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Journal of Virology, August 2001, p. 7692-7702, Vol. 75, No. 16
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.16.7692-7702.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Attenuation of Murray Valley Encephalitis Virus by Site-Directed
Mutagenesis of the Hinge and Putative Receptor-Binding Regions of
the Envelope Protein
Robert J.
Hurrelbrink* and
Peter C.
McMinn
Department of Microbiology, University of
Western Australia, Nedlands, Western Australia 6907, Australia
Received 12 March 2001/Accepted 16 May 2001
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ABSTRACT |
Molecular determinants of virulence in flaviviruses cluster in two
regions on the three-dimensional structure of the envelope (E) protein;
the base of domain II, believed to serve as a hinge during
pH-dependent conformational change in the endosome, and the
lateral face of domain III, which contains an integrin-binding motif
Arg-Gly-Asp (RGD) in mosquito-borne flaviviruses and is believed to
form the receptor-binding site of the protein. In an
effort to better understand the nature of attenuation caused by
mutations in these two regions, a full-length infectious cDNA clone of
Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was
employed to produce a panel of site-directed mutants with substitutions
at amino acid positions 277 (E-277; hinge region) or 390 (E-390; RGD
motif). Viruses with mutations at E-277 (Ser
Ile, SerAsn,
SerVal, and SerPro) showed various levels of in vitro and in vivo
attenuation dependent on the level of hydrophobicity of the
substituted amino acid. Altered hemagglutination activity observed for
these viruses suggests that mutations in the hinge region may
indirectly disrupt the receptor-ligand interaction, possibly by causing
premature release of the virion from the endosomal membrane prior to
fusion. Similarly, viruses with mutations at E-390 (AspAsn,
AspGlu, and AspTyr) were also attenuated in vitro and in vivo;
however, the absorption and penetration rates of these
viruses were similar to those of wild-type virus. This, coupled with
the fact that E-390 mutant viruses were only moderately inhibited by
soluble heparin, suggests that RGD-dependent integrin binding is
not essential for entry of MVE and that multiple and/or alternate
receptors may be involved in cell entry.
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INTRODUCTION |
Murray Valley encephalitis
virus (MVE) is a member of the Flavivirus genus
(family Flaviviridae) and is a small, lipid-enveloped virus
which contains a single-stranded positive-sense RNA genome. The genome
is approximately 11 kb in length and contains a single open reading
frame which is posttranslationally cleaved to generate three structural
(C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3,
NS4A, NS4B, and NS5) proteins. Viral genomic RNA also has a methylated
cap at its 5' terminus and forms a highly conserved stem-loop structure
at its 3' end (61). As for many flaviviruses, MVE
causes clinically significant disease in humans and, together with
Kunjin (KUN) virus, is responsible for almost all cases of flaviviral
encephalitis in mainland Australia (41).
In recent years, infectious cDNA clones have been produced for a number
of flaviviruses, including MVE (31, 39), enabling manipulation of the genome at the nucleotide level. Such clones have
been used to examine the glycosylation, cleavage, and function of the
prM and E (4, 20, 28, 33, 55, 57, 68), NS1 (53, 55, 57), NS2B/NS3 (9, 10, 54), and NS5
(34, 35, 36) proteins, as well as to generate viruses with
deletions in their 5' and 3' untranslated regions (6, 38, 43,
48). More recent work has seen the generation of chimeric yellow
fever viruses (YF), containing the prM and E genes of Japanese
encephalitis virus (JE) (8) or dengue virus type 2 (DEN-2)
(23). In primates, these chimeric viruses provide solid
protection against heterologous virus challenge and demonstrate great
potential for use as flavivirus vaccines (23, 24, 49, 50).
The envelope (E) protein of flaviviruses plays a significant role in
viral entry and possesses an interesting structural and functional
biology. It mediates attachment of the virus to host cells, as well as
fusion of the viral and cellular membranes after receptor-mediated
endocytosis. In addition, it is the major target of neutralizing
antibodies in the host and plays a significant role in both viral
tropism and pathogenesis. The three-dimensional structure of the
ectodomain of the E protein has been determined for tick-borne
encephalitis virus (TBE) (60), and it serves as a useful
model for other flaviviruses due to the high amino acid sequence
homology observed throughout the genus. The protein forms head-to-tail
dimers on the virion surface, and each monomer consists of three
domains, referred to as domain I (central domain), domain II
(dimerization domain), and domain III (immunoglobulin-like domain).
