Department of Genetics and Microbiology, University of Geneva Medical School, CMU, 9 avenue de Champel, 1211 Geneva 4, Switzerland 1
Author for correspondence: Laurent Roux.Fax +41 22 702 57 02. e-mail Laurent.Roux{at}medecine.unige.ch
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Abstract |
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Introduction |
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F and HN are trans-membrane glycoproteins of types I and II, respectively. They protrude from the viral envelope as homotrimers and homotetramers, respectively, and form the spikes that decorate the virions, as seen by electron microscopy. HN contains the cell receptor- binding site (neuraminic acid), as well as the neuraminidase activity, involved in detachment of the virion. F exhibits the fusogenic activity required in the process of infection, when the viral envelope fuses with the cell plasma membrane to deliver the viral genome into the cytosol (for a recent review of the Paramyxoviridae cell cycle, see Lamb & Kolakofsky, 1996 ).
The M protein (matrix protein) is so named because it can maintain the compact structure of the virion even after dissolution of the envelope by detergent. This structure falls apart only after high-salt treatment. The SeV M protein contains 348 amino acids (Blumberg et al., 1984 ). It is highly basic (pI 10·17), is partially phosphorylated on Ser70 (Sakaguchi et al. , 1997
) and is acetylated on Ala2 after cleavage of the initiating Met (Blumberg et al., 1984
). It is predicted to contain little secondary structure, namely 11%
- helix, 19% ß-pleated sheet and 70% random coil (Giuffre et al. , 1982
). It is found in infected cells and in virions in two isoforms defined by their reactivity for monoclonal antibodies (MAbs) (de Melo et al., 1992
). The minor isoform forms a subset (~30%) characterized by the maturation of a native epitope. SeV M, and presumably the M proteins of all paramyxoviruses (Peeples, 1991
, and references therein), has been shown to associate specifically with membrane fractions in flotation gradients (Stricker et al., 1994
). As is the case for M from other paramyxoviruses, SeV M is proposed to interact with at least one of the two viral glycoproteins (Yoshida et al., 1986
; Sanderson et al., 1993
, 1994
, 1995
). M also interacts with other M molecules, as evidenced by the formation of a crystalline array visualized by electron microscopy either in infected cells (Büechi & B ächi, 1982
) or after purification (Hewitt, 1977
; Hewitt & Nermut, 1977
; Heggeness et al., 1982
). Initially described as an aggregation, this interaction appears in fact to be a specific two-dimensional polymerization, as shown for vesicular stomatitis virus M (Gaudin et al., 1995
). Finally, M interacts with the nucleocapsid as evidenced by MN cross- linking (Markwell & Fox, 1980
) and by the co-capping of the nucleocapsid with the glycoproteins only in the presence of M (Yoshida et al., 1986
). Because of these multiple interactions, M has been proposed as the `bandleader' in charge of virion assembly at the site of virus-particle budding (Peeples, 1991
).
The assembly of virions of members of the Paramyxoviridae is generally proposed to take place at the plasma membrane, as the pre- budding structure is seen there before the newly formed virus particle pinches off. The two viral glycoproteins HN (or H) and F are expressed at the cell surface, anchored by their trans-membrane portion, with the large ectodomain on the outside and the short cytoplasmic tails on the inside (Nagai et al., 1975 ). At this stage, the two glycoproteins are thought to move freely in the lateral plane of the membrane (Nagai et al., 1976
; Markwell & Fox, 1980
). M forms a paracrystalline array at the inner face of the lipid bilayer (Bächi, 1980
; Büechi & Bächi, 1982
), which now concentrates the two glycoproteins via interactions with the cytoplasmic tail or the transmembrane portion of the glycoproteins. Concentration of these virus components is apparently achieved to the exclusion of the cellular surface proteins. The virally modified portion of the membrane now constitutes a recognition site for the viral nucleocapsid, which is found adhering to the M crystalline array (Büechi & Bächi, 1982
). At this stage, addition of antibodies against the viral glycoproteins to the infected cells leads to a co-capping of all the virus components (Tyrrell & Ehrnst, 1979
). This assembly model implies that M and the nucleocapsid travel to the plasma membrane before they can interact with the glycoproteins, which are transported via the default secretory pathway. Not much is known about the mechanism of transport of M and the nucleocapsids. The cytoskeleton may be involved here, as suggested by an affinity of M for actin (Giuffre et al. , 1982
) and by the absence of co-capping of the nucleocapsid and M with the glycoproteins upon treatment with cytochalasin B. Also, the reduced transport of M to the cell surface upon monensin treatment has led to the proposal that M is transported to the plasma membrane via interactions with the glycoprotein cytoplasmic tail (Sanderson et al., 1994
; for general reviews on the assembly and budding of paramyxoviruses and also on the M protein, see Dubois-Dalcq et al., 1984
; Peeples, 1991
; Ray et al., 1991
).
