
5
7
9
11
13
15
17
AND
19 -El
anti-El
-E2
Oh anti-E2
7-E2
b
135 ,-i
anti-El
anti-E2
FIG. 6. (a) Sucrose velocity analysis of E2 and El in RV-infected cells. RV-infected B-Vero ceils were pulse-labeled and chased for 0, 1, or 2 hr as indicated. The cells were washed and lysed, and the lysates spun to remove the nuclei. Each lysate was divided into two, and each aliquot was analyzed on a 5-20% isokinetic gradient as described under Methods. For each time point, fractions from one gradient were immunoextracted with monoclonal a-El and the other with a-E2ct. The immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography. Sedimentation is from right to left. (b) [35S]Labeled RV was solubilized in phosphate lysis buffer and analyzed on isokinetic gradients. El and E2 were immunoprecipitated from the fractions and analyzed by SDS-PAGE and fluorography as In (a).
ment more slowly (fractions 14/l 5) and no coprecipitation was seen in this region. Further experiments (see below) showed that this is the sedimentation position of monomeric E2 or El under these conditions. The conversion of E2i to E2, was easily visible, but there was no apparent difference between the E2 that migrated in the monomer and dimer positions. While it is possible that E2-El dimers are dissociating under the conditions in the sucrose gradient, as has been observed for other noncovalently linked oligomers, it clearly cannot be stated with certainty that only the E2 in heterodimers is converted to E2,. When the membrane proteins of isolated, labeled RV were solubilized and analyzed on similar gradients, we again found that about half of each protein migrated as monomers, with the rest distributed as dimers and larger oligomers (Fig. 6b). All of these oligomeric forms appeared to contain
FORSELL
the same ratio of El to E2 and presumably represent various degrees of dissociation of the viral membrane envelope. The viral nucleocapsid did not appear to dissociate into its component dimers under these conditions, as all the C protein was found in the pellet at the bottom of the gradient (not shown). In order to investigate the interaction of E2 and El in the absence of the complex array of membrane proteins that is the virion, and to see what happened to these two proteins when they were expressed on their own, we again employed the recombinant vaccinia system. Control experiments showed that, when expressed together from cDNA, E2 and El could be coprecipitated, albeit less efficiently than from infected cells (not shown). Lysates from labeled, wE2El-infected cells were analyzed in sucrose velocity gradients and the fractions immunoextracted with anti-El or anti-E2 antibodies. Again, El was found in two adjacent regions (Fig. 7a; E2El), almost identical to those seen in RV-infected cells, although slightly better separated in these experiments. Some E2 also coprecipitated with El in the faster sedimenting material. No processing of E2 is visible in these experiments. In general we have found that the conversion of E2i to E2, is slower when the protein is expressed from cDNA (unpublished observations); the reason for this is not clear. Reextraction of the gradient with anti-E2ct showed that E2 also sedimented in the same two regions (Fig. 7b; E2El). The total population of E2 showed very little processing, but this is probably because the cells had been continuously labeled for the 2 hr, rather than pulsed and chased. This would lead to a predominance of recently synthesized E2. When E2 and E 1 were expressed separately a different picture emerged. E2, expressed alone, migrated primarily as a single peak of monomer (Fig. 7b; E2); El, expressed alone, migrated as two peaks (Fig. 7a; El), one of monomer, the second (fractions 8/9) larger than the heterodimer. Mixing studies, in which lysates from cells infected with wE2 or wE1 were mixed before analysis on gradients, showed identical patterns to those seen with each protein alone (Fig. 7a, 7b; El + E2), demonstrating that E2 and El have to be expressed together in order to show heterodimer formation and coprecipitation and to prevent E 1 from forming the large oligomer. The formation of this large oligomer of E 1 was further examined by pulse-chase experiments with wE1 -infected cells (Fig. 8). From these data it can clearly be seen that El is initially found as a monomer (peak fractions 14/l 5) and that this is slowly converted into the faster sedimenting form (t,,, x 90 min). No intermediate-sized oligomer of El was seen, although the appearance of El in fractions all the way to the bottom of
OLIGOMERISATION
a
OF
RV PROTEINS
817
b
13
5
7
9
11
13
15
17
19
13
-El
E2El
cE2
5
7
9
11
13
15
17
19
CEl
E2El
tE2
El
E2
El+E2
El +E2
FIG. 7. Sucrose velocity gradient analysis of E2 and El oligomers. RK,, cells were infected with wE2E1, wE2, or wE1 and labeled for 2 hr. The cells were solubilized and then spun at 16,000 rpm for 10 min to remove the nuclei, and the lysates analyzed on 5-20s linear sucrose gradients. Half of the lysates from wE2- and wE1 -infected cells were mixed prior to analysis on one gradient (El + E2), while the remaining aliquots were analyzed on separate gradients (El, E2). The fractions from the gradient were immunoextracted with (a) monoclonal anti-El-23 (another dimer-precipitating anti-El monoclonal, similar to anti-El-17) and/or(b) anti-E2ct. NB the E2El and (El + E2) gradient fractions were extracted successively with anti-El and anti-E2. Sedimentation is from right to left.
the gradient suggests that even higher order aggregates than the main one may be being formed.
DISCUSSION Very little is known about the structure of the RV virion, except for its overall size and shape. Like the other Togaviridae, it appears to be a spherical particle; EM studies have shown an approximately 60-nm viriot-r, with a 30- to 35-nm core. We have found that the C protein in the virion is entirely in the form of disulfidelinked C, dimers. The implications of capsid protein dimerization for the process of self-assembly have been discussed (Rossman, 1984); it is clear from this analysis that, for the commonest T = 3 and T = 4 symmetries, dimers need to be able to take on more than one conformation in the mature virion. The presence of covalent bonds between the subunits of a C protein dimer would be expected to constrain the flexibility at
1
3
5
7
9
11
13
1s
17
IO
Oh
lh
2h FIG. 8. Formation of the high order oligomer of El. wE1 -infected RK,, cells were labeled for 30 min and then chased in complete medium for the indicated time. Lysates from the labeled cells were analyzed on isokinetic sucrose gradients and the fractions from each gradient immunoextracted with anti-El monoclonal antibodies. Immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography. Sedimentation is from right to left.
