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Journal of Virology, October 1998, p. 7885-7894, Vol. 72, No. 10
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Analysis of Constructed E Gene Mutants of Mouse
Hepatitis Virus Confirms a Pivotal Role for E Protein in
Coronavirus Assembly
Françoise
Fischer,1,
Carola F.
Stegen,2,
Paul S.
Masters,1,3,* and
William A.
Samsonoff1,3
Departments of Biomedical
Sciences1 and
Biological
Sciences,2 State University of New York at
Albany, and
Wadsworth Center for Laboratories and Research,
New York State Department of Health,3 Albany,
New York 12201
Received 20 April 1998/Accepted 8 July 1998
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ABSTRACT |
Expression studies have shown that the coronavirus small envelope
protein E and the much more abundant membrane glycoprotein M are both
necessary and sufficient for the assembly of virus-like particles in
cells. As a step toward understanding the function of the mouse
hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the
resulting mutations into the MHV genome by targeted recombination. Of
the four possible clustered charged-to-alanine E gene mutants, one was
apparently lethal and one had a wild-type phenotype. The two other
mutants were partially temperature sensitive, forming small
plaques at the nonpermissive temperature. Revertant analyses of these
two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that
there was a flaw in their assembly. Most significantly, when virions of
one of the mutants were examined by electron microscopy, they were
found to have strikingly aberrant morphology in comparison to the wild
type: most mutant virions had pinched and elongated shapes that
were rarely seen among wild-type virions. These results demonstrate an
important, probably essential, role for the E protein in coronavirus
morphogenesis.
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INTRODUCTION |
Coronaviruses are enveloped
positive-strand RNA viruses that contain, at a minimum, four structural
proteins (39). One of these, the nucleocapsid protein (N),
resides in the interior of the virion, encapsidating the large (27- to
32-kb) viral genome. Surrounding the nucleocapsid is an envelope
derived from the membrane of the compartment between the endoplasmic
reticulum and the Golgi, into which virion budding takes place.
Embedded in this envelope are the other three structural proteins: the
spike glycoprotein (S), the membrane glycoprotein (M), and the small
membrane or envelope protein (E; formerly m or sM). Two additional
structural proteins, a hemagglutinin-esterase glycoprotein (HE)
(7) and the product of the internal open reading frame (ORF)
of the N gene (I protein) (13, 38), are absent in a number
of coronavirus species or strains and are not essential for
infectivity.
For the membrane-bound proteins, the importance of S in receptor
binding and fusion and of M as the predominant constituent of
virion architecture has been understood for some time. The significance
of E, however, has come to be appreciated only relatively recently. E
originally had the status of a hypothetical product of a small ORF
revealed by cDNA sequencing (5, 40). Subsequently, it was
shown to be translated both in vitro (8, 25) and in vivo
(1, 16, 23, 25). This latter point, that E protein is
actually expressed during infection, could not be presumed a priori
because in mouse hepatitis virus (MHV) and avian infectious bronchitis
virus (IBV), the coding region for E is found as the most downstream
ORF in a bi- or tricistronic mRNA (5, 40). Next, contrary to
most earlier assumptions, it was shown for IBV (26), for
porcine transmissible gastroenteritis virus (16), and
finally for MHV (47) that the E protein is a coronavirus structural protein. This finding had probably eluded prior
analyses because of the small size (9 to 12 kDa) and low abundance of E protein in virions.
The potential role of E protein in viral infection remained speculative
until landmark studies by Vennema et al. (42) and Bos et al.
(4) examined the assembly of virus-like particles resulting
from the intracellular expression of MHV proteins in various
combinations. The surprising outcome of these investigations was that
the E and M proteins were shown to be both necessary and sufficient for
the formation and extracellular release of particles appearing
structurally identical to MHV virions (except for the absence of S
protein spike projections on their surface if this protein was not
coexpressed). This discovery established a new paradigm for
enveloped-virus assembly and motivated us to seek genetic evidence
supporting such a critical role for E protein in the whole virus.
The genetic manipulation of MHV and other coronaviruses currently is
less straightforward than for other positive-strand RNA viruses. For
the large genomes of members of this family, it has not yet been
possible to construct full-length cDNA clones from which, in principle,
infectious RNAs could be transcribed. We have been able to
generate site-directed mutations in MHV by targeted recombination
between transfected synthetic donor RNA species and recipient mutant
viruses that can be selected against (13, 14, 19, 22, 32,
33). In the present study, this technique was taken a step
further in that engineered mutants were identified by screening rather
than by selection, thereby allowing the construction of mutants that
were almost as defective as the parental virus from which they were
derived. We chose to create mutants of the E protein of MHV by
clustered charged-to-alanine mutagenesis, a genetic strategy that
comprehensively surveys the effects of localized surface alterations on
a given protein (2, 3, 44). It is based on the supposition
that clusters of charged amino acid residues are more likely to appear
on the surface, rather than the interior, of proteins and that
these often make strong contributions to protein-protein interactions.