With the availability of a reverse-genetic system for paramyxoviruses (reviewed in Conzelmann, 1996 ; Boyer & Haenni, 1994
) came the possibility of creating site-directed mutants to analyse the structurefunction relationships of the viral proteins. For this purpose, we have developed a system that allows the expression of mutated viral proteins in the context of an otherwise normal SeV infection. This is based on the preparation of mixed SeV stocks containing, in addition to the non-defective (ND) viral genome, a defective genome that is derived from a natural, transcribing, defective-interfering (DI) genome, designed to replicate at a high level so as to express extra proteins very efficiently (Mottet et al., 1996
). In this way, the SeV M protein, tagged with an influenza virus haemagglutinin (HA) protein epitope (HAM), was expressed during SeV infection along with wild-type M (Mwt). Next, mutant HAM proteins were generated that exhibited different properties with regard to their abilities to be incorporated into virus particles and to interfere with virion production (Mottet et al., 1996
). The present study extends our mutant collection and characterizes some of them in more detail. Our original identification of an amphiphilic
-helix that was proposed to be responsible for M attachment to the membrane has been revised. Attempts to identify the step in assembly at which the mutant M proteins interfere with the normal process of assembly are presented.
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Methods |
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Mixed SeV stocks.
Mixed SeV stocks refer to stocks containing DI genomes, supported in their replication and assembly by functions provided by the ND genome, in addition to the ND viral genome (Roux & Holland, 1979 ). In the present case, the DI genome consisted of a nucleocapsid containing the RNA vector (HA-M-CB119) carrying the open reading frame of the various M mutants. The methodology to generate these mixed SeV stocks has been described previously in detail (Calain & Roux, 1993
; Mottet et al., 1996
) and was not changed. The HAM virus stocks were characterized by the number of p.f.u. (in general ~108 p.f.u./ml; Sugita et al., 1974
) and by the ratio of DI vector to ND RNA upon infection (generally 20:1) estimated by Northern blot analysis as described previously (Calain & Roux, 1993
). In cells infected with these mixed virus stocks, the HAM protein expressed from the vector RNA co-exists with Mwt expressed from the ND genome. In general, due to the high replication efficiency of the RNA vector, which carries a copy-back feature, the HAM protein was found in excess over Mwt.
Antibodies and protein analysis.
The anti-M protein antibody preparations used were a rabbit serum raised against SDS-denatured M protein (-MSDS) (Mottet et al., 1986
; Tuffereau & Roux, 1988
) and MAbs 383 and 376 obtained from Claes Örvell (Laboratory of Clinical Virology, Huddinge Hospital, Huddinge, Sweden). The anti-HA epitope MAb (12CA5; referred to here as
-HA) (Field et al., 1988
) was obtained from Berkeley Antibody Co. A rabbit polyclonal serum raised against the whole purified virus (Rab-vir) was described by Mottet et al. (1986)
. Protein-isotope labelling (TransLabel, New England Nuclear), preparation of cellular extracts for immunoprecipitation (IP), IP and SDSPAGE were performed as described previously (Mottet et al., 1996
). Processing of the gels through PPO-DMSO enhancement or through Western blotting were exactly as described previously (Mottet et al., 1996
), except that blots were developed with the ECL system (Amersham). Virus particles in clarified cell supernatants were pelleted through a 25% glycerolTNE cushion (10 mM TrisHCl, 1 mM EDTA, 50 mM NaCl) for 2 h at 50000 r.p.m. before SDSPAGE.
M protein co-precipitation.