the intersubunit interface, tending to lock the dimer in one conformation or the other, especially if there is more than one such bond. Assuming that the RV nucleocapsid has T = 3 symmetry (as has been found for the structurally related alphaviruses (Fuller, 1987) there may be more than one C, dimer, differing in their disulfide linkages. We have found that the C protein first forms noncovalent dimers, which appear to be spontaneously linked by one or more disulfide bonds on removal of the protein from the reducing environment of the cytoplasm. This formation of IAA-resistant dimers in the infected cell may prove to be a useful marker for the budding of the virion; in our pulse-chase studies this covalently linked C, was observed to accumulate in the cell before release into the medium, in accord with studies which have suggested intracellular budding of RV. The dimerization of the capsid does not require virion assembly nor the presence of the other structural proteins; preliminary studies have shown that removal of the carboxy-terminal hydrophobic membrane anchor (Suomalainen et a/., 1990) does not affect dimerization (Baron, unpublished). It is not known if the formation of the disulfide link in the dimer is essential for viral function; the recent development of a fulllength clone of RV (Dominguez et al., 1990) will allow such questions to be addressed. The studies reported here also show that the membrane glycoproteins E2 and El form a heterodimer when expressed together. The ratios of structural proteins in RV have been previously reported as 5:1:5 (El :E2:C) (Vaheri and Hovi, 1972; Waxham and Wolinsky, 198513) or 1 :l : 1 (Bowden and Westaway, 1984). In our own initial studies (Baron and Forsell, unpublished) we have found a ratio of 1: 1 for El :E2, which is in agreement with the results of Bowden and Westaway (1984) and also with our finding that the two membrane glycoproteins form a heterodimer. Formation of
818
BARON
AND
this dimer in the infected cell appears to precede transport to the Golgi, as shown by the coprecipitation of E2 with El before processing of the N-linked glycans on E2. The heterodimer also formed when the two proteins were expressed from cloned cDNA; less dimer, and less processed E2, was found under these conditions, which may reflect some other interactions occurring in the RV-infected cells. The difference is not simply that the RV studies were done in Vero cells and the w studies in RK,, cells, since control studies have shown even less E2, in w-infected Vero cells. lmmunofluorescence studies on COS cells transfected with plasmids from which the RV structural proteins are expressed have shown that if El is expressed without E2, it is not transported to the Golgi, but instead accumulates in large cytoplasmic vesicles which appear to be derived from the intermediate compartment between the ER and Golgi (Baron eta/., submitted for publication). This may be connected with the formation of the large (bigger than dimer) oligomer that we observed in our sucrose gradient studies. No other labeled protein was found to coprecipitate with El when it was expressed without E2, suggesting that the larger aggregate is a homooligomer of El. The nature of this oligomer is unknown. The influenza haemagglutinin has been shown to form high molecular weight aggregates when it is synthesized in such a way that it is misfolded (Hurtley et al., 1989); this process, however, is extremely rapid, aggregates forming as soon as the protein is synthesized, and the protein binds the Iumenal protein BiP (Hurtley et al., 1989). The aggregation of RV El is clearly different in that it occurs slowly and no other labeled protein is found in the aggregate. The delay in its formation suggests that this aggregation event occurs after the protein reaches the transitional compartment, but this will require further investigation. The presence of E2 prevents the appearance of this oligomer (the studies reported here) and allows the transport of El to the Golgi (Baron et al., submitted for publication). E2 remains monomeric when expressed alone. It can be transported to the Golgi in the absence of El, albeit less efficiently than in the dimer (Baron et a/., submitted for publication). The envelope proteins of a number of viruses, and the components of other oligomeric proteins, must assemble correctly before they can leave the endoplasmic reticulum (reviewed in Hurtley and Helenius, 1989; Klausner, 1989). While E2 and El from RV appear to be dependent on correct oligomerization with each other for (efficient) transport through the Golgi and to the plasma membrane, both proteins can be transported out of the ER when ex-
FORSELL
pressed alone. In this respect RV seems more to resemble the alphaviruses, such as Semliki Forest virus, the p62 protein of which is exported (inefficiently) when expressed alone, while the El protein requires the formation of a heterodimer with the companion p62 protein before it is transported to the Golgi (Lobigs et a/., 1990). It is not known if the El protein in this case also accumulates in the pre-Golgi transitional elements. Surface expression studies (Baron et al,, submitted for publication) have shown that RV El does not come to the surface when expressed alone, while E2 comes inefficiently; if the proteins are expressed together they are both found at the cell surface. Hence, the proteins that reach the Golgi also come to the surface. ACKNOWLEDGMENTS We thank J. Wolinsky and R. Pettersson for the generous gift of antibodies. Special thanks go to Henrik Garoff for providing lab space and facilities. and to him, Johanna Wahlberg, and Maarit Suomalainen for critical reading of the manuscript. This research was funded by Grant B88-12X-08272-OlA from the Swedish Medical Research Council (MFR), the Swedish National Board for Technical Development (STUF) (87.0275 OP), and the Swedish Natural Science Research Council (NFR)(B-BV 9353-3019).
REFERENCES ABERNATHY, E. S., WANG, C.. and FREY, T. K. (1990). Effect of antiviral antibody on maintenance of long-term rubella virus persistent infection in vero cells. /. L%o/. 64, 5 183-5187. BARON, M. D., and GAROFF, H. (1990). Mannosidase II and the 135. kDa Golgi-specific antigen recognized by monoclonal antibody 53FC3 are the same dimeric protein. /. Biol. Chem. 265, 19,92819,931. BARON, M. D., EEIEL, T.. and SUOMALAINEN, M. Intracellular transport of rubella virus structural proteins. Submitted for publication. BOWDEN, D. S., and WESTAWAY, E. G. (1984). Rubella virus: Structural and nonstructural proteins. /. Gen. Viral. 65, 933-943. CLARKE, D. M., Loo, T. W., HUI, I., CHONG, P., and GILLAM, S. (1987). Nucleotide sequence and in vitro expression of Rubella virus 24s subgenomic messenger RNA encoding the structural proteins El, E2 and C. Nucleic Acids Res. 15, 3041-3057. DOMINGUEZ, G., WANG, C. Y., and FREY, T. K. (1990). Sequence of the genome RNA of rubella virus: Evidence for genetic rearrangement during togavirus evolution. Virology 177, 225-238. FULLER. S. D. (1987). The T = 4 envelope of Sindbis virus is organized by interactions with a complementary T = 3 capsid. Cell 48, 923934. GAROFF, H., KONDOR-KOCH, C., and RIEDEL, H. (1982). Structure and assembly of alphaviruses. Curr. Top. Microbial. lmmunol. 99, l-50. HEMPHILL, M. L., FORNG, R., ABERNATHY, E. S., and FREY, T. K. (1988). Time course of virus-specific macromolecular synthesis during rubella infection in vero cells. virology 162, 66-75. HO-TERRY, L., and COHEN, A. (1980). Degradation of rubella virus envelope components. Arch. Viral. 65, l-l 3. HO-TERRY, L., and COHEN, A. (1982). Rubella virion polypeptides: Characterization by polyacrylamide gel electrophoresis, isoelectric focusing and peptide mapping. Arch. Viral. 72, 47-54.
