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J Virol, February 1998, p. 1224-1234, Vol. 72, No. 2
Institut für Molekularbiologie,
Universität Zürich, Hönggerberg, CH-8093
Zürich, Switzerland
Received 24 July 1997/Accepted 16 October 1997
The cytoplasmic tail of the measles virus (MV) fusion (F) protein
is often altered in viruses which spread through the brain of patients
suffering from subacute sclerosing panencephalitis (SSPE). We
transferred the coding regions of F tails from SSPE viruses in an MV
genomic cDNA. Similarly, we constructed and transferred mutated
tail-encoding regions of the other viral glycoprotein hemagglutinin (H)
gene. From the mutated genomic cDNAs, we achieved rescue of viruses
that harbor different alterations of the F tail, deletions in the
membrane-distal half of the H tail, and combinations of these
mutations. Viruses with alterations in any of the tails spread rapidly
through the monolayer via enhanced cell-cell fusion. Double-tail
mutants had even higher fusion competence but slightly decreased
infectivity. Analysis of the protein composition of released mutant
viral particles indicated that the tails are necessary for accurate
virus envelope assembly and suggested a direct F tail-matrix (M)
protein interaction. Since even tail-altered glycoproteins colocalized
with M protein in intracellular patches, additional interactions may
exist. We conclude that in MV infections, including SSPE, the
glycoprotein tails are involved not only in virus envelope assembly but
also in the control of virus-induced cell fusion.
The surface of enveloped viruses is
covered with protrusions, or spikes, composed of multimeric
glycoproteins which bind to cellular receptors and are instrumental in
fusing the viral membrane with the cellular membrane. The spike
glycoproteins are connected to the internal virion proteins through
their cytoplasmic tails, and interactions between the tails and the
internal virion components trigger the formation of budding particles
of many viruses (20, 40, 42). Nevertheless, for some
viruses, tailless mutants can be produced (15, 16, 23, 25).
Several enveloped viruses propagate both by virus release and
reinfection and by cell-cell fusion. In a few retroviruses, including
human immunodeficiency virus, the glycoprotein tails regulate the
cell-cell fusion process (12, 14, 33, 51, 53). For the
propagation and pathogenicity of the paramyxovirus measles virus (MV),
virus-induced cell-cell fusion is also a prominent property. In the
brains of patients who died from the MV-induced syndrome subacute
sclerosing parencephalitis (SSPE), MV propagates apparently without
budding, probably by cell-cell fusion (reviewed in reference
1). In fact, MV may spread mainly in a
cell-associated form in human tissues. Even tissue culture-adapted MV
remains about 90% cell associated (47); therefore, virus
stocks are usually prepared from cell extracts.
The MV envelope protein complex, which is instrumental both for virus
attachment and cell fusion, is composed of two integral membrane
proteins, the hemagglutinin (H) and fusion (F) proteins, and a
membrane-associated protein, the matrix (M) protein (49). The type II transmembrane protein H mediates virus-cell attachment by
binding to the cell surface protein CD46 (11, 29) and is an
essential cofactor for fusion (9, 50). The H protein
transmembrane segment is preceded by a 34-amino-acid cytoplasmic tail.
The type I transmembrane protein F is synthesized as an inactive
precursor, F0, which is cleaved to generate the
disulfide-linked F2 and F1 subunits. Cleavage
of the F0 precursor occurs near a hydrophobic region, the
so-called fusion peptide. After virus release, possibly following the
contact of viral particles with the MV receptor (3), this
peptide may insert in the target membrane, initiating virus-cell
fusion. Another type of MV-induced membrane fusion is fusion of
MV-infected cells with neighboring cells, resulting in the formation of
syncytia. The F1 subunit is anchored in the membrane
through a hydrophobic segment which is followed by a 33-amino-acid
cytoplasmic tail.
The M protein lines the inner surface of the membrane and may play a
key role in assembly by concentrating the F and H proteins as well as
the ribonucleocapsid, the viral replicative unit, at sites of virus
assembly (8, 31).
MV is a strongly cell-associated virus. Released infectious particles
are pleomorphic and have low infectivity (21, 24, 28).
Current models of MV assembly and release rely mainly on the study of
related paramyxoviruses (26, 31). Biochemical and
microscopic investigations of Sendai virus (SeV) suggested that the
tails of both glycoproteins enhance M protein-membrane association
(35, 36). However, part of these findings could not be
reproduced (45).