Molecular determinants of virulence on the flavivirus E protein form
three distinct clusters and are likely to affect virulence by
disrupting the functional biology of the protein (60). The first cluster, located in a putative hinge region linking
domains I and II, appears to affect the pH-dependent conformational
change required for endosomal fusion. In MVE, mutations in this region at amino acid position 277 [E-277 (SerIle)] inhibit the
hemagglutination (HA) and fusion properties of the virus and cause a
loss of neuroinvasiveness (NI) in mice (46, 47).
Similarly, mutations in the second cluster, located at the tip of
domain II in a highly conserved "fusion peptide," also appear to
disrupt fusion. In contrast, mutations in the third cluster, located on
the lateral face of domain III, are predicted to disrupt receptor
binding. In some mosquito-borne flaviviruses, the presence of an
Arg-Gly-Asp (RGD) motif in this region has led some to suggest that
integrin binding may be important for virus entry (40).
The involvement of an RGD motif in cell entry has been described for a
number of viruses, including adenovirus type 2 (18, 73,
74), human rotavirus RV-5 (17), and foot and mouth
disease virus (FMDV) (32). Mutations in this motif have
been shown to affect virus infectivity and virulence in both MVE
(39, 40) and YF (69).
In this study, two panels of mutant MVE viruses with substitutions at
E-277 (Ser) or E-390 (Asp) were created to investigate the influence of
these mutations on virulence. For each virus, obvious effects on plaque
phenotype, growth kinetics in cell culture, genetic stability, and
virulence in mice were observed. Interestingly, mutations at E-277 or
E-390 did not affect the absorption or penetration rates of any of the
viruses nor did they affect the relative rates of inhibition of virus
binding by soluble heparin. However, some mutations caused a complete
loss of NI and/or a reduced ability to agglutinate red blood cells
(RBCs). Furthermore, some mutant viruses displayed either large- or
small-plaque phenotypes, correlating with altered growth kinetics in
Vero cells.
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MATERIALS AND METHODS |
Virus and infectious cDNA clone.
All mutant viruses were
derived from an infectious cDNA clone of MVE virus prototype strain
MVE-1-51 (designated pMVE-1-51). Thorough genotypic and phenotypic
characterization of virus derived from this clone (CDV-1-51) has been
described previously (31), and its complete genomic
sequence is known (GenBank accession no. AF161266). Site-directed
mutations of the E protein (E-277 or E-390) were engineered into clone
pMVE-1-51 by using the QuikChange Site Directed Mutagenesis (SDM) kit
(Stratagene). The nucleotide sequences of polyacrylamide gel
electrophoresis-purified sense primers used for SDM are shown in Table
1.
After SDM and transformation into a bacterial host, three separate cDNA
clones of each of the seven mutants were selected
and subjected to
sequence analysis over the entire E gene (approximately
15% of the
entire genome) for comparison to the known sequence
of MVE-1-51
(
31). This was performed both to confirm the site-specific
change and to ensure that other mutations had not been inadvertently
introduced during the cycling protocol. In every clone sequenced
(
n = 21), only the site-specific change was present. No
other
mutations were identified in any of the clones, confirming the
high fidelity of the
Pfu DNA polymerase enzyme used in the
cycling
reaction. One cDNA clone was selected for each mutant and used
as a template for the production of RNA and infectious
virus.
Virus recovery and stock preparation.
Site-directed mutants
of clone pMVE-1-51 were linearized with XbaI and used as
templates for subsequent transcription with T7 RNA polymerase. The
generation of genome-length RNA and its transfection into BHK-21 cells
by electroporation was performed as described previously
(31). Four to six days after electroporation, cell culture
supernatants showed obvious signs of cytopathic effect and were assayed
for virus by plaque assay on Vero cells. In order to generate stocks of
sufficient titer for subsequent assays, transfected BHK-21 cell culture
supernatants were passaged three times in C6/36 mosquito cells and
sequenced across the entire E gene as described below.
Sequence analysis.
Sequencing was performed using an
ABI-Perkin-Elmer automated sequencing system, which incorporates
fluorescently labeled dideoxynucleotides. All site-directed mutant
clones derived from clone pMVE-1-51 were subjected to sequence analysis
over the entire E gene. Mutant virus stocks (C6/36 cell culture
supernatants), as well as virus present in the brains of two
encephalitic mice infected with each mutant virus, were similarly
sequenced across the E gene. For these analyses, genomic RNA was
purified using QIAamp Viral RNA spin columns (Qiagen) and amplified by
reverse transcriptase PCR as described previously (31).
Sequencing primers were supplied by Life Technologies (Gibco BRL) and
were used at a final concentration of 0.8 pmol/ml. Details of primer
sequences are available by request.
Cell culture.