OLIGOMERISATION HOBMAN, T. C., and GILLAM, S. (1989). In vitro and in viva expression of Rubella virus glycoprotein E2: The signal peptide is contained in the C-terminal region of capsid protein. virology 173, 241-250. HURTLEY, S. M., BOLE, D. G., HOOVER-LILY, H., and HELENIUS, A. (1989). Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP). 1. Cell Biol. 108, 21 17-2 126. HURTLEY, S. M., and HELENIUS, A. (1989). Protein oligomerization in the endoplasmic reticulum. Annu. Rev. Cell Biol. 5, 277-307. KLAUSNER, R. D. (1989). Architectual editing: Determining the fate of newly synthesized membrane proteins. New Biol. 1, 3-8. LOEIGS, M., ZHAO, H., and GAROFF, H. (1990). Function of Semliki Forest virus E3 peptide in virus assembly: Replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E 1.1. Viral. 84, 4346-4355. OKER-BLOM, C. (1984). The gene order for rubella virus structural proteins is NHZ-C-E2-El -COOH. J. Viral. 51, 964-973. OKER-BLOM, C., KALKKINEN, N., ~RI&NEN, L., and PETTERSSON, R. F. (1983). Rubella virus contains one capsid protein and three envelope glycoproteins, El, E2a, and E2b. 1. Vim/. 46, 964-973. OKER-BLOM, C., ULMANEN, I., ~RI&NEN, L., and PET~ERSSON, R. F. (1984). Rubella virus 40s genome RNA specifies a 24s subgenomlc mRNA that codes for a precursor to structural proteins. J. Virol. 49, 403-408. ROSSMAN, M. G. (1984). Constraints on the assembly of spherical virus particles. Virology 134, l-l 1. SIMONS, K., HELENIUS, A., and GAROFF, H. (1973). Solubilization of the membrane proteins from Semliki Forest virus with Triton X-100. J. Mol. Biol. 80, 119-l 33. SUOMALAINEN, M., GAROFF, H., and BARON, M. D. (1990). The E2
OF RV PROTEINS
819
signal sequence of Rubella virus remains part of the capsid protein and confers membrane association in vitro. J. Viral. 64, 55005509. TAKKINEN, K., VIDGREN, G., EKSTRAND, J., HELLMAN, U., KALKKINEN, N., WERNSTEDT, C., and PEITERSSON, R. (1988). Nucleotide sequence of the Rubella virus capsid protein gene reveals an unusually high G/C content. J. Gen. Viral. 69, 603-6 12. TOIVONEN, V., VAINIONP~, R., SALMI, A., and HWPI& T. (1983). Glycopolypeptides of Rubella virus: Brief report. Arch. Viral. 77, 91-95. TRUDEL, M.. NADON, F., COMTOIS. R., PAYMENT. P., BONNEAU, A. M.. and LECOMTE, J. (1982). Identification of Rubella virus structural proteins by immunoprecipitation. J. Viral. Methods 5, 191-l 97. VAHERI, A., and How, T. (1972). Structural proteins and subunits of Rubella virus. J. Viral. 9, 1 O-l 6. VIDGREN, G., TAKKINEN, K., KALKKINEN, N., K%RI~~INEN, L., and PETTERSSON, R. (1987). Nucleotide sequence of the genes coding for the membrane glycoproteins El and E2 of Rubella virus. J. Gen. Viral. 68, 2347-2357. WAHLBERG, J. M., BOERE, W. A., and GAROFF, H. (1989). The heterodimerit association between the membrane proteins of Semliki Forest virus changes its sensitivity to mildly acidic pH during virus maturation. J. Viral. 63, 4991-4997. WAXHAM, M. N., and WOLINSKY, J. S. (1983). lmmunochemical identification of Rubella virus hemagglutinin. Virology 126, 194-203. WAXHAM, M. N., and WOLINSKY, J. S. (1985a). Detailed immunologic analysis of the structural polypeptides of Rubella virus using monoclonal antibodies. Virology 143, 153-l 65. WAXHAM, M. N., and WOLINSKY, J. S. (1985b). A model of the structural organization of Rubella virion$. Rev. lnf. Dis. 7(suppl. 1): s133-s139.
185, 811-819
(1991)
Oligomerisation
of the Structural
MICHAEL Department
of Molecular
Biology,
Karolinska Received
D. BARON’** Institute May
Centre 20,
Proteins
of Rubella Virus
KERSTIN FORSELL
AND
for Biotechnology,
199 1; accepted
August
Blickagdngen 23,
6, S- 14 1 52 Huddinge.
Sweden
199 1
Rubella virus contains, in addition to its RNA genome, a nucleocapsid protein (C) and two membrane proteins (E2 and El). We have studied the association of these proteins during viral assembly and when expressed from cDNA constructs. The C protein was found to dimerize very shortly after synthesis; this dimer became disulfide-linked in the virion. Formation of the dimer was independent of the presence of other RV proteins. The membrane glycoproteins formed an E2El heterodimer, a minor fraction of which was also found to be disulfide-linked in the virion. This heterodimer also formed when the two proteins were coexpressed from cloned cDNA. Formation of the heterodimer preceded the transport of E2 to the Golgi, as judged by modification of the protein by Golgi-located enzymes. In the absence of E2, the El protein was slowly converted to high molecular weight aggregates. Q fQQi Academic press, hc.
INTRODUCTION
tions in most cell lines (Abernathy et a/., 1990). The C protein, unlike that of alphaviruses, is not an autoprotease and is cleaved from the following proteins by signal peptidase (Clarke et al., 1987; Hobman and Gillam, 1989) leaving the C protein membrane-associated (Suomalainen et al., 1990). In addition, the C protein in isolated virus has been reported to be a disulfide-linked dimer (Waxham and Wolinsky, 1983); a fraction of E2 and El were also observed to be in disulfidelinked dimers in the same work. No such covalent linkages have been seen in the alphaviruses. The relative amounts of the structural proteins in the isolated virus are in some doubt, having been reported as either (5: 1:5) (El :E2:C) (Vaheri and Hovi, 1972; Waxham and Wolinsky, 1985b) or 1 :l :l (Bowden and Westaway, 1984). We have been studying the synthesis and transport of the viral structural proteins expressed from cloned cDNA (Suomalainen et al., 1990; Baron et al., submitted for publication) and have attempted to elucidate how these proteins interact during assembly of the virus in infected cells. We have found that the C protein spontaneously forms homodimers even if expressed in the absence of other RV proteins: these dimers appear to become disulfide-linked after virus budding or if exposed to an oxidizing environment. The El and E2 proteins form heterodimers; this oligomerization appears to be necessary to prevent later aggregation of El and for efficient transport of E2 and El from the ER to the Golgi.
Rubella virus (RV) is a membrane-enveloped, positive-stranded RNA virus, the only member of the genus Rubivirus in the Togavirus family. The virus contains three structural proteins (Bowden and Westaway, 1984; Ho-Terry and Cohen, 1980, 1982; Oker-Blom et a/., 1983; Toivonen et a/., 1983; Trudel et a/., 1982; Vaheri and Hovi, 1972; Waxham and Wolinsky, 1983) with molecular weights variously reported as between 33 and 38 kDa (the capsid or C protein), between 53 and 62 kDa (membrane protein El), and a broad band covering about 6 kDa in the region 43-51 kDa (membrane protein E2). The C protein forms a shell surrounding the genome (Vaheri and Hovi, 1972). This nucleocapsid is, in turn, surrounded by the membrane envelope, which contains El and E2 (Vaheri and Hovi, 1972; Waxham and Wolinsky, 1985b). The structural proteins are translated from a single, subgenomic message as a polyprotein in the order CE2-El (Oker-Blom, 1984). The subgenomic message is derived from the 3’-end of the viral genome (OkerBlom et al,, 1984); the 5’-end appears to encode a number of nonstructural proteins involved in viral replication (Dominguez et al., 1990). In these respects, RV resembles the alphaviruses (reviewed in Garoff eta/., 1982) in its replication strategy and (to some extent) in its structure. However, RV differs from the alphaviruses in several aspects of both replication and structure. Replication is much slower than in alphaviruses (Hemphill et a/., 1988) and the virus is able to form persistent infec’ To whom reprint ’ Present address: Ash Road, Pirbright,
requests Institute Woking,
should be addressed. for Animal Health Pirbright Surrey GU24 ONF. UK.