Direct biochemical analysis of the MV M protein-glycoprotein tail
interaction is difficult because the M protein strongly associates with
cellular membranes even in the absence of the glycoproteins
(7), as for the M proteins of Sendai and influenza viruses
(45, 52). In MV, an F tail-M protein interaction was postulated on the basis of observations made on assembly-defective viruses replicating in the brains of SSPE patients. In all SSPE-derived F genes analyzed so far, mutations in the regions coding for the cytoplasmic domain were detected; the corresponding F tails were shortened, elongated, or strongly altered (37). Conversely, M protein expression was frequently strongly impaired or abolished (1, 10).
We have used a novel genetic approach (32) to analyze the
function of the cytoplasmic tails of the MV glycoproteins. We rescued
nine MV mutants with altered glycoprotein tails and characterized their
mode of propagation and intracellular assembly and the protein composition of released virus particles.
Antibodies.
The following antibodies were used:
anti-MV goat polyclonal serum (courtesy of S. Udem), anti-Fcyt rabbit
anti-peptide serum (13), anti-F rabbit polyclonal
serum (courtesy of T. Varsanyi), anti-Hcyt rabbit anti-peptide serum
(3), anti-H rabbit polyclonal serum (courtesy of C. Örvell), anti-H mouse monoclonal ascites (39),
and anti-M mouse monoclonal ascites (39). The antibody directed against the F cytoplasmic tail (Fcyt) was raised against a
peptide corresponding to the 14 carboxyl-terminal amino acids of this
protein, NH2-(C)PDLTGTSKSYVRSL-COOH. The H antibody
(Hcyt) was raised against a peptide corresponding to the 14 H protein amino-terminal residues, NH2-SPQRDRINAFYKDN(C)-COOH. A
cysteine (C) was attached to the peptide termini to allow coupling to
keyhole limpet hemocyanin for rabbit immunization.
Plasmid constructs.
Plasmids peFHL and peH5 (Fig.
1A) are shuttle vectors for subcloning in
the full-length genomic plasmid p(+)MV (32). Plasmid peH5
contains a single ClaI site in the 5' untranslated region of
the H protein and a single EcoRI site at the border between the transmembrane domain and the ectodomain. These sites were previously introduced by silent point mutations (5).
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Measles Viruses with Altered Envelope Protein
Cytoplasmic Tails Gain Cell Fusion Competence
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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FIG. 1.
Structure of the MV genome and of the plasmids used for
virus rescue, and amino acid sequence of the cytoplasmic tails of the
MV glycoproteins. (A) Structure of p(+)MV and of the plasmid
intermediates used in the construction of mutant viruses. Plasmid
p(+)MV encoding the MV Edmonston B strain antisense genome is shown at
the top (32). Solid boxes represent the reading frames of
the six MV cistrons. The T7 promoter and relevant restriction sites are
indicated. The structures of the plasmids used for subcloning are shown
below. Shaded boxes represent gene segments coding for the altered F
and H tails. The nucleotide numbering is that used for EMBL accession
no. Z66517. (B) Predicted amino acid sequence of the standard (std) and
altered MV glycoprotein tails. Boldface letters represent residues
conserved within five members of the morbillivirus genus. Residues in
the SSPE-derived F protein tails differing from that of the Edmonston
strain are indicated by lowercase letters. TM, transmembrane; aa, amino
acids.
8, peHc14, and peHc24 were
constructed by subcloning a ClaI-EcoRI PCR
fragment into peH5 (Fig. 1A, bottom). PCR was performed with peH5 DNA
as the template; forward primers
5'-CCATCGATAATGGCCTTCTACAAAGATAACC,
5'-CCATCGATAATGAGCCATCCCAAGGGAAGTAGG, and
5'-CCATCGATAATGAACAGAGAACATCTTATGATT (restriction
sites in italics), respectively; and a reverse primer annealing
downstream from the region encoding the H transmembrane domain.
To construct plasmid peHcSeV, fusion PCR was performed. The SeV HN
tail-encoding region was amplified on the pGem4-SVHN (48) template with primers 5'-CCATCGATAATCATGGATGGTGATAGGGG
and 5'-GCAAAACATAAGGGGTGTCAACTTTACTTGA. Primer 5'-GACACCCCTTATGTTTTGCTGGC and a
primer annealing downstream of the region coding for the H
transmembrane domain were used to amplify the MV H transmembrane
encoding region. In the fusion step, the isolated PCR fragments with an
overlapping sequence of 19 nucleotides (underlined) were mixed and
amplified with the external primers, also used for the first
amplification round. The resulting fragment was digested with
ClaI and EcoRI and then subcloned into peH5.