Vero (ATCC CCL81 P130-P145), BHK-21 (ATCC
CCL10 P56-P59), and Aedes albopictus C6/36 (ATCC CRL1660
P5-P20) cells were grown in M199 medium supplemented with 2 mM
L-glutamine and 10% fetal calf serum and were
incubated at either 37°C (Vero, BHK-21) or 28°C (C6/36) in an
atmosphere containing 5% CO2. For plaque assays, subconfluent monolayers of Vero cells in 12-well tissue culture trays
were inoculated with virus and incubated for 1 h. Virus was then
removed, and cells were overlaid with methylcellulose containing 2%
fetal calf serum in M199 media. Cells were cultured for 4 to 6 days at
37°C (5% CO2) and stained with methylene blue to visualize plaques (1% [wt/vol] methylene blue, 10%
formaldehyde). To test for temperature sensitivity, plaque assays were
performed separately at 37°C and at an elevated temperature of
39°C, and the number and morphology of plaques were compared. For
virus growth assays, monolayers of Vero cells in
60-mm2 tissue culture dishes were infected with
virus at a multiplicity of infection between 1 and 5 (standardized for
each assay). Aliquots of cell culture supernatant (500 µl) were then
collected at 6, 12, 18, 24, and 30 h postinfection (p.i.) and
replaced with an equal volume of fresh media. The titer of virus in
each sample was subsequently determined by plaque assay. All virus
growth assays were performed in duplicate.
Absorption and penetration assays.
Monolayers of Vero cells
were infected in triplicate according to the adsorption assay method
described by Khromykh and Westaway (37) or the penetration
assay method described by Hung et al. (30). For adsorption
assays, approximately 100 PFU of virus was added to each well and
allowed to adsorb for 30, 60, or 90 min at 37°C. Cells were then
washed twice with phosphate-buffered saline, overlaid with growth
medium, and incubated for 4 to 6 days as per standard plaque assay. For
penetration assays, approximately 100 PFU of virus was added to each
well and allowed to adsorb for 30, 60, or 90 min at 37°C. Cells were
then incubated in acid glycine (pH 3.0) for 3 min to inactivate
noneclipsed virus. Acid glycine was aspirated, and cells were overlaid
with growth medium as outlined for adsorption assays above. The rate of
adsorption or penetration was calculated as the ratio of the average
number of plaques at 30 or 60 min p.i. relative to the average number of plaques at 90 min p.i. and was expressed as a percentage.
HA assays.
Virus from infected C6/36 cell supernatants was
used as a source of hemagglutinin. HA assays were performed using a
modified protocol of Clark and Casals (14) as described
previously (46). Titers were recorded as the reciprocal of
the highest dilution which yielded complete agglutination of gander RBCs.
Heparin inhibition assays.
Inhibition of virus binding by
soluble heparin was performed using modified protocols based on those
described by Chen et al. (13), Hung et al.
(30), and Lee and Lobigs (39). Approximately 100 PFU of virus was preincubated in Hank's borate-buffered saline (Gibco BRL) containing 200 µg of heparin (Sigma)/ml at 4°C for 1 h. Virus was then inoculated onto monolayers of prechilled Vero cells (30 min at 4°C) and incubated for a further 1 h at 4°C
to allow virus binding. The inoculum was then removed and cell
monolayers were washed with cold phosphate-buffered saline prior to the
addition of M199/MC and incubation at 37°C under standard conditions
for 4 to 6 days. Plaques were visualized as per standard plaque assay. Percent inhibition by soluble heparin was calculated according to the
following formula: (a 
b)/a,
where a is the number of plaques on cells incubated with
untreated virus and b is the number of plaques on cells
incubated with heparin-treated virus.
Virulence in mice.
Litters of five 21-day-old ARC/Swiss mice
(Animal Resources Centre, Murdoch, Western Australia, Australia) were
injected intracranially (i.c.) or intraperitoneally (i.p.) with 10 or
50 µl, respectively, of a 10-fold dilution of virus. Mice were
examined daily for signs of morbidity, and deaths were recorded.
"Humane end points" were employed to minimize distress in
experimental animals, a method which does not significantly alter 50%
lethal dose values in models of viral encephalitis (75).
The 50% humane end point dose (HD50) was
calculated for each group by following the 50% lethal dose method
described by Reed and Muench (59). Mean time to death was
determined by injecting mice (10 per group) i.c. or i.p. with 103 PFU of virus and recording the survival of
mice over a period of 21 days. Statistical significance was determined
using a paired Student's t test.
For growth in mouse brain assays, groups of 30 18-day-old ARC/Swiss
mice were injected i.c. with 10
3 PFU of virus. At
selected times, three mice from each group were
anesthetized
(penthrane) and their brains were removed and snap
frozen in liquid
nitrogen. Tissues were then stored at
80°C until
required. Prior to
titration by standard plaque assay, tissues
were thawed, weighed, and
manually homogenized before being prepared
as 10% (wt/vol) suspensions
in Hanks' borate-buffered saline.