Such interactions are expected to be destabilized by the replacement of
each member of a charge cluster with the small aliphatic side chain of
alanine, a substitution which ought to have benign consequences
with respect to normal protein folding. Clustered
charged-to-alanine mutagenesis has yielded a remarkably high proportion
of conditional-lethal mutants (including temperature-sensitive lethal
mutants) in studies of actin (44), 
-tubulin
(34), the poliovirus polymerase (11) and the
vaccinia virus G2R protein (17); however, some proteins may
be intractable to this approach (45). We constructed MHV E
protein mutants by applying a fixed algorithm in which, for each
instance where two or more charged amino acids appeared within a
sliding window of 5 residues, a mutant was constructed that replaced all of these charged residues with alanine (44).
Two of the mutants obtained were partially temperature sensitive and were dramatically defective in virion assembly.
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MATERIALS AND METHODS |
Virus and cells.
Wild-type, mutant, and revertant virus
stocks of all MHV strains were propagated in mouse 17 clone 1 (17Cl1)
cells. The sources of strains MHV-1 and MHV-3 were described previously
(30). It should be noted that our laboratory strain of MHV-3
differs from the Bicêtre strain of MHV-3 (9). MHV-2
and MHV-DVIM were generously provided by Ehud Lavi (University of
Pennsylvania School of Medicine) and Kathryn Holmes (University of
Colorado Health Sciences Center), respectively. Plaque titer
determinations and plaque purifications were carried out with mouse L2
cells, which were also maintained in spinner culture for RNA
transfection via electroporation, as described previously
(29).
Cloning and sequencing of the E gene of various MHV strains.
A region encompassing the E genes of MHV-1, MHV-2, MHV-3, and MHV-DVIM
RNA isolated either from infected cells or from purified virus was
amplified by reverse transcription followed by PCR (RT-PCR), with
primers corresponding to nucleotides (nt) 147 to 164 of gene 5a and nt
244 to 261 of the M gene. Although these primers were based on the
known sequence of MHV-A59, a 710-bp PCR product was successfully
obtained for each of the four other strains and was cloned into a pCR
TA cloning vector (Invitrogen). A consensus nucleotide sequence was
determined from three or four clones for each MHV strain.
Plasmid constructs.
T7 transcription vectors generated for
the transfer of mutations into the E gene of MHV were derived from
pCFS8 (14), which encodes a pseudo-defective interfering
(DI) RNA that includes the complete S gene and all of the MHV-A59
genome downstream of S through the poly(A) tail. As detailed previously
(14), in this region pCFS8 contains two differences from the
authentic MHV sequence: (i) a coding-silent change in the S gene that
eliminates a HindIII site, and (ii) a phenotypically
silent 19-nt tag at the start of gene 4 that contains an
XbaI site.
Clustered charged-to-alanine mutations 1 to 4 were initially created in
pCFS1 (14), a precursor of pCFS8 that contains the segment
extending from the MluI site at nt 3262 of the S gene through the KpnI site at nt 394 of the M gene (see Fig. 3).
Mutations 1 and 2 (see Fig. 2) were each generated by a single step of
PCR mutagenesis followed by replacement of the
BstEII-EcoRV fragment of pCFS1 with the
BstEII blunt-end fragment of the mutant PCR product to yield
pCFS9 and pCFS10, respectively. Mutations 3 and 4 were generated by
splicing overlap extension PCR mutagenesis (18), and the
BstEII-EcoRV fragment of each mutant PCR product was used to replace the corresponding segment of pCFS1 to yield pCFS11
and pCFS15, respectively. Mutations 1 to 4 were then transferred into
the pseudo-DI vector by incorporation of the
MluI-EagI fragment of pCFS9 or the
XbaI-EcoRV fragment of pCFS10, pCFS11, or pCFS15 in place of the corresponding fragment of pCFS8 to produce pFF45-pFF48, respectively.
Second-site reverting mutations, in conjunction with the original
mutations of mutant 2 (Alb154), were also introduced into MHV via
transcription vectors derived from pCFS8. For the construction of
these, total RNA isolated from cells infected with Alb154RevA, Alb154RevB, Alb154RevD, or Alb154RevI was reverse transcribed under
standard conditions (35) with a random primer,
p(dN)6 (Boehringer Mannheim). The 170-bp region from gene
5a through the M gene (see above) of each cDNA was amplified by PCR,
restricted with BstEII and EcoRV, and, together
with the XbaI-BstEII fragment of pCFS8,
incorporated in place of the XbaI-EcoRV fragment
of pCFS8 in a three-way ligation to produce plasmids pFF61 to pFF64.
Standard techniques were used for all recombinant DNA manipulations
(
35). The composition of all constructed plasmids was
verified by restriction analysis; all PCR-generated regions and
newly
created junctions of each plasmid were verified by DNA sequencing
by
the method of Sanger et al. (
36) with modified T7 DNA
polymerase
(Sequenase; U.S. Biochemical Co.) or by automated sequencing
with
an Applied Biosystems 373A or 377 DNA sequencer.
Targeted recombination and identification of recombinant mutant
viruses.