Plasmids peFHLP, peFHLF, and peFHLI were described previously
(37). peF(cSeV)HL was generated by subcloning a
PstI-PacI PCR fragment encoding the SeV F
cytoplasmic tail into peFHL. PCR was performed with
pGem4-SVF0 (48) as the template and primers 5'-AAAACTGCAGACTCAAAAGGTCAATGC and
5'-CCCTTAATTAATATACAGATCTCAACGGAT (restriction
sites in italics).
Plasmids p(+)MV-Hc8, p(+)MV-Hc14, p(+)MV-Hc24, and
p(+)MV-HcSeV, containing the full-length antigenomic copy of the H-tail mutant MV genomes, were constructed by three-way ligations of a
PacI-SpeI fragment containing the mutated H gene
from plasmid peHc8, peHc14, peHc24, or peHcSeV, respectively,
with a SpeI-SacII fragment and a
SacII-PacI fragment of p(+)MV.
Plasmids p(+)MV-Fc+28, p(+)MV-Fc16, p(+)MV-Fc24, and p(+)MV-FcSeV
coding for the F tail mutant MV genomes were constructed by three-way
ligations of a NarI-PacI fragment carrying the
mutated F gene coding region from plasmid peFHLP, peFHLF, peFHLI, or
peF(cSeV)HL, respectively, with a PacI-SacII
fragment and a SacII-NarI fragment of p(+)MV.
Plasmids p(+)MV-Hc14/Fc+28, p(+)MV-Hc14/Fc24, and
p(+)MV-Hc14/FcSeV, containing the MV tail double-mutant genomes were constructed by ligation of a PacI-SacII fragment
of p(+)MV-Hc14 with the corresponding
SacII-PacI fragment of p(+)MV-Fc+28,
p(+)MV-Fc24, and p(+)MV-FcSeV, respectively.
All generated full-length plasmids coding for a mutated antigenomic
copy of the MV genome conform to the rule-of-six (17). The
sequences were confirmed by dideoxynucleotide sequencing, and the
nucleotide numbering is that used for EMBL accession no. Z66517.
Cells, viruses, and virus assays. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum (DMEM-5) for Vero (African green monkey kidney) cells, 10% fetal calf serum (DMEM-10) for HeLa T4 (human cervical carcinoma) and 293 (human embryonic kidney) cells, or DMEM-10 containing 1 mg of G418 per ml for the helper cell line 293-3-46.
The Edmonston B strain based standard MV and all mutant derivatives were rescued, propagated, and purified basically as described previously (32). Propagation and purification of the recombinant vaccinia virus expressing the T7 RNA polymerase (vTF7-3) were performed as described previously (6). The biological activities of the viruses were determined by a plaque assays as described previously (32) or by 50% end-point dilution assays performed as follows. Serial dilutions (10-fold) of virus samples were made in DMEM-5, and 50 µl of each dilution was used to infect eight replica 6-mm wells containing 7,500 Vero cells in 50 µl of DMEM-5 overnight at 37°C. The next day, 150 µl of DMEM-5 was added, and 4 days postinfection, the wells were stained with 0.1% crystal violet in 20% ethanol. Virus-induced cytopathic effects were scored, and the log10 50% tissue culture infective dose (TCID50) was calculated by the method of Kärber (18).Fusion assays. Cell fusion assays were performed basically as described previously (6). Briefly, HeLa T4 cells were infected with vTF7-3 at a multiplicity of infection (MOI) of 5 and subsequently colipofected with 3.5 µg of an H protein-expressing plasmid and with 1.5 µg of an F plasmid. Syncytium formation was quantified 20 h posttransfection.
Metabolic labelling, immunoprecipitation, and endo H digestion. Vero cells (106 cells) in 35-mm wells were infected with standard MV or MV mutants at an MOI of 0.5 for 2 h at 37°C. At 16 to 20 h postinfection, the cells were starved for 30 min in Met- and Cys-free DMEM, pulsed for 1 h in the presence of 100 µCi of Tran35S-label (ICN), and chased for the indicated time with DMEM-10. Alternatively, the same number of HeLa T4 cells were infected with vTF7-3 and lipofected as described above.