Titers were expressed as PFU per
gram of
tissue.
For protection assays, groups of 21 18-day-old ARC/Swiss mice were mock
inoculated or were inoculated with between 10
4
and 10
1 PFU of clone-derived virus by the i.p.
or i.c. route. At 17 days
p.i., surviving mice in each group were
challenged with 10
4 i.p.
HD
50s of clone-derived wild-type virus
(CDV-1-51). Mice
were observed for a further 21 days for signs of
morbidity, and
deaths were recorded. All mouse experiments were
undertaken using
protocols approved by the University of Western
Australia Animal
Experimentation Ethics Committee. Mice were kept on a
clean litter
of sawdust and given food and water ad
libitum.
Protein structure graphics.
The three-dimensional structure
of a soluble ectodomain fragment of the TBE E protein (Brookhaven
Protein Databank [PDB] entry 1SVB) (60) was used as the
basis for all diagrammatic representations of the E protein. Such
representations were made using RASMOL molecular visualization software
(version 2.6) (62). Homology modeling of the MVE E protein
was performed using Swiss-PDB-Viewer software (version 3.6b3)
(21), where the primary amino acid sequence of the MVE E
protein was threaded onto the known structure of the TBE E protein and
submitted to the Swiss-Model (ExPASy) server in Geneva, Switzerland,
for energy minimization and subsequent generation of a final model.
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RESULTS |
Mutagenesis of the hinge region at E-277.
Four amino acid
substitutions at E-277, varying in terms of their size and
hydrophobicity, were selected for this study. At E-277, the hydrophilic
Ser residue was converted to a strongly hydrophobic Ile residue
(SI) in order to reconstitute the attenuated virus BHv1 from an
earlier study (46). This was performed to ascertain
whether a single amino acid change in the E protein was responsible for
the observed phenotype, which included a low NI in mice and an
inability to agglutinate RBCs. In addition, a second mutant from the
McMinn et al. study (46) was reconstituted, since the
SerAsn (SN) mutation in this virus (BHv2) had no
observable effect on viral phenotype and represented a conservative
amino acid change. Two other substitutions at E-277 were also made to produce novel viruses not previously reported. These substitutions, SerPro (SP) and SerVal (SV), were chosen
because they were intermediate in terms of their hydrophobicity between
the Ile and Asn residues of the first two mutants. It was predicted
that such changes would have various effects on virus phenotype and that these effects may correlate with the level of hydrophobicity of
the substituted amino acid.
Phenotypes of E-277 (hinge) mutant viruses in vitro.
The in
vitro phenotypes of the four hinge mutant viruses (designated
CDV-1-51v1, -v2, -v3, and -v4; Table 2)
were compared and contrasted to that of clone-derived wild-type virus
(CDV-1-51; see reference 31) by a number of means,
including plaque morphology, growth kinetics in Vero cells, temperature
sensitivity, adsorption and penetration rates, and HA activity. With
the exception of CDV-1-51v2 (SN), no change to the wild-type
(CDV-1-51) plaque morphology was observed for any of the E-277 mutant
viruses (see notes to Table 2). CDV-1-51v1 (SI), CDV-1-51v3
(SV), and CDV-1-51v4 (SP) all displayed a normal plaque
phenotype, contrasting with the large-plaque phenotype observed for
CDV-1-51v2 (SN).
More obvious differences between the phenotypes of the E-277 mutant
viruses became apparent when their relative growth kinetics
in Vero
cells were compared. As shown in Fig.
1a,
these differences
were most prominent at 12 h p.i., with CDV-1-51v1
(S
I), CDV-1-51v4
(S
P), and CDV-1-51v3 (S
V)
exhibiting the lowest titers at this
time point. Interestingly,
CDV-1-51v2 (S
N) displayed a slightly
higher titer than parental
virus at this and all other time points,
possibly reflecting an
improved replicative ability in Vero cells.
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FIG. 1.
Growth kinetics of hinge (A) and RGD (B) mutant viruses
in Vero cells. Monolayers were infected at a multiplicity of infection
of 5, and samples of cell culture supernatant were collected at the
times indicated. Viral titers were determined by plaque assay on Vero
cells. All assays were performed in duplicate.
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To further analyze in vitro phenotype, the temperature sensitivities of
each of the hinge mutant viruses were determined by
plaque assay at 37 and 39°C (Table
2). Little variability in
titer was observed for any
of the mutant viruses, suggesting that
the introduced mutation had
little to no effect on E protein stability
at the elevated temperature.