E gene mutations were transduced into the MHV genome by
the targeted recombination method previously used to incorporate
mutations into the S (14), M (10), and N
(13, 32, 33) genes, gene 4 (14), and the 3'
untranslated region (19, 22). Mutant viruses were obtained
as progeny recombinants arising from transfection of synthetic
pseudo-DI RNAs from HindIII-truncated pFF45-48 and pFF61-64 into cells infected with the temperature-sensitive and thermolabile N gene mutant, Alb4. Details of RNA synthesis and transfection were as described previously (14, 29).
Of the four clustered charged-to-alanine mutants, only mutant 2 was
selected as a recombinant capable of forming large (i.e.,
wild-type-size) plaques at 39°C, the nonpermissive temperature
for
Alb4. Three candidate plaques for this mutant were analyzed
by RT-PCR
with a primer pair flanking the locus of the Alb4 deletion
and with a
primer pair flanking the gene 4 tag (see Fig.
3). The
repair of the
Alb4 deletion was ascertained by the size of the
PCR product from the
former (
22). The PCR product from the latter
was digested
with
XbaI to determine the presence of the gene 4
tag.
Following sequence verification, a recombinant from this
set was
designated Alb153.
Since targeted recombination with donor RNA containing the other three
mutations did not yield large-plaque mutants at 39°C,
a screening
approach was used for their identification. Following
heat treatment to
counterselect Alb4 parental virus (
22), surviving
plaques
obtained at 33°C were combined in multiple pools of 10
plaques and
RNA was isolated from a cell monolayer infected with
each pool and was
analyzed by RT-PCR. Recombinants were detected
with primer pairs in
which one primer originated within the heterologous
19-nt gene 4 tag or
in which one primer originated within the
region that is deleted in the
Alb4 N gene. Plaques from pools
giving a positive PCR signal for one of
these markers were subsequently
analyzed by RT-PCR.
Individual candidates for mutant 3 (20 plaques) and mutant 4 (40 plaques) were then screened for the gene 4 tag or for repair
of the
Alb4 deletion with primer pairs flanking or originating
within these
markers, as above. Positive candidates were then
screened by RT-PCR
amplification of the E gene and tested for
the presence of an
FspI site created by the mutations in mutant
3 or mutant 4 (see Fig.
2). One mutant of each type was found
in this search, and
following sequence verification, they were
designated Alb154 (mutant 3)
and Alb183 (mutant 4). A similar
screening procedure was used to search
for mutant 1. In this case,
the RT-PCR product derived from the E gene
was tested for the
loss of the
EcoRV site that should be
disrupted by one of the
mutations in this mutant (see Fig.
2). In
multiple trials, no
recombinant meeting this criterion was found.
Final confirmation of the composition of each of the three successful
recombinants was obtained by sequencing purified genomic
RNA to
establish the presence of the E gene mutations, the gene
4 tag, and the
region that is absent in the Alb4 N gene deletion.
MHV recombinants
were purified by polyethylene glycol precipitation
followed by two
cycles of equilibrium centrifugation on preformed
potassium
tartrate-glycerol gradients, and viral genomic RNA was
isolated exactly
as described previously (
13,
22). For the
isolation of total
cytoplasmic RNA from virus-infected 17Cl1 cell
monolayers, a Nonidet
P-40 gentle lysis procedure was used (
21).
Direct RNA
sequencing was carried out by a modification of a dideoxy
termination protocol (
12,
32).
Revertant analysis.
Independent spontaneous revertants of
mutant 3 (Alb154) and mutant 4 (Alb183) were isolated starting from
individual plaques obtained at 39°C from passage 2 stocks. Each
plaque was serially passaged 10 times (mutant 3) or 11 times (mutant 4)
at 37°C at an estimated multiplicity of 0.2 PFU/cell. Following the
final passage, revertants were isolated as viruses forming large
(wild-type-size) or intermediate-size (between mutant size and
wild-type size) plaques at 39°C. Mutations responsible for reversion
were determined by sequencing of the E gene and other genes in RNA from
cells infected with purified plaques or in purified virion RNA.
Electron microscopy.
Samples of mutant 4 (Alb183) and the
isogenic wild-type strain Alb129, freshly passaged at 33°C, were
harvested, clarified of cell debris by centrifugation for 10 min at
1,200 × g, and used directly for electron microscopy
without further concentration or purification (see Fig. 7 and 8). In a
separate experiment (see Table 2), mutant 4 and wild type were freshly
passaged at 33 and at 39°C and clarified and 8-ml samples were then
concentrated by centrifugation for 2 h at 151,000 × g through 1.5 ml of 50 mM Tris-maleate (pH 6.5)-1 mM
EDTA-10% glycerol onto a cushion of 100% glycerol in a Beckman SW41
rotor at 4°C. Viral pellets were resuspended in 1 ml of 50 mM
Tris-maleate (pH 6.5)-1 mM EDTA. Drops (40 µl) of the
different viral suspensions were placed on dental wax, and previously
glow-discharged, Formvar-coated copper grids were placed on the drops
for 2 to 4 min. Excess liquid was removed by wicking with filter paper,
and the grids were immediately washed by floating on one or two drops
of 10 mM ammonium acetate. After the final wash, the grids were briefly
floated on 2% sodium phosphotungstate (pH 7.0), the excess stain
liquid was removed by wicking, and the samples were viewed in a Zeiss
(LEO) 910 transmission electron microscope operating at 80 keV.