Immunoprecipitation with a polyclonal goat anti-MV antibody was performed as described previously (9). For endo-
-N-acetylglucosaminidase H (endo H) analysis,
immunoprecipitated samples were adjusted to 75 mM sodium citrate (pH
5.6) and incubated in the presence or absence of 1 mU of endo H
(Boehringer Mannheim) for 4 h at 37°C.
Immunoblotting. Vero cells infected with MV mutants as described above were harvested 20 h postinfection in lysis buffer (50 mM Tris [pH 8.0], 62.5 mM EDTA, 1% Nonidet P-40, 0.4% deoxycholate). Samples from equivalent numbers of cells were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 1% bovine serum albumin-1% skim milk powder in TBST (10 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween 20) for 2 h at room temperature and then incubated with different specific rabbit anti-MV protein antibodies (anti-Fcyt at 1:4,000 or anti-Hcyt at 1:3,000) overnight at 4°C. The proteins were visualized after incubation with horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin G (IgG) (1:3,000; Dakopatts) for 1 h at room temperature by using the enhanced chemiluminescence (Amersham) system.
FACScan analysis. HeLa T4 cells (5 × 105 cells) were infected with vTF7-3 and lipofected with H-expressing plasmids as described above. H protein surface expression was determined 24 h posttransfection. Cells in DMEM-10 were incubated in a total volume of 50 µl with a 1:100 dilution of a monoclonal anti-H antibody for 1 h at 4°C, washed, and incubated with a 1:50 dilution of R-phycoerythrin-conjugated rabbit anti-mouse Ig (Serotec) for 30 min at room temperature. Flow cytometry was performed after fixation of the cells in phosphate-buffered saline (PBS) containing 1% paraformaldehyde.
One-step growth analysis.
Vero cells (2 × 105 cells) in 15-mm wells were infected with MV mutants at
an MOI of 3 for 2 h at 37°C. Inocula were collected to control
virus adsorption. The cells were washed twice with PBS, overlaid with
500 µl of DMEM-5, and incubated at 32.5°C to optimize virus release
(47). Every 12 h, cell-free virus and cell-associated
virus were collected and stored at 
30°C. The cell-free samples were
prepared by clarifying supernatants of infected cells for 2 min at
8,000 rpm in an Eppendorf model 5415C centrifuge. Cell-associated virus
was recovered by scraping infected cells into 500 µl of DMEM-5. The
infectivity of the samples was determined by TCID50
measurement.
Isolation of viral particles. Vero cells (107 cells) were infected at an MOI of 0.1 for 2 h at 37°C. At 20 h postinfection, the cells were starved for 30 min in Met- and Cys-free DMEM. Continuous labelling was performed in the presence of 1 mCi of Tran35S-label in Met- and Cys-free DMEM supplemented with 1/20 volume of DMEM-10 at 32.5°C. For velocity centrifugation, culture supernatants were collected 2 days after infection, clarified, and pelleted in an SW41 rotor at 30,000 rpm for 2 h through 20% sucrose onto a 60% cushion prepared in TNE buffer (10 mM Tris [pH 7.5], 100 mM NaCl, 1 mM EDTA [pH 7.5]). Purified particles were layered on top of a 20 to 60% step gradient and centrifuged for 18 h in an SW41 rotor at 35,000 rpm, and 10 fractions of equal volume were collected. Viral particles were pelleted from diluted fractions in a TLA 45 rotor for 1 h at 45,000 rpm, resuspended in lysis buffer, and directly subjected to SDS-PAGE.
Confocal microscopy.
Vero cells (105 cells) on
glass coverslips were infected at an MOI of 0.1 for 3 h at 37°C.