To determine if observed differences
in growth kinetics were due to
altered cell entry, the ability
of each of the hinge mutant viruses to
bind to and penetrate host
cells was determined by adsorption and
penetration assay. As shown
in Table
2, no significant differences in
the ability of E-277
mutant viruses to bind to and penetrate host cells
were observed.
Adsorption rates (ratios) ranging from 43 to 49% were
observed
at 30 min p.i., increasing to a range of 72 to 81% by 60 min
p.i.
Similarly, penetration rates ranging from 47 to 51% were observed
at 30 min p.i., increasing to a range of 75 to 82% by 60 min p.i.
Since differences in the binding and penetration of each of the hinge
mutant viruses were not evident, the ability of each
of these viruses
to fuse with RBCs at acidic pH was determined
by HA assay. In
flaviviruses, HA is a pH-dependent interaction
between viral and
erythrocyte membranes that is thought to involve
the mixing of membrane
lipids (
56). As such, it serves as a
measure of the
ability of a virus to fuse efficiently with host
cell membranes,
specifically those of the endosomal membrane during
fusion. Infected
C6/36 cell supernatants, ranging in titer between
10
7 and 10
8 PFU/ml, were
used as a source of HA and assayed for HA activity
over the pH range
5.9 to 7.2. Using an optimum pH of 6.6 (determined
for CDV-1-51), the
HA titers of each of the hinge mutant viruses
were determined as shown
in Table
3. In addition, the HA titers
of
two other MVE variants, BHv1 and BHv2 [E-277 (S
I) and E-277
(S
N), respectively; see reference
46], were
determined, serving
as positive and negative controls for altered HA
activity. Interestingly,
both CDV-1-51v1 (S
I) and BHv1
(S
I) failed to agglutinate RBCs
at the lowest dilution tested
(1:2) over the entire pH range examined
(data not shown), confirming
that a single amino acid substitution
at E-277 had been responsible for
the observed phenotype of BHv1
in a previous study (
46).
Furthermore, the HA titers for both
CDV-1-51v2 (S
N) and BHv2
(S
N) were also identical at 1:320,
similar to that observed for
clone-derived wild-type virus (1:160).
The HA titers of the remaining
two mutants, CDV-1-51v3 (S
V) and
CDV-1-51v4 (S
P), were
both 1:40; however, the optimal pH for
HA was increased from 6.6 to 6.8 for both viruses. Hydrophobic
amino acid substitutions at E-277
therefore caused either a complete
loss of HA activity [CDV-1-51v1
(S
I)] or a marked reduction of
this activity coupled with an
increase in optimal pH [CDV-1-51v3
(S
V) and CDV-1-51v4
(S
P)]. In contrast, a hydrophilic amino
acid substitution had
no effect on HA activity [CDV-1-51v2 (S
N)].
Phenotypes of E-277 (hinge) mutant viruses in vivo.
As an
extension of the in vitro analyses described above, the in vivo
phenotype of each of the hinge mutant viruses was examined by
determining HD50 and mortality profiles and
comparing average survival times after i.p. or i.c. challenge. In
addition, the growth kinetics of each virus in infected mouse brain was
also determined.
As shown in Table
4,
HD
50 values for each of the mutant viruses ranged
between 2 and 5 PFU by the i.c. route, resulting in
100% mortality and
a mean time to death in the range of 4.4 to
5.5 days. In comparison to
the profile for CDV-1-51 (i.c. HD
50 of 1 PFU;
100% mortality; time [mean ± standard deviation] to
death,
4.6 ± 0.5 days), each of the E-277 mutant viruses was
subsequently
deemed to be of high neurovirulence (NV). With the
exception of
CDV-1-51v2 (S
N), the NI (determined by i.p.
inoculation) of each
of the hinge mutant viruses was found to be
markedly different
to that of CDV-1-51. The highly neuroinvasive
CDV-1-51v2 (S
N)
had an i.p. HD
50 of 4 PFU,
a mortality of 90%, and a mean time
to death of 7.3 ± 1.3 days,
similar to the profile observed for
clone-derived wild-type virus (i.p.
HD
50 of 5 PFU; mortality of
100%; time to death,
7.2 ± 0.9 days). In contrast, CDV-1-51v1
(S
I) was found to
be of low NI, with an i.p. HD
50 greater than
10
4.5 PFU (no observed mortality at the lowest
dilution of virus tested).
The remaining two mutants, CDV-1-51v3
(S
V) and CDV-1-51v4 (S
P),
were found to be of
intermediate NI, with HD
50 values of 620 and
580 PFU, mortalities of 40 and 50%, and mean times to death of
8.5 ± 1.3 and 10.2 ± 3.6 days, respectively.