Micrographs were recorded at a magnification of ×25,000, and the
negatives were enlarged photographically.
Nucleotide sequence accession numbers.
The coding regions of
the E genes of MHV-1, MHV-2, MHV-3, and MHV-DVIM have been assigned
GenBank accession no. AF051146, AF051147, AF051148, and AF051149,
respectively.
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RESULTS |
Comparison of MHV E protein sequences.
As a preliminary step
in establishing the importance of different regions and residues of the
E protein, we determined the sequences of the E genes of MHV-1, MHV-2,
MHV-3, and MHV-DVIM. An alignment of the resulting deduced amino acid
sequences with that of our reference strain, MHV-A59, and with those
previously determined for MHV-S and MHV-JHM, was performed to obtain a
sampling of the range of sequence variation allowed for the E
protein (Fig. 1). This comparison presumes, and all existing
interstrain recombinational evidence supports the idea, that these E
proteins are functionally interchangeable.
As shown in Fig.
1, the MHV E proteins
are all quite small, comprising 83 to 88 residues (9.6 to 10.2 kDa),
and exhibit a
high degree of sequence identity (89 to 98%). Almost
one-third
of the protein is made up of a relatively long hydrophobic
region,
a putative membrane anchor spanning residues V10 through I37.
Although the topology of the E protein has not been unequivocally
established, evidence from a study of the E protein of transmissible
gastroenteritis virus (
16) suggests that the molecule is in
a C-exo N-endo configuration. The membrane anchor is highly conserved
among the seven strains of MHV and is flanked by two invariant
charged
residues (D8 and K38). Four absolutely conserved cysteine
residues
(C23, C40, C44, and C47), which are potential sites for
palmitoylation
(
47) or interchain disulfides, are found within
the membrane
anchor or adjacent to it on the carboxy side. The
only hydrophilic
portion of the molecule occurs near the carboxy
terminus. Although the
charged residues here are highly conserved,
this segment represents the
most divergent part of the E protein,
most notably at the extreme
carboxy terminus, which shows a 5-amino-acid
extension (IIQTL) in many
of the strains.
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FIG. 1.
Amino acid sequence comparison of the E proteins of
seven MHV strains. The deduced MHV-1, MHV-2, MHV-3, and MHV-DVIM E
sequences were determined in the present work. The E sequences of
MHV-A59 (8), MHV-JHM (40), and MHV-S (31,
46) have been reported previously (GenBank accession numbers
M16602, X04997, and M64835, respectively). Spaces in the alignment
indicate positions at which the amino acid is identical to that of
MHV-A59; dots denote every 10th residue.
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Generation of clustered charged-to-alanine mutants of the MHV E
protein.
To address whether the E gene is an essential gene, we
first attempted, by targeted RNA recombination, to create a null mutant of MHV-A59 in which the E gene was replaced by the gene for the green
fluorescent protein. While we could successfully construct multiple
recombinants in which gene 4 was replaced by the green fluorescent
protein gene (14), we were not able to generate such a
deletion in the E gene despite repeated efforts. Although this
negative result could not be interpreted unequivocally, it implied that
ablation of the E gene would be lethal to the virus.
We thus sought next to construct conditional lethal E gene mutants by
using the strategy of clustered charged-to-alanine mutagenesis
(
2,
3,
44). Owing to the small size and limited hydrophilicity
of the
E protein, the algorithm of Wertman et al. (
44) suggested
only four possible sets of mutations, all of them falling in the
carboxy-terminal one-third of the molecule (Fig.
2). The four
clustered charged-to-alanine
mutants were constructed in transcription
vectors that also contained a
phenotypically silent marker in
gene 4 (
14) and were then
introduced into the MHV genome by
targeted recombination. Synthetic
mutant donor RNAs were independently
transfected into cells infected
with the N gene deletion mutant
Alb4 (Fig.
3), and released progeny viruses were
harvested and
analyzed for the desired mutations. Since we were
interested in
generating temperature-sensitive mutants, plaques of
progeny virus
were first assayed under nonpermissive conditions
(39°C), either
directly or with prior heat treatment, to eliminate
mutants capable
of forming large (i.e., wild-type-size) plaques at this
temperature.
Only one of the four possible mutants, mutant 2 (designated Alb153),
was found to fall into this category and its
apparently wild-type
phenotype was not characterized further. Although
viruses forming
large plaques at 39°C were identified among progeny
from the other
three donor RNAs, these proved to be either revertants
of Alb4
or recombinants that had repaired the Alb4 deletion but did not
contain the upstream E gene mutations.
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FIG. 2.
Strategy for mutagenesis of the MHV-A59 E gene. The
putative membrane anchor, charged amino acid residues, and four
possible clustered charged-to-alanine mutants are indicated above the E
protein sequence. Beneath are shown the base changes made in
transcription vectors to change charged residues to alanines. The
EcoRV site near the end of the E coding sequence and the
FspI site created by the mutations in mutant 3 and mutant 4 are underlined.
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FIG. 3.