After infection, the cells were shifted to 32.5°C. At 50 h
postinfection, the cells were permeabilized and fixed for 5 min in
methanol at 20°C, blocked in PBSB (0.5% bovine serum albumin in
PBS) for 10 min, and then incubated consecutively with the following
antibodies diluted in PBS containing 0.5% Triton X-100: monoclonal
anti-M (1:200), tetramethylrhodamine-5-isothiocyanate-conjugated goat
anti-mouse IgG (Cappel; 1:50), polyclonal serum against H or F,
respectively (1:100 each), and donkey anti-rabbit IgG coupled to
fluorescein isothiocyanate (Chemicon; 1:100). The coverslips were
mounted with the antifadant Citifluor AF1 (Cancer Research Campaign),
and immunoreactivity was visualized by confocal microscopy (Bio-Rad
MRC-600 scanner) in conjunction with a Zeiss Axiophot fluorescence
microscope in 0.5-µm optical sections. Image stacks, confirmed by
single excitation to prevent spillover artifacts, were obtained in
double-excitation mode and recombined with Imaris and Selima
image-processing software (Bitplane AG, Technopark Zurich,
Switzerland).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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To analyze the function of the envelope protein tails, we truncated, elongated, or substituted them. In the F gene, cDNAs of natural mutants isolated from SSPE patients were available (37), whereas H gene deletion mutants were produced by PCR-based mutagenesis. Also, two mutants in which the SeV glycoprotein tails replaced the homologous MV domains were constructed.
A schematic diagram of the cytoplasmic tail mutants is presented in
Fig. 1B. The Fc+28 protein has a tail elongated by 28 amino acids due
to a mutation of the stop codon incurred in the brain of SSPE patient P
(37). The Fc16 and Fc24 proteins have premature stop
codons due to nonsense mutations in the brains of patients F and I,
respectively (10, 37). In the FcSeV protein, we substituted
the 33-amino-acid MV F protein tail by the 43-residue SeV F protein
tail, which is not sequence related. In addition, three H proteins with
tail deletions of different lengths, Hc8, Hc14, and Hc24, were
constructed. Finally, in the HcSeV protein, we substituted the
34-residue MV H tail with the 35-amino-acid SeV tail.
Intracellular transport of tail-altered glycoproteins. The intracellular transport of the altered proteins was characterized in a vaccinia virus T7 RNA polymerase (VV-T7) expression system (9). Transfected cells were pulse-labelled for 1 h with [35S]Met plus [35S]Cys, and the proteins were immunoprecipitated with a polyclonal anti-MV serum after a 3-h chase. As expected, the immunoprecipitated F proteins differed in their mobility (Fig. 2A) and were resolved into several bands. The upper bands correspond to distinct glycosylation stages of the F0 precursor (6, 13). The approximately 40-kDa band represents the nonglycosylated F1 subunit. The observed mobilities correlated well with the predicted size of all the proteins. As observed previously for standard F0 precursors (13), treatment with endo H showed that most oligosaccharides on all F0 precursors had high sensitivity toward this enzyme (Fig. 2A, lanes +), indicating that F0 endo H resistance cannot be used to monitor F protein transport. That the relative transport efficiency of the different F proteins is similar is suggested by the comparable extents of F0 protein cleavage (Fig. 2A).
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The membrane proximal residues of the H tail are necessary for fusion-helper function. The function of the altered envelope proteins was tested by a cell-cell fusion assay (9). F protein-mediated membrane fusion of human or primate cells is dependent on coexpression of the H protein (50). Coexpression of altered F proteins with standard H protein induced fusion to a similar extent to that found with the standard F protein (Fig. 2A, bottom), confirming that intracellular transport was maintained and indicating that fusion function was not impaired.
The fusion assay for the H constructs indicated that the fusion-helper efficiency of Hc8 and Hc14 was comparable to that of standard H
protein (more than 90% of the nuclei in syncytia) whereas Hc24 and
HcSeV manifested either no or a significantly reduced fusion-helper
activity, respectively (Fig. 2B, bottom). Lower fusion-helper activity
can result either from inefficient fusion-helper function or from
decreased levels of cell surface expression. The latter was subjected
to FACScan analysis with a monoclonal antibody directed against the H
ectodomain. We found that Hc24 and HcSeV were expressed at levels
approaching 100% or more than 70% of that of the standard H protein,
respectively. Therefore, surface expression does not account at all
(Hc24) or not entirely (HcSeV) for the drop in the fusogenic
activity of the proteins. The fact that HcSeV and Hc24 but not
Hc14 lost most of their fusion-helper function indicates that
residues proximal to the membrane are crucial for the fusion process.
Viruses with altered glycoprotein tails can be rescued. We then attempted rescue of MVs that harbor envelope proteins with altered cytoplasmic tails by using a recently developed reverse genetic system (32). We constructed full-length antigenomic cDNAs that carry the same mutations as the T7 expression plasmids described above. We transfected these plasmids or the standard plasmid p(+)MV (Fig. 1A, top) in helper cells. With two exceptions, syncytia were observed 3 to 5 days after transfection, suggesting virus rescue.