When analysis of the growth kinetics of hinge mutant viruses in
infected mouse brain was performed, attenuation was found
to be similar
to that observed in vitro. As shown in Fig.
2a,
mean peak titers ranging from
10
8.3 to 10
8.7 PFU/g were
obtained for most viruses by day 3 p.i. In contrast,
the titer of
CDV-1-51v1 (S
I) lagged approximately 10-fold behind
that of the
other viruses (including CDV-1-51) at the same time
point, having a
titer of 10
7.4 PFU/g. On completion of the assay
(day 5 p.i.), titers for CDV-1-51v3
(S
V), CDV-1-51v4
(S
P), and CDV-1-51v1 (S
I) were observed to
be 20-, 8-, and 13-fold lower, respectively, than those for both
CDV-1-51 and
CDV-1-51v2 (S
N).
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FIG. 2.
Growth kinetics of hinge (A) and RGD (B) mutant viruses
in infected mouse brain. Mice were inoculated i.c. with 103
PFU of virus, and brains were collected at the times indicated. Viral
titers were determined by plaque assay of 10% homogenates on Vero
cells. All assays were performed in duplicate.
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Mutagenesis of the RGD motif at E-390.
Three amino acid
substitutions at E-390 were selected for this study. At E-390, the
negatively charged Asp residue was converted to an uncharged Asn
residue [E-390 (DN)] in order to reconstitute an attenuated
virus (P5/Ab10) from a previous study (40). Phenotypic characterization of P5/Ab10 was limited to the observation that the
virus had low NI in mice (40). As such, the E-390
(DN) mutant created in this study was used to further
characterize the nature of the attenuation and to ascertain whether a
single amino acid change in the E protein was responsible for the
observed phenotype. The second mutant, E-390 (DE), was
constructed for two reasons: first, because it represented a
conservative amino acid change, and second, because other flaviviruses
such as West Nile virus (WNV) (72) and KUN
(15) possess an RGE as opposed to an RGD motif in this
region of the protein. Substitution of a Glu residue at the third
position of an RGD motif has been shown to abolish RGD-dependent
integrin binding by a range of viruses, including FMDV
(44), chimeric hepatitis B virus (64), and echovirus type 9 (76). The last mutant, E-390 (DY),
was constructed because it represented the substitution of a large
hydrophobic amino acid residue into a strongly hydrophilic region of
the protein. Like the DN and DE substitutions above, such
a mutation would be predicted to significantly disrupt RGD-dependent
integrin binding if this were important in the attachment and
penetration of MVE into host cells.
Phenotypes of E-390 (RGD) mutant viruses in
vitro
As for the panel of hinge mutant viruses,
the in vitro phenotypes of each of the RGD mutant viruses (CDV-1-51v5,
-v6, and -v7; Table 2) were compared and contrasted in terms of their plaque morphology, growth kinetics in Vero cells, temperature sensitivity, adsorption and penetration rates, and HA activity. All
three RGD mutants displayed plaque morphologies different from that of
clone-derived wild-type virus. CDV-1-51v5 (DN) and CDV-1-51v7
(DY) exhibited a small-plaque morphology, while CDV-1-51v6 (DE) exhibited a large-plaque morphology (see notes to
Table 2). These differences were found to be reflective of differences in the relative growth kinetics of each virus in Vero cells. As shown
in Fig. 1b, obvious differences in replicative ability were evident at 6 h p.i., with CDV-1-51v5 (DN) and CDV-1-51v7
(DY) displaying significantly lower titers at this time point
(100- to 500-fold lower titers than CDV-1-51). In contrast, the titer of CDV-1-51v6 (DE) at this and all other time points was similar to that observed for CDV-1-51. At 12 h p.i., the titers of both CDV-1-51v5 (DN) and CDV-1-51v7 (DY) continued to lag
behind those of both CDV-1-51 and CDV-1-51v6 (DE) by the same
order of magnitude, narrowing to 50- and 10-fold differences,
respectively, by 24 h p.i.
Further analysis of in vitro phenotype was performed by temperature
sensitivity testing of each of the RGD mutant viruses
(by plaque assay
at 37 and 39°C). As for the panel of hinge mutant
viruses, little
variability in titer was observed for any of the
RGD mutants (Table
2).
In addition, no significant differences
in the adsorption or
penetration rates of these viruses into host
cells (Table
2) or their
ability to agglutinate RBCs (Table
3)
were
observed.