Incorporation of clustered charged-to-alanine mutations
into the MHV genome by targeted recombination. Sets of mutations
engineered into subclones pCFS9, pCFS10, pCFS11, and pCFS15 were
transferred to pCFS8 (14) to produce transcription vectors
pFF45, pFF46, pFF47, and pFF48, respectively, which encode pseudo-DI
RNAs comprising a 5' segment of the MHV genome fused, at the start of
the S gene, to the entire 3' end of the genome. E gene mutations are
represented by a star; the solid rectangle indicates the 19-nt tag at
the start of gene 4. Restriction sites shown are those relevant to
plasmid construction, as detailed in Materials and Methods. Brackets
designate gene fragments rather than entire genes.
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Other clustered charged-to-alanine mutants had to be identified by
molecular screening, because there was no other clear basis
for
differentiating them from the Alb4 parent, which is also temperature
sensitive. Since we expected that the E mutants would be temperature
sensitive but not thermolabile (or not as thermolabile as Alb4
[
22]), progeny viruses from targeted recombination
were heat
treated (
22) to enrich for potential recombinants.
In retrospect,
this may have hindered rather than helped subsequent
screening.
Pools of candidate recombinants, obtained as progeny plaques
at
the permissive temperature of 33°C, were assayed by RT-PCR, and
individual plaques from pools that gave positive signals were
then
analyzed further, as described in Materials and Methods.
This process
yielded a recombinant of mutant 3 (Alb154) and a
recombinant of mutant
4 (Alb183). A recombinant representing mutant
1 was never obtained
among the numerous candidates screened, suggesting
that this mutant, if
not lethal, was more impaired than the Alb4
parent. Analysis of mutants
2, 3, and 4 by RT-PCR and by direct
sequencing of purified
genomic RNA revealed that each had the
constructed set of four
or five point mutations in the E gene,
resulting in the conversion of
two or three charged residues to
alanines (Fig.
4). The sequence shown in Fig.
4 for
Alb183 was
obtained with considerable difficulty owing to the small
amount
of genomic RNA that could be purified from this virus,
possibly
because of virion fragility (see below). The mutations in this
virus were verified in total cellular RNA isolated from Alb183-infected
cells and also in revertants of Alb183. As expected, the Alb4
N gene
deletion in each of the three recombinants had been repaired,
and in
addition, the gene 4 tag from the donor RNA was present
(data not
shown). This distribution of markers was consistent
with each
recombinant having been generated by a single crossover
event upstream
of gene 4 (Fig.
3).
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FIG. 4.
Portions of genomic RNA sequence within the E
genes of clustered charged-to-alanine mutant 2 (Alb153), mutant 3 (Alb154), and mutant 4 (Alb183). The lane markers indicate the
terminating dideoxynucleoside triphosphate for each reaction. The
segments of sequence show the derived positive-sense RNA sequence with
its corresponding translation adjacent. Stars denote mutated
nucleotides.
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At elevated temperatures, the mutant 3 and mutant 4 recombinants,
Alb154 and Alb183, each produced significantly smaller plaques
than
those of wild-type MHV-A59 or Alb129, an isogenic recombinant
that
contains the gene 4 tag but no other mutations (
14).
As
indicated in Table
1, this
partially temperature-sensitive phenotype
was evident at both 37 and 39°C. At either of these temperatures,
each mutant produced
plaques that were less than one-third the
diameter of wild-type
plaques, although they were not as small
as plaques of the N gene
deletion mutant Alb4.
Revertant analysis.
The sizes of plaques of mutants 3 and 4 differed sufficiently from those of plaques of the wild type to serve
as the basis for the isolation of revertants. Following either 10 or 11 serial passages at the nonpermissive temperature of virus stocks
originating from independent plaques of mutant 3 or mutant 4, spontaneous revertants were selected as viruses able to form plaques
detectably larger than those of the mutant from which they were
derived. In most cases, revertants formed wild-type-size plaques
at 39°C; in some cases, the revertant plaque size was intermediate
between those of the wild type and the mutant. It should be noted that despite the selective pressure applied over multiple passages, not
every stock yielded a revertant.
For mutant 3, nine independent revertants were isolated. Sequencing of
the E genes of these revealed that each contained a
single second-site
mutation in the E protein and that they could
be divided into five
groups (Fig.
5). Similarly, 14 independent
revertants of mutant 4 were isolated and were divided into
eight
groups (Fig.
5). In most cases, each mutant 4 revertant also
contained
a single second-site mutation. The two exceptions to this
observation,
mutant 4 RevC and mutant 4 RevM, each contained an
additional
mutation that was most probably of no significance. For
mutant
4 RevC, the S62I mutation that it had in common with mutant
4
RevG must have been sufficient for reversion; likewise, for mutant
4 RevM, the S62C mutation that it had in common with mutant 4
RevK and
mutant 4 RevP must have been sufficient for reversion.
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FIG. 5.
Revertant analysis of mutant 3 (Alb154: K63A, K67A) and
mutant 4 (Alb183: D60A, R61A, K63A). For each mutant or revertant, only
amino acid residues differing from the wild-type MHV-A59 residue (top
line) are shown; second-site mutations in the revertants are in
boldface type.