Plasmids p(+)MV-Hc24 and p(+)MV-HcSeV did not support virus rescue.
The most likely explanation for the failure is the reduced fusion-helper activity of the altered proteins Hc24 and HcSeV. This
may prevent not only efficient cell-cell fusion but also virus-cell
fusion and thus impede virus propagation.
To investigate whether viruses with alterations in both envelope
protein tails could be rescued, we constructed cDNAs in which the
plasmid encoding the shortest H tail (Hc14) was combined with
plasmids coding for three altered F tails (Fc+28, Fc24, or FcSeV).
All three plasmids supported virus rescue.
The MV-specific proteins expressed in cells infected with each of the
nine rescued mutant viruses were analyzed by immunoprecipitation of
pulse-labelled proteins (Fig. 3). Altered
F proteins migrated slight differently from their standard
counterparts, whereas the difference in the mobility of the H proteins
was less pronounced due to the poorer resolution of the gel in this
molecular weight range. The reason for the N protein expression
imbalance in MV-Hc8 is not known.
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8, MV-Hc14, MV-Fc+28, MV-Hc14/Fc+28, and
standard MV, whereas the Hcyt antibody recognized only H proteins from
infection with MV-Fc+28, MV-Fc16, MV-Fc24, MV-FcSeV, and standard
MV (data not shown). We also verified by pulse-chase analysis that the
intracellular processing of the mutated envelope proteins was
comparable to that of the corresponding standard protein and equivalent
to that observed in the VV-T7 system (data not shown). In summary, all
rescued viruses produced proteins of the expected size and
characteristics. No indication for the selection of revertants could be
observed. The fact that not even F tail revertants, which may have
arisen via single point mutations, were observed suggests that
tail-mutated viruses do not have significant selective disadvantages in
cultured cells.
Mutant viruses reach high titers.
To gain insight into the
mode of propagation of the rescued viruses, we performed a one-step
growth analysis (MOI, 3). Figure 4 shows
the growth curves of the relevant single and double mutants (without
the F and H mutants with the shortest deletions, MV-Fc16 and
MV-Hc8). Cell-free and cell-associated viruses were harvested at
12-h intervals.
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76% of nuclei were found in syncytia.
14/FcSeV achieved a higher
titer than its F tail-altered progenitor MV-FcSeV. This may be
explained either by a compensatory effect of the H tail deletion in
MV-Hc14/FcSeV or by an undefined lesion in the MV-FcSeV genome,
which was eliminated when the P-M-F insert was transferred to the
Hc14 genomic plasmid background.
In summary, these results suggest that the glycoprotein tails may not
be required for virus release in Vero cells. However, it must be
considered that MV budding is inefficient and that substantial particle
release may occur even in standard infections at cell death, without
proper budding.
Viruses with altered glycoprotein tails gain cell fusion
competence.
We then asked if alteration of the glycoprotein tails
had other effects on viral propagation, as may have been expected from the characteristics of SSPE-derived MVs. Indeed, microscopic
observation of the morphology of cells infected with the mutants
revealed a markedly different series of events from that for standard
virus. Figure 5 shows the cytopathic
effects (CPE) monitored 12 h (Fig. 5A to E), 36 h (Fig. 5F to
J), or 60 h (Fig. 5K to O) after infection with different MVs.
Standard virus (Fig. 5B, G, and L) caused nearly no CPE during the
first 36 h of infection. Then, within the next 12 h, small
syncytia formed, rapidly rounded up, and later detached. Syncytia
induced by MV-Fc+28 (Fig. 5C, H, and M) and MV-Fc24 (Fig. 4, CPE
panel) grew marginally faster than those induced by standard infection,
whereas syncytia induced by MV-FcSeV mutant grew slowly, correlating
with slower virus growth (not shown).
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14/Fc+28 in
Fig. 5E, J, and O and in the CPE panels in Fig. 4). At 36 h postinfection, almost all the cells were fused in very large syncytia (>100 nuclei), which progressively detached and lysed. Viruses with
mutations only in the H tail had an intermediate form of CPE, that is,
extensive cell fusion but with slower kinetics (MV-Hc14 [Fig. 5D,
I, and N]). These data indicate that alterations of the glycoprotein
tails, especially deletion of the membrane-distal 14 residues of the H
tail, result in enhanced cell-to-cell fusion.