Infected BHK cells were disrupted into Heggeness in vitro assembly buffer (Heggeness et al., 1982 ) (9·2 mM phosphate buffer, pH 7·2, 0·7% sodium deoxycholate, 2·14 mM Triton X-100, 2 mM PMSF and 1% aprotinin with or without 1 M NaCl) and incubated for 18 h at room temperature. After sonication for 10 s (Branson Sonic Sonifer B-12, lowest speed), cell extracts were spun for 45 s at 12000 r.p.m. in a microfuge. The supernatants were then processed for IP by using the anti-M MAb 383 or
-HA antibody. The immunoprecipitates were finally analysed by Western blotting and probed with MAb 383 (diluted 1:25000).
Flotation gradients.
Infected BHK cells were harvested 18 h post-infection and resuspended in 10% sucrose containing 2 mM PMSF and 1% aprotinin. After Dounce homogenization (20 strokes), the cell debris was pelleted (2 min, 1000 r.p.m., 4 °C) and the supernatants (1·2 ml), made to a final sucrose concentration of 70%, were loaded at the bottom of SW60 tubes (Beckman) and overlaid with 2·5 ml 65% sucrose and 0·8 ml 10% sucrose. After centrifugation to equilibrium (40000 r.p.m., 18 h, 4 °C), ten 500 µl fractions were collected, diluted to 1·5 ml with IP buffer and processed for IP with the Rab-vir rabbit serum. The immunoprecipitates were then analysed by Western blotting and probed with -HA and MAb 383. Alternatively, the fractions were diluted to 4 ml with TE (20 mM TrisHCl, pH 7·5, 2 mM EDTA) and centrifuged for 2 h at 4 °C and 55000 r.p.m. in an SW60 rotor (Beckman) to pellet the nucleocapsids. These were resuspended in TE plus 0·2% Sarkosyl, extracted with phenolchloroform to isolate the nucleocapsid RNA and recovered by ethanol precipitation. The RNAs were then processed by agarose gel electrophoresis and Northern blotting as described previously (Mottet et al., 1990
; Stricker et al., 1994
). The probe was 32P- labelled anti-M transcript synthesized in vitro from M-pGem4 (de Melo et al., 1992
).
Immunofluorescent staining and confocal microscopy.
LLCMK2 cells were plated on poly-l-lysine-coated coverslips (coated with a 5 µg/ml solution for 15 min and dried under UV). After growth overnight, cells were infected and fixed 18 h later with 4% paraformaldehyde for 30 min at room temperature. After three washes with PBS lacking Ca2+ and Mg2+ (PBS -, 15 min at room temperature), cells were incubated with the primary antibodies, -HA and
-MSDS (diluted 1200 and 1100, respectively, in 3% BSA and 0·3% Triton X-100), for 2 h at room temperature. After three more washes with PBS-, rhodamine-conjugated anti-mouse antibodies and FITC-conjugated anti-rabbit antibodies were added and the cells were incubated for 2 h at 4 °C in the dark. The procedure was completed by three final PBS- washes and the coverslips were mounted in 20% glycerol plus 0·2 g/ml polyvinyl alcohol. Specimens were observed with a Zeiss confocal laser- scan fluorescent inverted microscope (LSM 410, Carl Zeiss) (for a detailed description of the confocal microscope see Tuchweber et al. , 1996
) through an oil plan-neofluar 40x/1·3 objective. Images of 512x512 pixels, stored on an erasable optical disk (Sony), were imported into MicrographX Designer and printed at 1420 d.p.i. on glossy paper on an Epson Stylus Colour 800 printer.
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Results |
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Secondly, the possibility that the single-band PAGE migration could result from a conformational change induced by the Val113 substitution was investigated by monitoring the maturation of the minor native epitope on the M protein, which matures with time (de Melo et al., 1992 ; see Introduction). In contrast to the situation for M and HAMwt, maturation of the native epitope of HAM30 could not be demonstrated (not shown), a result that supports an alteration in the folding of the protein induced by the substitutions in HAM30.