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Almost all of the second-site reverting mutations of both mutant 3 and
mutant 4 fell at one of three positions close to the
original clustered
charged-to-alanine mutations in the E gene,
and almost all of these
(S55A, S62I, S62C, and Y66C) occurred
in both revertant sets (Fig.
5).
Surprisingly, the two revertants
that deviated from this pattern,
mutant 3 RevI and mutant 4 RevA,
each had a relatively conservative
amino acid substitution within
the putative membrane anchor (V31A and
T27A, respectively). This
may suggest either that compensatory
structural changes within
the membrane domain of E protein can be
transmitted to the ectodomain
or else that monomer-monomer interactions
within the membrane
can make up for a loss of interaction between
ectodomain moieties.
To ascertain whether the observed second-site mutations were indeed
responsible for reversion, we analyzed in more detail
a subset of
mutant 3 revertants representative of each position
at which
second-site mutations were found. Initially, we sequenced
the entire M
gene of each member of this group, mutant 3 RevA,
mutant 3 RevB, mutant
3 RevD, and mutant 3 RevI, to check for
the possible presence of
mutations in this protein, which potentially
interacts directly with
the E protein (
4,
42). No mutations
were found in any of the
M genes of these revertants. Next, we
attempted to introduce each
reverting mutation, in conjunction
with its cognate set of clustered
charged-to-alanine mutations,
into the MHV genome. This was
carried out by targeted recombination
between the appropriate donor RNA
construct and the Alb4 N gene
deletion mutant. For mutant 3 RevA,
mutant 3 RevB, and mutant
3 RevD, recombinants were obtained that
contained both the mutant
3 clustered charged-to-alanine mutations and
the corresponding
second-site mutation. Since all of these produced
large plaques
at 39°C, this demonstrated that the E gene second-site
mutation
found in each of these revertants was sufficient to compensate
for the original mutations of mutant 3. However, for reasons that
we
currently do not understand, repeated attempts to generate
a
recombinant corresponding to mutant 3 RevI were unsuccessful.
This
result, coupled with the unexpected membrane anchor location
of the
second-site mutation in mutant 3 RevI, prompted us to sequence
the
remaining structural genes (S and N) of this virus as well
as the
packaging signal domain within gene 1b (
15). No mutations
other than that in the E protein (V31A) were found. Thus, although
we cannot rule out the possibility of a compensating mutation
in a
nonstructural gene, it is likely that the V31A mutation accounted
for
the reversion of mutant 3 RevI.
Taken together these analyses confirmed that for both mutant 3 and
mutant 4 one or more of the clustered charged-to-alanine
mutations
constructed in the E gene was responsible for the observed
plaque size
phenotype. Moreover, the results demonstrated that
a single second-site
amino acid change within the E protein was
able to reverse the
phenotypic effect of the original mutation(s).
No intergenic
suppressors of the mutant 3 or mutant 4 mutations
have yet been found.
Phenotype of E protein mutants.
Difficulties encountered in
the preparation of purified virions of mutant 3 and mutant 4 led us to
suspect that there might be defects in their structural integrity. To
test the thermolability of these mutants, stocks of mutant 3, mutant 4, and their isogenic wild-type counterpart, Alb129, were grown at 33°C
and also at 39°C. Released viruses were then heat treated for various
intervals at 40°C and pH 6.5 (22), and survivors were
measured by plaque titer determination at 37°C. As shown in Fig.
6A, virions of the two mutants were at
least an order of magnitude more thermolabile than were wild-type
virions when grown at 33°C. By 24 h of heat treatment, the
infectious titers of mutant 3 and mutant 4 had dropped 15- and 78-fold,
respectively, more than that of the wild type. In contrast, when
grown at 39°C, both mutant 3 and mutant 4 were much less
strongly affected by heat treatment and did not differ significantly
from the wild type, which showed roughly the same heat sensitivity at
39°C as at 33°C (Fig. 6B). However, the infectious titers of stocks
of the two mutants grown at 39°C were markedly lower than those of
stocks grown at 33°C. One explanation consistent with these data is
that for the two mutants grown at 33°C, most virions were assembled
in a flawed manner that was sufficient for their stability at the
lower, permissive temperature but led to a loss of viability upon heat
treatment. Conversely, for mutant viruses grown at 39°C, only a
relatively small fraction of virions assembled successfully, but these
had passed some threshold of structural stability and were no more
susceptible to heat treatment than were wild-type virions. Further work
is required to more precisely elucidate the basis for the
thermolability differences observed in Fig. 6. It should be noted that
both the plaque size and the frequency of survivors of heat treatment
among mutants 3 and 4 grown at 39°C ruled out the possibility that
these survivors were revertants.
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|
FIG. 6.
Thermal inactivation of mutant 3 (Alb154: K63A, K67A),
mutant 4 (Alb183: D60A, R61A, K63A), and the isogenic wild-type strain
(Alb129). (A) Passage 3 stocks of each virus were grown at 33°C and
thermally inactivated at 40°C (pH 6.5) for the indicated times
(22). Titers of surviving viruses were determined at 37°C.