METHODS Cells and virus B-Vero and RK,, cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal
Laboratory,
811
0042.6822/91
$3.00
Copyright Q 1991 by Academic Press, Inc All rights of reproduction in any form reserved.
812
BARON AND FORSELL
calf serum, 10 mll/l HEPES (AI-2-hydroxyethylpiperazine-N’-2-ethane sulfonic acid), 100 units liter-’ penicillin, 100 pg liter-’ streptomycin. RV infection was as described (Suomalainen et a/., 1990). The Therien strain of RV was used throughout; the cloning of the structural region of this strain has been described (Suomalainen et a/., 1990; Takkinen et a/., 1988; Vidgren et a/., 1987). The construction of recombinant vaccinia virus expressing the complete structural protein polyprotein (wRVS4), E2(wE2), El(wEl), or the E2El part of the polyprotein (wE2El) is described elsewhere (Baron et al., submitted for publication). The same techniques were used to construct the recombinant wC17, which contains a cDNA clone containing the first 1500 bases of the structural protein coding sequence (Suomalainen et a/., 1990). All media were from GIBCO; all cell culture plasticware were from Costar or NUNC (Denmark). Radiolabeling
of RV and RV-infected
cells
Radiolabeled virus was prepared as previously described (Suomalainen et al., 1990). RV-infected cells were labeled 24 hr postinfection; for studies on infected cells, 35-mm dishes of cells were routinely starved of methionine by incubating for 30 min in methionine-free MEM containing 10 mM HEPES and 0.2% bovine serum albumin. This medium was then replaced by 0.5 ml of the same medium containing 50 &i [35S]methionine (SJl515, Amersham, UK) and incubated (unless otherwise indicated) for 15 min (pulse). The labeling medium was removed and replaced with 2 ml of complete medium for the indicated time (chase time). Labeled cells were washed once with cold PBS (15 mM NaPO,, pH 7.4, 150 mM NaCI) and then solubilized in 250 ~1 of Tris lysis buffer (50 mll/l Tris-Cl, pH 7.4, 150 mM NaCI, 2 mM EDTA, 1% (v/v) Nonidet P-40 (NP-40) 20 pg ml-’ phenylmethylsulfonyl fluoride (PMSF)) or in the same volume of phosphate lysis buffer (50 mM NaPO,, pH 7.4, 0.4 1\/1 NaCI, 5 rnM EDTA, 10 mM ATP, 1% NP-40, 20 pg ml-’ PMSF). Where indicated, the lysis buffer contained 10 mM iodoacetamide (IAA). Lysates were spun for 2 hr at 17,000 rpm in a Beckman JAI 8.1 rotor, and the supernatants used for immunoprecipitation (Wahlberg et a/., 1989). Samples were analyzed by polyacrylamide gel electrophoresis after denaturation in sodium dodecyl sulphate (SDS-PAGE) as previously described (Baron and Garoff, 1990). Quantification of immunoprecipitated proteins was performed by excising gel slices and determining the [35S] content by scintillation counting (Wahlberg et al., 1989).
Antibodies The antibodies used in these studies were: polyclonal rabbit anti-RV(the gift of R. Pettersson, Ludwig Institute for Cancer Research, Stockholm, Sweden); polyclonal rabbit anti-E2ct, a polyclonal serum that recognizes the last 12 amino acids of E2 (Baron eT al., submitted for publication); monoclonal anti-C-l, antiE2-1, anti-El -10, 17, 19 and 23 (the generous gift of J. Wolinsky, University of Texas Health Science Center, Houston, TX). Labeling
of vaccinia-infected
cells
Confluent or near confluent monolayers of RK,, cells were infected with recombinant or wild-type vaccinia virus at a multiplicity of infection of -5. The cells were labeled as for RV-infected cells but 5 hr postinfection and using a 30-min pulse. Labeled cells were lysed and RV proteins immunoprecipitated as normal. Sucrose velocity gradients Samples (100-300 ~1) were layered onto 1 1.5-ml 5-20% linear gradients of sucrose in 50 mM Tris-Cl, pH 7.5, 100 mM NaCI, 2 mlM EDTA, 0.1% NP-40 and centrifuged for 28-30 hr at 40,000 rpm in a Beckman SW41 rotor. Fractions (50 drops) were collected from the bottom of the tube and labeled proteins immunoprecipitated with various antibodies. RESULTS Virion C protein
is a disulfide-linked
dimer
It has been reported (Waxham and Wolinsky, 1983) that there are disulfide linkages between the structural proteins in the RV virion. However, in those studies no attempt was made to prevent the formation of disulfide bonds between free cysteine residues during solubilization and immunoprecipitation. In order to confirm this observation, therefore, we first pretreated the virus with iodoacetamide and then solubilized it in the presence of the same reagent. The viral proteins were precipitated with the respective monospecific antibodies and the resultant proteins analyzed by SDS-PAGE after denaturing in the presence or absence of dithiothreitol (Fig. 1). It is clear from these studies that the C protein in the virus is present as a disulfide-linked dimer, since it is found entirely in this form (C,) despite the presence of a high concentration of IAA during lysis (Fig. 1, lane 4). The membrane glycoprotein El, but not E2, showed increased mobility in the gel if disulfide bond reduction was omitted, as has previously been reported (Waxham and Wolinsky, 1983); this difference
OLIGOMERISATION +DR 123456
-DTT
FIG. 1. Disulfide bond linkages in RV. [35S]Methionine-labeled RV was solubilized in the presence of 10 mM IAA and the proteins were immunoprecipitated by using monoclonal antibodies anti-C-l (lanes 1 and 4) or anti-El -17 (lanes 2 and 5) or a polyclonal antibody to the carboxy-terminal of E2 (anti-E2ct) (lanes 3 and 6). Half of each im,munoprecipitate was denatured in SDS in the presence (lanes l-3) and half in the absence (lanes 4-6) of 50 mM dithiothreitol (DTT). The samples were analyzed by SDS-PAGE and fluorography. The two panels are from different parts of the same gel, exposed identically.