A drastic change in the tertiary structure of HAM30 was confirmed by comparison of its tryptic digestion pattern with that of HAMwt (Fig. 3 ). Here, only fragments including the conserved N terminus were detected since the Western blot was probed with
-HA, reacting with the HA epitope at the N terminus of M. The pattern appears different in the number of discrete bands, their relative position and intensity. The difference in the relative positions of some bands (for instance, X1/X2 and Y1/Y2) could be explained by the conservation (Y1) or the loss (Y2) of the substitution at position 113 being responsible for the PAGE migration change. However, this explanation would not hold for the difference between the full-length proteins and their respective larger digest fragments (X1 and Y1). Neither could the extra band (Y3) and the relative intensity of the others (X1/Y1 or X2/Y2) be explained in this way. In conclusion, the substitution at Val113 appears to induce a general conformational change in HAM that correlates with its dysfunction.
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HAM30 does not co-precipitate M, but binds to membrane fractions
M function has been defined as the ability to be incorporated into virions. This, however, implies a series of steps through which the protein has to progress before ending up in virus particles. Exclusion from virus particles may thus reflect a blockage of any one of the steps, the identification of which could be informative about the path followed.
Self-assembly is one of the steps through which M passes, and this was investigated by selective immunoprecipitation. Extracts of cells infected with mixed virus stocks expressing Mwt as well as HAMwt or HAM30 were immunoprecipitated with -HA or
-M antibodies. The proteins selected were then analysed by Western blots probed with
-M antibody (Fig. 4
). In a mixed infection expressing HAMwt (Fig. 4a
, lane 2), immunoprecipitation with
-HA resulted in the recovery of Mwt together with HAMwt, reflecting an association between the two proteins. This association was quantitative, as shown by the equivalent recovery of Mwt with
-M (Fig. 4a
, lane 1), which precipitates both proteins directly. Moreover, Mwt did not co-precipitate with HAMwt in high salt (Fig. 4a
, lane 4), conditions known to disassemble Mwt (Heggeness et al., 1982
; de Melo et al., 1992
). In contrast, no co-precipitation of Mwt with HAM30 was apparent with the
-HA antibody (Fig. 4b
, lane 2). This shows that HAM30 did not associate with Mwt in the way that HAMwt did, an indication that it has lost the ability to form at the least a homodimer.
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HAM30 reduces nucleocapsidmembrane association
We showed previously that more than 70% of the full-length (ND) nucleocapsids was found in association with the membrane fractions (Stricker et al., 1994 ). In contrast, defective (DI) nucleocapsids were found mainly (>>75%) in the free fractions. This result correlated with the efficiency with which each category of nucleocapsids was found in virus particles, so that the degree of membrane association was considered relevant to the process of virus assembly. The difference in association between the two populations of nucleocapsids was interpreted as reflecting competition for the M protein, known to be a limiting factor in virion production (Kingsbury, 1974
). These earlier experiments were performed with mixed virus stocks composed of naturally derived DI genomes encoding no extra viral proteins. In Fig. 5
, the degree of ND and DI nucleocapsid association was analysed in mixed virus infections where the DI genomes encoded M proteins in excess, either functional (HAMwt and HAM21) or non-functional (HAM30 ). HAM21 was included here because it appeared to perform even better than HAMwt (Mottet et al. , 1996
). As before, the majority of ND nucleocapsids (88%) were membrane-bound in the absence of DI genomes (Fig. 5b
, lanes 1 and 2). With HAMwt expression, i.e. in the presence of DI genomes as well (Fig. 5b
, lanes 3 and 4), the fraction of bound ND nucleocapsids appeared to be slightly reduced, possibly due to competition with DI genomes in a situation where M was less limiting, since it was expressed from both ND and DI genomes. DI nucleocapsids, however, were still mostly membrane-free (66%). With HAM21 expression, competition between ND and DI nucleocapsids appears to shift even more in favour of the latter (Fig. 5b
, lanes 5 and 6). With HAM30, however, the trend was altered (Fig. 5b
, lanes 7 and 8). ND nucleocapsid binding still decreased, but now DI nucleocapsid binding was also drastically reduced, with the net result that a clear reduction of total nucleocapsid association with membrane fractions was observed, a trend that was verified in three separate experiments (not shown). This property of HAM30 correlates with a decrease in total virus production noted before in mixed virus infections where non-functional M mutants were expressed (HAM 5, HAM11, HAM30; Mottet et al., 1996
). It is unfortunate that ND genomes could not be visualized because of high replication interference by DI genomes, although the amounts could be scored over the background by phosphorimager scanning. Adding excess ND virus contributed to an increase in the ND:DI ratio, so that ND genomes were visible. Unfortunately, under these conditions, the Mwt:HAM 30 ratio also increased such that the interference by HAM30 on nucleocapsid binding was lost (not shown).