Intracellular M-F-H protein colocalization does not require intact glycoprotein tails. We recently observed (8) that infection of cells with an M protein-deficient MV leads not only to faster cell-to-cell propagation but also to a homogeneous distribution of the viral glycoproteins over the entire cell surface, rather than to their concentration in patches as in MV standard infections. To investigate the effect of alterations of the glycoprotein cytoplasmic tails on viral envelope assembly, the intracellular localization of the envelope proteins was characterized by confocal microscopy.
We compared a standard MV infection (Fig. 6A to C and G to I) with an infection with the shortest viable H protein tail mutant, MV-Hc14 (Fig. 6D and J), the F protein tail exchange mutant
MV-FcSeV (Fig. 6E and K), and the double mutant MV-Hc14/FcSeV (Fig.
6F and L). In a standard infection, the F protein (Fig. 6A), the H
protein (Fig. 6G), and the M protein (Fig. 6B and H) were concentrated in bright patches. Superimposition of the two image pairs revealed colocalization of the glycoproteins with M protein (Fig. 6C and I).
Internal patches (Fig. 6C) and plasmalemma-proximal patches (Fig. 6C)
were observed. Extensive colocalization of the glycoproteins with M
protein was observed in cells infected with every mutant virus,
including MV-Hc14 (Fig. 6D and J), MV-FcSeV (Fig. 6E and K), and the
double tail mutant MV-Hc14/FcSeV (Fig. 6F and L). This indicates
that assembly of the viral envelope can occur even when the
glycoproteins have altered tails.
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The tails determine envelope protein incorporation efficiency. To further characterize the effects of F and H protein tail alterations on virus assembly, we analyzed the composition of released MV particles. To obtain sufficient particle yields, supernatants of infected cells had to be collected late in infection, when cell lysis was already important. It is thus unknown how many of the analyzed particles were virions which completed budding and how many were released from dying cells. Particles were prepared by pelleting onto a sucrose cushion and subsequent purification by equilibrium centrifugation.
Figure 7 shows the protein composition and sucrose gradient migration characteristics of particles from standard MV (Fig. 7A), MV-Hc14 (Fig. 7B), MV-FcSeV (Fig. 7C), and
MV-Hc14/FcSeV (Fig. 7D). In the standard MV infection, the five
major structural MV proteins (labelled from the top H, P, N, F1, and H)
represented the large majority of the protein detected and were
concentrated in fraction 7 and in the two neighboring fractions. A
sixth strong band, migrating slightly above F1 (labelled X), may
represent cellular actin (47).
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14/FcSeV
(Fig. 7D).
Moreover, although the viral glycoproteins were produced and
transported at similar ratios in infected cells (Fig. 3), differences in the envelope protein composition of the mutant particles were observed. In standard MV particles (Fig. 7A), the N/H/F1/M
signal ratio determined by densitometry was approximately 1:1:1:1. In MV-Hc14 particles, it was 10:3:5:5 (Fig. 7B), reflecting a slightly less efficient incorporation of all viral envelope proteins, in particular of H. A protein ratio in MV-FcSeV particles was difficult to
determine because of the high background of cellular proteins (Fig.
7C), but selective loss not only of F protein but also of M protein was
evident. Finally, in MV-Hc14/FcSeV the amount of cellular proteins
was greatest and that of viral envelope proteins was smallest. We
attempted to analyze mutant virus particles by electron microscopy, but
the analysis of standard MV particles (43) confirmed their
extremely pleomorphic nature (21, 24, 28), rendering a
prospective comparative study difficult to interpret. The above data
indicate that the glycoprotein tails are important determinants of
virus particle assembly and that the F tail may determine the
efficiency of M protein incorporation in the envelope.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Our results indicate that in MV infections an important function of the glycoprotein tails is to regulate cell fusion. Accurate MV envelope assembly also depends on the glycoprotein tails; the envelopes of tail-altered viruses are adulterated with cellular proteins, but these viruses remain infectious.
Glycoprotein tails and envelope assembly. We observed that glycoproteins with altered tails are incorporated in the MV envelope less efficiently than are standard glycoproteins and that the envelopes of tail-altered MVs are adulterated with cellular proteins. The presence of the homotypic glycoprotein tails was previously shown to contribute to the specificity of envelope assembly in several viruses (22, 27, 30, 38). It is thus possible that adulteration of the MV envelope is a consequence of the absence of the homologous interactions between the glycoproteins and internal proteins, as demonstrated for a retroviral envelope (46).