in mobility is probably due to the presence of intrachain disulfide bonds in El. In addition, a fraction of El and E2 was found as the reported disulfide-linked dimers (d) (Fig. 1, lanes 5 and 6). Although these have been described as El-El and El-E2 dimers (Waxham and Wolinsky, 1983) they were both precipitated in equal proportions by either anti-El or anti-E2; this suggests that these dimers may be part of larger complexes containing both El and E2, and therefore precipitated by either antibody. It is clear from Fig. 1 that El and E2 are coprecipitated by either anti-El or anti-E2 even when the proteins are not covalently linked. This coprecipitation has been reported (although not discussed) before with some anti-El monoclonals (Waxham and Wolinsky, 1983, 1985a,b); it is abolished by denaturing El and E2 before immunoprecipitation (Waxham and Wolinsky, 1985a; our own unpublished observations). Anti-E2 monoclonals failed to coprecipitate noncovalently linked El and precipitated virion E2 poorly; denaturation of virion proteins in SDS resulted in improved precipitation of E2 by monoclonal anti-E22 (not shown). These data suggest that the epitope seen by this monoclonal is at least partly concealed in the heterodimer. Nevertheless, it must be a strong epitope since all of the anti-E2 monoclonals we tested showed the same phenotype. The RV C protein forms noncovalent
homodimers
The C protein is cytoplasmic, so disulfide links cannot form until the protein moves out of the cytoplasmic compartment; this will happen when the virus buds, whether that budding occurs at the plasma membrane or at an intracellular membrane. We therefore performed pulse-chase studies on RV-infected B-Vero
OF RV PROTEINS
813
cells to determine the time after synthesis when disulfide-linked C protein could be seen. We were surprised to find that, when cells were solubilized in our normal Tris-based lysis buffer, C protein appeared entirely as a disulfide-linked dimer even after only the 15-min labeling period (e.g. Fig. 2a). However, when 10 mM IAA was included in the lysis buffer, no disulfide-linked dimer was seen (Fig. 2a). The formation of disulfidelinked C protein in the cell therefore appeared to be artifactual, apparently promoted by oxidizing conditions in the lysis buffer. Those conditions were specific to the Tris-based lysis buffer; when we tried a phosphate-based lysis buffer also in use in our lab, no covalently linked dimer was seen whether or not IM was present during lysis (not shown). We therefore used the phosphate-based lysis buffer in all other experiments, except when we specifically wanted to use the formation of the disulfide bond as an assay for dimerization. The efficiency of disulfide bond formation between C protein monomers was such that it was clear that the C protein in the cytoplasm must already be in a noncovalent dimer or other oligomer. (Note that the concentration of IAA used was sufficient to suppress entirely the formation of disulfide bonds between the proteins in the dimer, and we are therefore confident that this concentration was sufficient to inhibit spontaneous disulfide bond formation during lysis of the isolated virus.) When RV-infected cells were pulse-labeled and chased for up to 22 hr (Fig. 2b), it became possible to observe the formation of MA-resistant covalent dimers (C,); these were detectable at 2 hr chase and clearly visible at 4 hr chase. At this time, labeled C protein was detected in medium virus. Quantification showed that the rate of release of C protein into the medium was roughly the same as the rate of loss of C, from the cell between 8 and 22 hr, suggesting that the C, in the cell is a precursor to C protein in released virus, which was, as we had shown above, all in the C, state. The labeled C protein in the cell remained predominantly in the noncovalently linked form, although it is not clear at this stage whether this is all free C protein or includes intracellular virus, since we have no measure of the rate at which virion C protein becomes disulfide-linked after budding. The amount of labeled C protein released into the medium after 22 hr was only a small fraction of the total labeled during the pulse. We also consistently found an apparently disulfidelinked form of C protein (marked C*, Fig. 2b) at all time points. This form migrated more slowly on SDS-PAGE than the viral form of the C protein dimer and was never seen in the released virion; unlike C,, it disappeared from the cells at about the same rate as noncovalently linked C protein (fllP = 8 hr). The state of the C protein
BARON
814
AND
FORSELL Cell lysate
b chase
a chase(min)
+Dll RV
0
60
60
RV
0
60
2
0
4
Medium
8
22
C’ --c
-DTT 0
(h)
0
RV
2
4
8
22
4-c2 - DTT
60
-c2 +DlT -c FIG. 2. Drmerization of C protein. (a) Thirty-five-millimeter plates of RV-infected B-Vero cells were labeled as described under Methods and chased for the indicated times. The cells were solubilized in Tris lysis buffer with or without 10 mM IAA and the lysate was extracted with anti-c-l, The immunoprecipitated proteins were analyzed by SDS-PAGE after denaturing in the presence or absence of 50 mM DTT. (b) Sixty-millimeter plates of RV-infected cells were labeled with [35S]methionine and chased for the indicated trmes. after which the cells were solubilized in the presence of 10 mM WA. At 2, 4. 8. and 22 hr of chase the medium was also collected, and virus was pelleted by centrifugation and solubilized. C protein was immunoprecipitated from the cell lysates and analyzed by SDS-PAGE after denaturing in the presence or absence of 50 mM DTT. The positions of the monomeric C protein, the C, and C* dimers, and the 70.kDa protein (P) are shown. RV, labeled virus marker. Note that the Medium part of the gel was exposed to X-ray film for four times as long as the cell lysate part
in C*, and its importance in the assembly of the virus, is unclear. It is possible that it is an intermediate in the formation of the virion, but equally possible that it is a dead-end, nonproductive form of C protein, possibly attached to some other, unlabeled protein. The initial time point (0 hr chase) always contained a high molecular weight protein (marked P; Fig. 2b) of apparent molecular weight 70 kDa. This might be incompletely processed precursor polyprotein or a cell protein coprecipitating with the C protein. Preliminary experiments suggest that it is not one of the hsp70 family, since the recovery of the protein is not affected by the presence or absence of Mg/ATP in the lysis buffer or the enzyme apyrase; recovery of P is enhanced by the presence of IAA in the lysis buffer (not shown). In order to determine whether the appearance of disulfide-linked C protein was a function of virion formation or of the C protein alone, we used recombinant vaccinia virus expressing either the whole structural polyprotein (wRVS4) or the capsid plus the first half of E2 (wC17; the E2 segment was included to ensure an as near normal as possible interaction of the C protein with the ER membrane). These viruses were used to infect RK,, cells, a common host for RV that tolerated vaccinia infection better than B-Vero cells did. The infected cells were labeled, chased for 0 or 4 hr, and analyzed for the presence of oligomeric C protein by lysis in Tris lysis buffer with and without IF% As can be seen in Fig. 3, lysis in the absence of IAA allowed essentially all the C protein to become disulfide-linked, whereas the presence of IAA in the lysis buffer prevented the formation of covalent bonds between C protein monomers. In the infected cell, IA/-resistant dimers were visible after 2-4 hr chase (Fig. 2). When the C protein was
expressed from cDNA, however, no such covalent dimer was seen, even after 4 hr chase. The dimerization of the C protein, therefore, appears to occur spontaneously, with or without virion formation, and in the presence or absence of the rest of the structural proteins; the formation of IAA-resistant dimers, on the other hand, appears to occur only during productive infection with RV. Sucrose velocity gradient analysis of the C protein expressed from wRVS4 (Fig. 4) was used to determine the size of the native oligomer. Labeled, wRVS4-infected cells were lysed in the presence or absence of IAA; C protein migrated in the same position on the gradient (fractions 12/l 3) whether or not disulfide bonds were allowed to form, indicating that the olivvc17
IAA Chase
-73 +-+-+-+-
(h)
+DlT
-DlT
FIG. 3. Dimerization of C protein expressed from recombinant vaccinia virus. RK,, cells were infected with wC17 or wRVS4 and labeled with [?]methionine as described under Methods. After 0 or 4 hr of chase, cells were solubilized in the presence or absence of 10 mM IA4 and C protein was immunoprecipitated. The precipitated protein was analyzed by SDS-PAGE after denaturing in the presence or absence of 50 mM DlT. RV, labeled virus marker.