Phenotypes of E-390 (RGD) mutant viruses in
vivo
As for the hinge mutant viruses described
above, the in vivo phenotypes of each of the RGD mutant viruses
(CDV-1-51v5, -v6, and -v7) were compared and contrasted to that of
CDV-1-51. The results are shown in Table 4. HD50 values
ranged between 1 and 18 PFU by the i.c. route, resulting in 100%
mortality and a mean time to death between 4.3 and 5.5 days. Each of
the E-390 mutant viruses was therefore deemed to be of high NV
according to the criteria outlined by McMinn et al. (HD50
of less than 20 PFU) (46).
With the exception of CDV-1-51v6 (D
E), the NI of each of the RGD
mutant viruses was found to be markedly different from that
of
CDV-1-51. The highly neuroinvasive CDV-1-51v6 (D
E) had an
i.p.
HD
50 of 2 PFU, a mortality of 100%, and a mean
(± standard
deviation) time to death of 6.7 ± 0.8 days, similar
to the profile
observed for CDV-1-51 (i.p. HD
50
of 5 PFU; mortality of 100%;
mean time to death of 7.2 ± 0.9 days). In contrast, both CDV-1-51v5
(D
N) and CDV-1-51v7
(D
Y) were found to be of low NI, with i.p.
HD
50 values of greater than
10
4.5 PFU (no observed mortality at the lowest
dilution of virus
tested).
Analysis of the growth kinetics of RGD mutant viruses in infected mouse
brain showed a slightly different pattern from that
observed in vitro.
As shown in Fig.
2b, the titer of CDV-1-51v6
(D
E) had already
reached a peak of 10
9.4 PFU/g by day 3 p.i.,
approximately eightfold higher than that
observed for CDV-1-51
(10
8.5 PFU/g) at the same time point. In
contrast, the titers of CDV-1-51v5
(D
N) and CDV-1-51 (D
Y)
lagged behind that of CDV-1-51, reaching
10
7.7
and 10
6.1 PFU/g, respectively. On completion of
the assay (day 5 p.i.),
titers for all three of the RGD mutant
viruses were two- to fourfold
lower than that of CDV-1-51
(10
9.7 PFU/g), ranging from
10
9.1 to 10
9.5 PFU/g.
Inhibition of virus binding by soluble heparin.
Previous work
on DEN-1, DEN-2, and MVE had highlighted a potential role for
glycosaminoglycans (GAGs) in host cell entry, based on their ability to
inhibit virus binding (13, 27, 30, 39). To ascertain
whether clone-derived MVE virus or attenuated variants with mutations
at E-277 or E-390 were similarly susceptible to inhibition by GAGs,
mutant viruses were preincubated with soluble heparin (200 µg/ml) for
1 h at 4°C prior to their inoculation onto Vero cells for
a further 1 h at the same temperature. The inoculum was then
removed, and virus titers were determined by plaque assay. As shown in
Fig. 3, inhibition of virus infectivity was approximately 40% for CDV-1-51 and ranged between 34 and 46% for
the E-277 mutant viruses. Similar inhibition was observed for the E-390
mutant viruses, ranging between 38 and 50%. Given that data generated
for DEN-2 (27, 30) had shown a much higher inhibition of
virus infectivity (greater than 75%) at the same heparin dose, these
results suggested that MVE was only moderately inhibited by
heparin. Furthermore, the introduced mutations at E-277 and E-390
appeared to have little to no effect on the heparin binding ability of
any of the viruses.
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FIG. 3.
Inhibition of virus binding by soluble heparin. Rates of
inhibition were calculated using the following formula:
(a b)/a, where
a is the number of plaques on cells incubated with
untreated virus and b is the number of plaques on cells
incubated with heparin-treated virus. CDV-1-51 is shown in black, E-277
mutant viruses are in white, and E-390 mutant viruses are in gray.
Average results from two separate experiments are shown.
|
|
Stability of introduced mutations during in vitro passage.
Due
to selective pressures conferred by an attenuating mutation, it is
possible that the amino acid sequence of a mutant virus may revert to
that of wild-type virus. Alternatively, second-site mutations may
arise, serving in some way to stabilize or compensate for the
attenuating mutation. To determine if the attenuation of viruses with
mutations at E-277 or E-390 resulted in a selective pressure for
reversion (or the appearance of second-site mutations), the stability
of each of the mutants produced in this study was determined in vitro
by examining the nucleotide sequence of the E protein at different
passage levels.
Reverse transcriptase PCR-generated cDNA of each virus was sequenced
after one passage (P1) in C6/36 cells and again after
two additional
passages in the same cell line (P3). In all four
of the hinge mutant
viruses, the sequence of the E protein was
unchanged. Similarly, in two
of the three RGD mutants [CDV-1-51v5
(D
N) and CDV-1-51v6
(D
E)], the E protein sequence was also unaffected.