All mutant viruses retained high levels of infectivity, indicating that adulteration of their envelope did not cause major problems at cell entry. This may not be so surprising, since even the well-structured vesicular stomatitis virus (VSV) particles can tolerate insertion of foreign glycoproteins (up to 35% of the own envelope glycoprotein weight) without losing much infectivity (19, 38). An important observation is that the F tail determined not only the efficiency of F protein incorporation into the envelope but also that of the M protein. This may be due to a direct interaction, since it was recently shown that a hybrid VSV glycoprotein with an F tail directed the incorporation of the MV M protein in the envelope of the MV-VSV chimera whereas a standard VSV glycoprotein did not (44). Since even tail-altered glycoproteins colocalized with M protein in intracellular patches, additional interactions may exist. These interactions could be direct, involving the transmembrane domains of the two glycoproteins, or indirect, involving membrane rafts (41) in which the two viral glycoproteins may be enriched.Glycoprotein tails and cell fusion. The F tail and/or the M protein are invariably altered in the brains of SSPE patients. This observation led to the suggestion that impairment of the F tail-M protein interaction may favor MV propagation in brain cells (1). In line with the above suggestion, we demonstrate here that the modification of the F or H glycoprotein tail, respectively, in the background of a standard genome results in increased fusion competence of the mutated viruses. Double tail mutants have the highest fusion competence, which is comparable to that of a rescued M-less virus (8).
Why are MVs with altered F tails, but not those with altered H tails, found in the brains of SSPE patients? The reason lies in the structure of the F and H genes. In the F coding region, single point mutations introducing premature stop codons can lead to the formation of proteins with shorter tails (37). In the H coding region, two combined point mutations (one eliminating the initiator methionine codon and a second introducing a new one) or a deletion would be needed to produce a tail-altered H protein. Thus, MV variants with truncated F tails are likely to appear much more frequently than those with altered H tails. The enhanced cell fusion of SSPE-derived MV strains and that of the glycoprotein tail mutants produced in this study can be explained by considering a regulatory role of the M protein on fusion function. M may lock the F and H protein oligomers in a fusion-inefficient conformation, which may be necessary to limit fusion of intracellular membranes once the fusion peptide has been liberated. Upon virus release, an external stimulus may result in a conformational change of the M-F-H complex, which then becomes fusion competent. A fusion-refractory state of the envelope protein complex has been previously postulated based on morphological observations of particles released by another paramyxovirus, Newcastle disease virus La Sota strain. Immature, noninfectious Newcastle disease virus La Sota particles possess a regular arrangement of envelope protein spikes (34) which may reflect the M protein organization at the inner surface of the lipid bilayer (4). It was proposed that the regular arrangement of the spike proteins coincides with a fusion-refractory conformation. Upon maturation this regular arrangement disappears and fusion-competence is established (34). The cytoplasmic tails of the envelope proteins of certain retroviruses have also been shown to regulate fusion efficiency. In particular, the expression of truncated forms of simian immunodeficiency virus envelope proteins in CD4-positive cells caused enhanced syncytium formation (33, 51, 53). Moreover, removal of a short peptide of the Moloney murine leukemia virus and of a type D retrovirus during viral maturation renders the envelope protein competent for fusion (2, 14). We suggest that regulation of fusion competence via the envelope protein tails is a widespread mechanism, especially relevant for the propagation of certain cell-associated enveloped viruses.| |
ACKNOWLEDGMENTS |
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We thank Roland Naef for help with confocal microscopy, Martin A. Billeter for critical reading of the manuscript and continuous support, and Clas Örvell, Tamas Varsanyi, and Erling Norrby for generous gifts of antibodies. Plasmids pGem4-SVF0 and pGem4-SVHN were kind gifts of Laurent Roux.
This work was supported by grants 31-29343.90 (START) and 31-45900.95 from the Swiss National Science Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Molekularbiologie, Abteilung I, Universität Zürich, Hönggerberg, CH-8093 Zürich, Switzerland. Phone: 41 1 633 2492. Fax: 41 1 371 7205. E-mail: cattaneo{at}molbio1.unizh.ch.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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J Virol, February 1998, p. 1224-1234, Vol. 72, No. 2
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