(B) Same as panel A but with virus stocks grown at 39°C. The titers
of virus stocks prior to heat treatment are indicated at the bottom.
|
|
For mutant 4, the more defective of the two E protein mutants, we next
sought further indications of structural impairment.
When virions of
mutant 4 were examined by electron microscopy,
a remarkable difference
from virions of the isogenic wild-type
control, Alb129, was seen.
Negatively stained preparations of
wild type had an appearance typical
for MHV and other coronaviruses
(Fig.
7).
With few exceptions, each virion was a roughly spherical
particle 80 to
100 nm in diameter, with a more densely staining
center and a complete
halo of surface spikes projecting approximately
20 nm beyond the
periphery of the particle. By contrast, although
some virions of mutant
4 conformed to this description, most particles
of this E protein
mutant deviated dramatically from the typical
coronavirus structure, as
exemplified in Fig.
8. The majority
of
mutant 4 virions exhibited a more narrow, tubular appearance.
These
elongated virions were often pinched at multiple points,
producing
dumbbell-shaped forms. Even the more spherical particles
of mutant 4 frequently contained two or three lobes. Between 80
and 90% of mutant
4 virions, whether grown at 33 or at 39°C, could
be classified as
having an abnormal morphology, whereas no more
than 15% of wild-type
virions fell into this category (Table
2).
The estimated volumes of mutant 4 virions were, on average, similar
to those of wild-type virions but
varied much more from the mean
value. As can be seen in Fig.
8,
despite the abnormality of virion
shape, the mutant showed no
detectable aberration in its distribution
of surface spikes. These
observations provided striking evidence
for the critical role of the
coronavirus E protein in virion morphogenesis,
clearly corroborating
the results obtained by expression studies
that established the
requirements for virus-like particle formation
(
4,
42).
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|
FIG. 7.
Electron microscopy of wild-type (Alb129) virions
released from infected mouse 17Cl1 cells at 33°C. Freshly passaged
virus was viewed by electron microscopy following negative staining
with sodium phosphotungstate.
|
|
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|
FIG. 8.
Electron microscopy of virions of E protein mutant 4 (Alb183: D60A, R61A, K63A) released from infected mouse 17Cl1 cells at
33°C. Freshly passaged virus was viewed by electron microscopy
following negative staining with sodium phosphotungstate.
|
|
View this table:
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|
TABLE 2.
Virion morphology of wild type (Alb129) and E protein
mutant 4 (Alb183: D60A, R61A, K63A) grown at 33 and
39°Ca
|
|
| |
DISCUSSION |
In this study, we have constructed the first coronavirus E gene
mutants used then to show that the E protein of MHV is fundamental to
the correct assembly of virions. Initially, we determined the sequences
of the E proteins of a number of strains of MHV. This added to the
accumulated evidence that, even though deletion, frameshift, or
transcriptionally inactive MHV mutants have been identified in the HE
gene (28), gene 2 (37), gene 4 (31, 41, 43,
46), and gene 5a (31, 46), the E gene is always found
to be intact. Furthermore, comparison of our data with other available
MHV E sequences showed that this protein is highly conserved. Most of the relatively limited set of divergent residues tend to
be grouped in the hydrophilic carboxy-terminal one-third of the
molecule. Within this region, however, charged amino acids almost
always remain invariant. Heterogeneity also occurs near the carboxy
terminus of the E protein in other coronavirus species for which more
than one sequence is known. In particular, it has been noted that this
portion of the avian infectious bronchitis virus E protein (the product
of gene 3c) not only shows primary sequence differences among a number
of strains but also varies in length by as many as 16 residues
(25).
The overall conservation of the MHV E protein may be taken to argue
that the E protein is critical, if not essential, for the virus. Thus,
it is probably not remarkable that we were unable to construct a
deletion mutant in which E would have been replaced with a heterologous
gene. However, the limitations of targeted recombination do not allow
us to draw a definitive conclusion from this negative result, and it is
conceivable that complete removal of the E gene might not be lethal.
The gene for the 6K protein of Semliki Forest virus, which in some
respects may be analogous to E, can be deleted to produce mutants that
are severely impaired but still viable (24, 27). It will be
possible to more decisively test whether the E gene is absolutely
essential if an infectious cDNA of the MHV genome becomes available.
The conservation of charged residues in the MHV E molecule supported
the underlying logic of our effort to seek conditional lethal E gene
mutants by the strategy of clustered charged-to-alanine mutagenesis. Of
the four possible mutants that we attempted to create, we were
able to obtain three, and two of these exhibited an impaired
phenotype relative to wild-type MHV. These two, mutant 3 (Alb154) and
mutant 4 (Alb183), are the first mutants we have constructed by
targeted RNA recombination that were identified by screening rather
than by selection, and they thus represent a broadening of the range
and usefulness of this method for genetic studies. Numerous
independent, spontaneous revertants of the two E mutants were isolated
and analyzed. As expected, since the original mutants contained
multiple mutations, all the revertants resulted from second-site
mutations. Each was found to contain a single relevant reverting
mutation in the proximity of the original E gene clustered
charged-to-alanine mutations. We had hoped that some of the second-site
revertants would map elsewhere than in the E gene and thus provide
evidence for sites of interaction between E and another viral protein,
but we have not yet found a revertant of this type. Based on the
studies of Vennema et al. (42) and Bos et al.