OLIGOMERISATION 2
4
6
8
10
12
14
16
18
-C
-c2
FIG. 4. Sucrose velocity gradient analysis of C protein oligomer. WRVSCinfected RK,, cells were labeled for 2 hr and lysed in the presence or absence of 10 mM IAA. The cells were solubilized and then spun at 16,000 rpm for 10 min to remove the nuclei, and the lysates analyzed on 5-20s linear sucrose gradients as described under Methods. C protein was immunoprecipitated from each fraction and the immunoprecipitates were analyzed on SDS-PAGE after denaturation in the absence of DTT. Sedimentation is from right to left.
gomer is stable; the C protein migrated only slightly slower than the 4.5 S Semliki Forest virus spike protein heterodimer (Simons et a/., 1973; Wahlberg et al., 1989) which was used as a convenient marker, and migrated at fraction 1 1 in these gradients. From these data we conclude that the C protein exists as a dimer and that this dimer forms spontaneously both in infected cells and when the C protein is expressed from cDNA.
OF
RV PROTEINS
815
CX-El-17, 1 or 2 hr chase); indeed, immunoextraction with a monoclonal anti-E2 which only recognized monomeric E2 (E2-1) did not precipitate either El or E2, (Fig. 5a), suggesting that it is the E2 in the heterooligomer that is processed. Extended chase times (Fig. 5b) showed that essentially all the initial form of E2 (E2J was converted to E2, after 4 hr. As with the C protein, only a small fraction of labeled El or E2 was released into the medium as virus, even after prolonged chase. The size and formation of the heterooligomer were further studied by using sucrose gradients. RV-infected cells were pulse-labeled and chased for 0, 1, or 2 hr, and the cells solubilized in lysis buffer. These lysates were analyzed on isokinetic gradients as for the C protein. Gradient fractions were immunoextracted with anti-E2 or anti-El antibodies and the immunoprecipitated proteins analyzed by SDS-PAGE. As can be seen in Fig. 6a, E2 and El were found in two adjacent regions. In one of these (peak at fraction 12) the two proteins coprecipitated, indicating the presence of the heterooligomer. By comparison with the sedimentation of the SFV heterodimer and the RV C protein homodimer it is clear that El and E2 are forming a heterodimer. About half of either protein was found to sedia
a-El-1
7
wE2ct
a-E2-1
E2 and El form a heterodimer Our studies on the virion membrane proteins had shown that El and E2 could be coprecipitated by antibodies to either protein (Fig. l), suggesting that El and E2 form a noncovalent oligomer of some kind. However, this complex could be the result of interactions in the virion envelope, and we therefore sought to determine at what stage oligomers formed in the infected cell. In pulse-chase studies we found that E2 and El were coprecipitated by either anti-El or anti-E2 antibodies after 30 min chase (Fig. 5a). We consistently found that El-17 (used here) and other anti-El monoclonal and polyclonal antibodies precipitated El from RV-infected cells poorly at early times after synthesis, suggesting that the epitopes recognized in the mature protein are formed some time after completion of its synthesis; since these antibodies did precipitate El in the absence of E2 (see below), the epitope(s) did not require the formation of the dimer itself. Surprisingly, since previous reports have suggested that E2 in infected cells does not show size heterogeneity (Bowden and Westaway, 1984) E2 showed a time-dependent conversion into the broad band typical of the virion E2 (E2,) (Fig. 5a); this was especially noticeable for the E2 that coprecipitated with El (Fig. 5a,
+E2i
b
Hours of chase
lysate
medium
024822M24822 -El 1
=v
--c
Hours of chase
lysate
medium
024822M24822
E2iW
FIG. 5. Oligomerization of E2 and El, (a) Thirty-five-millimeter plates of RV-infected B-Vero cells were pulse-labeled and chased for the indicated time. Lysates were divided into three aliquots and immunoextracted with monoclonals a-El -17 or a-E2-1, or polyclonal a-E2ct, as indicated. The immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography. (b) The experiment was performed essentially as in Fig. 2b, except that the lysate or medium was divided into two and immunoextracted with a-El-17 (top) or cY-E2ct (bottom). M, labeled virus marker.
BARON
816 a
1
3
5
7
9
11
13
15
17
AND
19 -El
anti-El
-E2
Oh anti-E2
7-E2
b
135 ,-i
anti-El
anti-E2
FIG. 6. (a) Sucrose velocity analysis of E2 and El in RV-infected cells. RV-infected B-Vero ceils were pulse-labeled and chased for 0, 1, or 2 hr as indicated. The cells were washed and lysed, and the lysates spun to remove the nuclei. Each lysate was divided into two, and each aliquot was analyzed on a 5-20% isokinetic gradient as described under Methods. For each time point, fractions from one gradient were immunoextracted with monoclonal a-El and the other with a-E2ct. The immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography. Sedimentation is from right to left. (b) [35S]Labeled RV was solubilized in phosphate lysis buffer and analyzed on isokinetic gradients. El and E2 were immunoprecipitated from the fractions and analyzed by SDS-PAGE and fluorography as In (a).
ment more slowly (fractions 14/l 5) and no coprecipitation was seen in this region. Further experiments (see below) showed that this is the sedimentation position of monomeric E2 or El under these conditions. The conversion of E2i to E2, was easily visible, but there was no apparent difference between the E2 that migrated in the monomer and dimer positions. While it is possible that E2-El dimers are dissociating under the conditions in the sucrose gradient, as has been observed for other noncovalently linked oligomers, it clearly cannot be stated with certainty that only the E2 in heterodimers is converted to E2,. When the membrane proteins of isolated, labeled RV were solubilized and analyzed on similar gradients, we again found that about half of each protein migrated as monomers, with the rest distributed as dimers and larger oligomers (Fig. 6b). All of these oligomeric forms appeared to contain
FORSELL
the same ratio of El to E2 and presumably represent various degrees of dissociation of the viral membrane envelope. The viral nucleocapsid did not appear to dissociate into its component dimers under these conditions, as all the C protein was found in the pellet at the bottom of the gradient (not shown). In order to investigate the interaction of E2 and El in the absence of the complex array of membrane proteins that is the virion, and to see what happened to these two proteins when they were expressed on their own, we again employed the recombinant vaccinia system. Control experiments showed that, when expressed together from cDNA, E2 and El could be coprecipitated, albeit less efficiently than from infected cells (not shown). Lysates from labeled, wE2El-infected cells were analyzed in sucrose velocity gradients and the fractions immunoextracted with anti-El or anti-E2 antibodies. Again, El was found in two adjacent regions (Fig. 7a; E2El), almost identical to those seen in RV-infected cells, although slightly better separated in these experiments. Some E2 also coprecipitated with El in the faster sedimenting material. No processing of E2 is visible in these experiments. In general we have found that the conversion of E2i to E2, is slower when the protein is expressed from cDNA (unpublished observations); the reason for this is not clear. Reextraction of the gradient with anti-E2ct showed that E2 also sedimented in the same two regions (Fig. 7b; E2El). The total population of E2 showed very little processing, but this is probably because the cells had been continuously labeled for the 2 hr, rather than pulsed and chased. This would lead to a predominance of recently synthesized E2. When E2 and E 1 were expressed separately a different picture emerged. E2, expressed alone, migrated primarily as a single peak of monomer (Fig. 7b; E2); El, expressed alone, migrated as two peaks (Fig. 7a; El), one of monomer, the second (fractions 8/9) larger than the heterodimer. Mixing studies, in which lysates from cells infected with wE2 or wE1 were mixed before analysis on gradients, showed identical patterns to those seen with each protein alone (Fig. 7a, 7b; El + E2), demonstrating that E2 and El have to be expressed together in order to show heterodimer formation and coprecipitation and to prevent E 1 from forming the large oligomer. The formation of this large oligomer of E 1 was further examined by pulse-chase experiments with wE1 -infected cells (Fig. 8). From these data it can clearly be seen that El is initially found as a monomer (peak fractions 14/l 5) and that this is slowly converted into the faster sedimenting form (t,,, x 90 min). No intermediate-sized oligomer of El was seen, although the appearance of El in fractions all the way to the bottom of
OLIGOMERISATION
a
OF
RV PROTEINS
817
b
13
5
7
9
11
13
15
17
19
13
-El
E2El
cE2
5
7
9
11
13
15
17
19
CEl
E2El
tE2
El
E2
El+E2
El +E2
FIG. 7. Sucrose velocity gradient analysis of E2 and El oligomers. RK,, cells were infected with wE2E1, wE2, or wE1 and labeled for 2 hr. The cells were solubilized and then spun at 16,000 rpm for 10 min to remove the nuclei, and the lysates analyzed on 5-20s linear sucrose gradients. Half of the lysates from wE2- and wE1 -infected cells were mixed prior to analysis on one gradient (El + E2), while the remaining aliquots were analyzed on separate gradients (El, E2). The fractions from the gradient were immunoextracted with (a) monoclonal anti-El-23 (another dimer-precipitating anti-El monoclonal, similar to anti-El-17) and/or(b) anti-E2ct. NB the E2El and (El + E2) gradient fractions were extracted successively with anti-El and anti-E2. Sedimentation is from right to left.