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Discussion |
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In the context of SeV infection, HAM30 is shown here (i) to bind to total membrane fractions, (ii) not to co- precipitate Mwt, which indicates its inability to self- assemble, (iii) to reduce nucleocapsidmembrane association and (iv) not to be transported to the plasma membrane. These phenotypic traits can help to define the path that M follows en route to the virus particle. HAM30 is able to attach to the total membrane fraction, with an apparent inability to form a homodimer. Attachment to the membrane could therefore be achieved by individual M proteins, which could then self-assemble once bound to membrane. When expressed alone from pGem plasmids, about 50 and 20%, respectively, of M wt and HAM30 protein was bound to membrane (Mottet et al., 1996 ). For both proteins, this percentage increased to almost 100% in SeV infection (not shown). Co- operation with the other viral components could therefore drive M towards a more extensive association. Co-expression of F and M was reported to reinforce M membrane association and to promote its diffusion from a perinuclear location towards the cell edge (Sanderson et al., 1994
). Although the participation of F in the binding of M to the membrane is a matter of debate (Stricker et al., 1994
), the participation of other viral proteins in a more extensive membrane association and/or in the transport of M to the cell membrane is a valid assumption. As M can polymerize in vitro in the absence of other viral proteins in a concentration- dependent manner (Gaudin et al., 1995
; Heggeness et al., 1982
), attachment to the membrane may serve to increase the local M concentration, so as to prime self- association. If HAM30, which cannot interact with M wt, binds efficiently to the membrane, it could interfere with self-assembly of Mwt, disturbing formation of the leaflet structure. This would in turn result in the observed decrease in virus budding (Mottet et al., 1996
).
If this scheme is valid, and it does not contradict what has been proposed up to now, an intriguing observation comes from the ability of HAM30 to interfere with nucleocapsid binding to the total membrane fraction (Fig. 5) even though HAM 30 is not efficiently transported to the cell plasma membrane (Fig. 6
). This suggests that the interaction of nucleocapsids with the membrane could take place in the perinuclear region, where HAM30 is located (Fig. 6
). The proposal that partial or even full assembly could take place on intra-cytoplasmic membranes is not new. Ö rvell & Grandien (1982)
have already shown by immunofluorescent staining studies that the majority of SeV M never reaches the plasma membrane but rather remains associated with the ER membrane. The same observation has been made by Bellini et al. (cited in Dubois-Dalcq et al., 1984
) for the measles virus M protein. Comparison of the budding of SeV particles during non-defective or mixed virus infections leads to the proposal that non-defective nucleocapsids could associate with M in perinuclear regions to promote effective transport of M (Stricker et al., 1994
). Finally, budding of rhabdoviruses in the ER cisternae has been documented (Fekadu et al., 1982
), an observation that could imply intra-cytoplasmic assembly of this category of viruses, which are normally shown to bud from the cell surface. As attractive as it looks, this model is, however, disproved by the great difficulty of detecting complete assembly structures anywhere but at the plasma membrane, and the present data are only suggestive of the possibility that viral assembly could take place internally.
One reason for the weak expression of the mutant phenotype certainly comes from the co-expression of Mwt and HAM30 in the present system of mixed-virus infections. Indeed, attempts to recover a recombinant virus expressing only HAM30 failed, in contrast to the successful recovery of recombinant SeV expressing HAM21 (not shown). The preparation of a cell line that conditionally expresses Mwt (as done by Tashiro et al., 1996 ) is presently being done, in order to allow recovery of recombinant SeV HAM 30 so as to be able to follow the abortive assembly mediated by HAM30 alone. If successful, this approach will allow us to use the trans-dominant negative M mutants in defining more clearly the step(s) at which assembly aborts and, hopefully, to identify the various steps of virus assembly.
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Acknowledgments |
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References |
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Received 26 April 1999;
accepted 20 July 1999.