(4), we favor the notion of direct intermolecular contacts
between E protein and M protein, but it is possible that E protein can
bring about virion budding without such an association. Alternatively,
the lesions in mutant 3 and mutant 4 may be such that they cannot be
repaired by a compensating change in any protein other than E, while
other portions of the molecule may interact with the M protein.
The localization of all reverting mutations within the E gene strongly
indicated that the phenotypes of mutant 3 and mutant 4 were indeed
caused by the mutations (or a subset of the mutations) originally
constructed in these recombinants and were not due to hypothetical
secondary mutations that could have arisen during their isolation. This
conclusion was reinforced by the demonstration for a subset of mutant 3 revertants that reintroduction of a reverting mutation in conjunction
with its cognate set of clustered charged-to-alanine mutations resulted
in a wild-type phenotype. Taken together, these results show that at
least one hydrophilic region outside the membrane can play an important
role in E protein function, but a role for oligomeric associations
between membrane anchor regions may also be inferred by the surprising
loci of two of the reverting mutations: V31A (in mutant 3 RevI) and
T27A (in mutant 4 RevA). Although these changes are relatively small,
their significance may be that threonine and valine are more rigid and
bulky than alanine. Hence, the mutation to alanine may increase the
flexibility of the membrane domain to allow an adjustment in the
contact with the membrane domain of another E monomer that compensates
for the destabilization introduced by the clustered charged-to-alanine mutations in the ectodomain. Alternatively, we can hypothesize that the
residues V31 and T27 are not actually located within the membrane
domain but just downstream of it and that they directly interact with
the carboxy terminus of the E protein. This would suggest that the
membrane anchor of the E protein is 17 rather than 28 amino acids,
consistent with the fact that transmembrane segments of Golgi proteins
are generally shorter than those of plasma membrane proteins and that
15 residues are sufficient to cross the Golgi membrane (6).
Considerably more information about the E protein is required before we
can understand the structural basis for the action of all of the
reverting mutations.
The phenotypic properties of the E mutants, especially mutant 4, plainly substantiate the importance of the MHV E protein in virion
morphogenesis. The precise role of E in the formation of the
viral particle, however, remains to be clearly defined. The
thermolability data of Fig. 6 do not enable us to distinguish between E
as an active structural constituent of the membrane envelope or as an
accidental passenger that appears in the budded virion but has already
performed its function at a prior step of assembly. For virions
assembled at 33°C, the mutant E protein may adopt a defective
conformation upon heating. On the other hand, the mutant E protein may
have already acted in a defective manner establishing improper
associations among M protein monomers that disrupt virions upon
elevation of temperature. Likewise, the electron micrographs of mutant
4 (Fig. 8) revealed a distinct contrast with wild-type virions (Fig.
7), but further experiments remain to establish the events that
distorted the assembly process for the mutant. Pertinent to the range
of sizes of the mutant particles, we do not yet know if every aberrant
particle contained a nucleocapsid or if every particle contained only
one nucleocapsid. Many of the mutants of the 6K protein of Sindbis
virus have been found to package multiple nucleocapsids within single
membrane envelopes (20). One potential role that has been
proposed for the coronavirus E protein is that it is the factor that
pinches off the "neck" of the nascent virion particle in the final
stages of budding (42). We might speculate, then, that the
multiple pinches observed in many of the mutant 4 particles (Fig. 8)
could be examples of such a function acting in inappropriate places. We
hope that additional genetic studies, complemented by protein expression studies (4, 10, 42), will establish a basis to
evaluate the mode of action of E protein and approach the outstanding questions on the membrane orientation and state of oligomerization of
this molecule, its effect on the budding compartment, and its possible
interactions with other viral and cellular proteins.
| |
ACKNOWLEDGMENTS |
We are grateful to Cheri Koetzner for expert technical assistance
and to Patrick van Roey for helpful discussion of the revertant data.
We thank Tim Moran and Matthew Shudt of the Molecular Genetics Core
Facility of the Wadsworth Center for the synthesis of oligonucleotides and for automated DNA sequencing. We thank Kathryn Holmes and Ehud Lavi
for providing MHV strains. All electron microscopy was performed at the
Electron Microscopy Core Facility of the Wadsworth Center.
This work was supported in part by Public Health Service grant AI 39544 from the National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: David Axelrod
Institute, Wadsworth Center, NYSDOH, New Scotland Ave., P.O. Box 22002, Albany, NY 12201-2002. Phone: (518) 474-1283. Fax: (518) 473-1326. E-mail: masters{at}wadsworth.org.
Present address: LaboRétro, INSERM U412, ENS, 46 allee
d'Italie, 69364 LYON cedex 07, France.
Present address: Physiologisch-chemisches Institut, Universitaet
Tuebingen, Germany.
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Journal of Virology, October 1998, p. 7885-7894, Vol. 72, No. 10
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Abstract
Introduction