the gradient suggests that even higher order aggregates than the main one may be being formed.
DISCUSSION Very little is known about the structure of the RV virion, except for its overall size and shape. Like the other Togaviridae, it appears to be a spherical particle; EM studies have shown an approximately 60-nm viriot-r, with a 30- to 35-nm core. We have found that the C protein in the virion is entirely in the form of disulfidelinked C, dimers. The implications of capsid protein dimerization for the process of self-assembly have been discussed (Rossman, 1984); it is clear from this analysis that, for the commonest T = 3 and T = 4 symmetries, dimers need to be able to take on more than one conformation in the mature virion. The presence of covalent bonds between the subunits of a C protein dimer would be expected to constrain the flexibility at
1
3
5
7
9
11
13
1s
17
IO
Oh
lh
2h FIG. 8. Formation of the high order oligomer of El. wE1 -infected RK,, cells were labeled for 30 min and then chased in complete medium for the indicated time. Lysates from the labeled cells were analyzed on isokinetic sucrose gradients and the fractions from each gradient immunoextracted with anti-El monoclonal antibodies. Immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography. Sedimentation is from right to left.
the intersubunit interface, tending to lock the dimer in one conformation or the other, especially if there is more than one such bond. Assuming that the RV nucleocapsid has T = 3 symmetry (as has been found for the structurally related alphaviruses (Fuller, 1987) there may be more than one C, dimer, differing in their disulfide linkages. We have found that the C protein first forms noncovalent dimers, which appear to be spontaneously linked by one or more disulfide bonds on removal of the protein from the reducing environment of the cytoplasm. This formation of IAA-resistant dimers in the infected cell may prove to be a useful marker for the budding of the virion; in our pulse-chase studies this covalently linked C, was observed to accumulate in the cell before release into the medium, in accord with studies which have suggested intracellular budding of RV. The dimerization of the capsid does not require virion assembly nor the presence of the other structural proteins; preliminary studies have shown that removal of the carboxy-terminal hydrophobic membrane anchor (Suomalainen et a/., 1990) does not affect dimerization (Baron, unpublished). It is not known if the formation of the disulfide link in the dimer is essential for viral function; the recent development of a fulllength clone of RV (Dominguez et al., 1990) will allow such questions to be addressed. The studies reported here also show that the membrane glycoproteins E2 and El form a heterodimer when expressed together. The ratios of structural proteins in RV have been previously reported as 5:1:5 (El :E2:C) (Vaheri and Hovi, 1972; Waxham and Wolinsky, 198513) or 1 :l : 1 (Bowden and Westaway, 1984). In our own initial studies (Baron and Forsell, unpublished) we have found a ratio of 1: 1 for El :E2, which is in agreement with the results of Bowden and Westaway (1984) and also with our finding that the two membrane glycoproteins form a heterodimer. Formation of
818
BARON
AND
this dimer in the infected cell appears to precede transport to the Golgi, as shown by the coprecipitation of E2 with El before processing of the N-linked glycans on E2. The heterodimer also formed when the two proteins were expressed from cloned cDNA; less dimer, and less processed E2, was found under these conditions, which may reflect some other interactions occurring in the RV-infected cells. The difference is not simply that the RV studies were done in Vero cells and the w studies in RK,, cells, since control studies have shown even less E2, in w-infected Vero cells. lmmunofluorescence studies on COS cells transfected with plasmids from which the RV structural proteins are expressed have shown that if El is expressed without E2, it is not transported to the Golgi, but instead accumulates in large cytoplasmic vesicles which appear to be derived from the intermediate compartment between the ER and Golgi (Baron eta/., submitted for publication). This may be connected with the formation of the large (bigger than dimer) oligomer that we observed in our sucrose gradient studies. No other labeled protein was found to coprecipitate with El when it was expressed without E2, suggesting that the larger aggregate is a homooligomer of El. The nature of this oligomer is unknown. The influenza haemagglutinin has been shown to form high molecular weight aggregates when it is synthesized in such a way that it is misfolded (Hurtley et al., 1989); this process, however, is extremely rapid, aggregates forming as soon as the protein is synthesized, and the protein binds the Iumenal protein BiP (Hurtley et al., 1989). The aggregation of RV El is clearly different in that it occurs slowly and no other labeled protein is found in the aggregate. The delay in its formation suggests that this aggregation event occurs after the protein reaches the transitional compartment, but this will require further investigation. The presence of E2 prevents the appearance of this oligomer (the studies reported here) and allows the transport of El to the Golgi (Baron et al., submitted for publication). E2 remains monomeric when expressed alone. It can be transported to the Golgi in the absence of El, albeit less efficiently than in the dimer (Baron et a/., submitted for publication). The envelope proteins of a number of viruses, and the components of other oligomeric proteins, must assemble correctly before they can leave the endoplasmic reticulum (reviewed in Hurtley and Helenius, 1989; Klausner, 1989). While E2 and El from RV appear to be dependent on correct oligomerization with each other for (efficient) transport through the Golgi and to the plasma membrane, both proteins can be transported out of the ER when ex-
FORSELL
pressed alone. In this respect RV seems more to resemble the alphaviruses, such as Semliki Forest virus, the p62 protein of which is exported (inefficiently) when expressed alone, while the El protein requires the formation of a heterodimer with the companion p62 protein before it is transported to the Golgi (Lobigs et a/., 1990). It is not known if the El protein in this case also accumulates in the pre-Golgi transitional elements. Surface expression studies (Baron et al,, submitted for publication) have shown that RV El does not come to the surface when expressed alone, while E2 comes inefficiently; if the proteins are expressed together they are both found at the cell surface. Hence, the proteins that reach the Golgi also come to the surface. ACKNOWLEDGMENTS We thank J. Wolinsky and R. Pettersson for the generous gift of antibodies. Special thanks go to Henrik Garoff for providing lab space and facilities. and to him, Johanna Wahlberg, and Maarit Suomalainen for critical reading of the manuscript. This research was funded by Grant B88-12X-08272-OlA from the Swedish Medical Research Council (MFR), the Swedish National Board for Technical Development (STUF) (87.0275 OP), and the Swedish Natural Science Research Council (NFR)(B-BV 9353-3019).
REFERENCES ABERNATHY, E. S., WANG, C.. and FREY, T. K. (1990). Effect of antiviral antibody on maintenance of long-term rubella virus persistent infection in vero cells. /. L%o/. 64, 5 183-5187. BARON, M. D., and GAROFF, H. (1990). Mannosidase II and the 135. kDa Golgi-specific antigen recognized by monoclonal antibody 53FC3 are the same dimeric protein. /. Biol. Chem. 265, 19,92819,931. BARON, M. D., EEIEL, T.. and SUOMALAINEN, M. Intracellular transport of rubella virus structural proteins. Submitted for publication. BOWDEN, D. S., and WESTAWAY, E. G. (1984). Rubella virus: Structural and nonstructural proteins. /. Gen. Viral. 65, 933-943. CLARKE, D. M., Loo, T. W., HUI, I., CHONG, P., and GILLAM, S. (1987). Nucleotide sequence and in vitro expression of Rubella virus 24s subgenomic messenger RNA encoding the structural proteins El, E2 and C. Nucleic Acids Res. 15, 3041-3057. DOMINGUEZ, G., WANG, C. Y., and FREY, T. K. (1990). Sequence of the genome RNA of rubella virus: Evidence for genetic rearrangement during togavirus evolution. Virology 177, 225-238. FULLER. S. D. (1987). The T = 4 envelope of Sindbis virus is organized by interactions with a complementary T = 3 capsid. Cell 48, 923934. GAROFF, H., KONDOR-KOCH, C., and RIEDEL, H. (1982). Structure and assembly of alphaviruses. Curr. Top. Microbial. lmmunol. 99, l-50. HEMPHILL, M. L., FORNG, R., ABERNATHY, E. S., and FREY, T. K. (1988). Time course of virus-specific macromolecular synthesis during rubella infection in vero cells. virology 162, 66-75. HO-TERRY, L., and COHEN, A. (1980). Degradation of rubella virus envelope components. Arch. Viral. 65, l-l 3. HO-TERRY, L., and COHEN, A. (1982). Rubella virion polypeptides: Characterization by polyacrylamide gel electrophoresis, isoelectric focusing and peptide mapping. Arch. Viral. 72, 47-54.
OLIGOMERISATION HOBMAN, T. C., and GILLAM, S. (1989). In vitro and in viva expression of Rubella virus glycoprotein E2: The signal peptide is contained in the C-terminal region of capsid protein. virology 173, 241-250. HURTLEY, S. M., BOLE, D. G., HOOVER-LILY, H., and HELENIUS, A. (1989). Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP). 1. Cell Biol. 108, 21 17-2 126. HURTLEY, S. M., and HELENIUS, A. (1989). Protein oligomerization in the endoplasmic reticulum. Annu. Rev. Cell Biol. 5, 277-307. KLAUSNER, R. D. (1989). Architectual editing: Determining the fate of newly synthesized membrane proteins. New Biol. 1, 3-8. LOEIGS, M., ZHAO, H., and GAROFF, H. (1990). Function of Semliki Forest virus E3 peptide in virus assembly: Replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E 1.1. Viral. 84, 4346-4355. OKER-BLOM, C. (1984). The gene order for rubella virus structural proteins is NHZ-C-E2-El -COOH. J. Viral. 51, 964-973. OKER-BLOM, C., KALKKINEN, N., ~RI&NEN, L., and PETTERSSON, R. F. (1983). Rubella virus contains one capsid protein and three envelope glycoproteins, El, E2a, and E2b. 1. Vim/. 46, 964-973. OKER-BLOM, C., ULMANEN, I., ~RI&NEN, L., and PET~ERSSON, R. F. (1984). Rubella virus 40s genome RNA specifies a 24s subgenomlc mRNA that codes for a precursor to structural proteins. J. Virol. 49, 403-408. ROSSMAN, M. G. (1984). Constraints on the assembly of spherical virus particles. Virology 134, l-l 1. SIMONS, K., HELENIUS, A., and GAROFF, H. (1973). Solubilization of the membrane proteins from Semliki Forest virus with Triton X-100. J. Mol. Biol. 80, 119-l 33. SUOMALAINEN, M., GAROFF, H., and BARON, M. D. (1990). The E2
OF RV PROTEINS
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signal sequence of Rubella virus remains part of the capsid protein and confers membrane association in vitro. J. Viral. 64, 55005509. TAKKINEN, K., VIDGREN, G., EKSTRAND, J., HELLMAN, U., KALKKINEN, N., WERNSTEDT, C., and PEITERSSON, R. (1988). Nucleotide sequence of the Rubella virus capsid protein gene reveals an unusually high G/C content. J. Gen. Viral. 69, 603-6 12. TOIVONEN, V., VAINIONP~, R., SALMI, A., and HWPI& T. (1983). Glycopolypeptides of Rubella virus: Brief report. Arch. Viral. 77, 91-95. TRUDEL, M.. NADON, F., COMTOIS. R., PAYMENT. P., BONNEAU, A. M.. and LECOMTE, J. (1982). Identification of Rubella virus structural proteins by immunoprecipitation. J. Viral. Methods 5, 191-l 97. VAHERI, A., and How, T. (1972). Structural proteins and subunits of Rubella virus. J. Viral. 9, 1 O-l 6. VIDGREN, G., TAKKINEN, K., KALKKINEN, N., K%RI~~INEN, L., and PETTERSSON, R. (1987). Nucleotide sequence of the genes coding for the membrane glycoproteins El and E2 of Rubella virus. J. Gen. Viral. 68, 2347-2357. WAHLBERG, J. M., BOERE, W. A., and GAROFF, H. (1989). The heterodimerit association between the membrane proteins of Semliki Forest virus changes its sensitivity to mildly acidic pH during virus maturation. J. Viral. 63, 4991-4997. WAXHAM, M. N., and WOLINSKY, J. S. (1983). lmmunochemical identification of Rubella virus hemagglutinin. Virology 126, 194-203. WAXHAM, M. N., and WOLINSKY, J. S. (1985a). Detailed immunologic analysis of the structural polypeptides of Rubella virus using monoclonal antibodies. Virology 143, 153-l 65. WAXHAM, M. N., and WOLINSKY, J. S. (1985b). A model of the structural organization of Rubella virion$. Rev. lnf. Dis. 7(suppl. 1): s133-s139.