Interestingly, the E gene of CDV-1-51v7 (D
Y)
contained a single
point mutation at E-497, resulting in a
Thr
Ile (T
I) substitution.
This mutation was found in both
the P1 and P3 viruses and must
therefore have arisen prior to or during
initial amplification
of the virus in BHK-21 cells after
transfection.
During titration of E-277 and E-390 virus stocks by plaque assay on
Vero cells, virus with a normal plaque phenotype was observed
at a
frequency of approximately 0.5% (1 plaque in 200) in the
P3 stock of
CDV-1-51v5 (D
N). To ascertain whether this or any
other mutant
virus stock contained revertants at low frequency,
the P3 stock of each
virus was passaged three times more in either
C6/36 cells (P4-P6) or
Vero cells (VP1-VP3). With the exception
of CDV-1-51v5 (D
N), all
viruses retained their respective plaque
phenotypes during passage in
both cell lines. As shown in Fig.
4,
however, the CDV-1-51v5 (D
N) mutant (which retained its
small-plaque
phenotype throughout passage in C6/36 cells) reverted over
the
course of three passages in Vero cells to a normal plaque
phenotype.
This normal plaque phenotype constituted approximately 10%
of
the viral population at VP1, 80% of the viral population at VP2,
and 100% of the viral population at VP3. Nucleotide sequencing
of
the E gene of CDV-1-51v5 (D
N) at VP3 confirmed that the Asn
(N)
residue at E-390 had reverted to the Asp (D) residue of the
wild-type
virus. Similar sequencing of the VP3 stocks of CDV-1-51v6
(D
E)
and CDV-1-51v7 (D
Y) revealed no reversion of the mutated
E-390
residue to that of the wild type.
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FIG. 4.
Plaque phenotype of CDV-1-51v5 (DN) after
multiple passage in Vero cells. A third passage C6/36 stock was
progressively passaged to generate Vero passage 1 (VP1) through passage
3 (VP3) stocks. (A) Original C6/36 P3 stock; (B) CDV-1-51v5 (DN)
VP1; (C) CDV-1-51v5 (DN) VP2; (D) CDV-1-51v5 (DN) VP3.
|
|
Reversion of the CDV-1-51v5 (D
N) mutant may be suggestive of an
overall intolerance of MVE to mutations at E-390, evidenced
by
reversion of the CDV-1-51v5 (D
N) mutant in cell culture and
the
appearance of a second-site mutation at E-497 of the CDV-1-51v7
(D
Y) mutant. Such pressure was unlikely for the CDV-1-51v6
(D
E)
mutant because the amino acid change was conservative and
because
the replicative ability of the virus was unaffected (see in
vitro
and in vivo phenotypes
above).
| |
DISCUSSION |
Mutations in the hinge region.
The effects of mutations in the
hinge region of the MVE E protein on mouse NI highlight the importance
of this region in viral virulence. Previous studies with JE, YF, or
JE/YF chimeras have shown that mutations in the polar interface linking
domains I and II cause significant attenuation. This interface, shown
diagrammatically in Fig. 5, includes
residues E-52 to E-54 (hereafter referred to as hinge 1), E-129 to
E-136 (hinge 2), E-191 to E-200 (hinge 3), and E-266 to E-284 (hinge 4;
MVE numbering based on the TBE model described by Rey et al.
[60]). A range of different mutations in these regions
can cause a loss of either NI or NV. For example, a mutation in hinge 1 of JE [E-52 (QR/K)] causes a loss of NI (26),
while a similar mutation in YF [E-52 (GR)] contributes to a
loss of NV (63). Mutations in hinges 2 and 4 also
contribute to a loss of NI and/or NV in JE [E-270 (IS) and
E-138 (EK)] and JE/YF chimeras [E-138 (EK)] (2,
7, 8, 11, 68).
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FIG. 5.
The hinge region of the MVE E protein, based on the
known three-dimensional structure of the TBE E protein
(60). In MVE, the polar interface linking domains I and II
includes residues E-52 to E-54 (hinge 1 [green]), E-129 to E-136
(hinge 2 [orange]), E-191 to E-200 (hinge 3 [red]), and E-266 to
E-284 (hinge 4 [yellow]). Hinges 1 through 4 correspond to regions
D0-a, e-E0, H0-f, and
 B-I0, respectively, according to the nomenclature
described by Rey et al. (60). Residue E-277, the residue
selected for SDM, is highlighted in black.
|
|
In addition to those described above, mutations in hinge 4 also cause a
loss or reduction of NI in MVE [E-277 (S
I, V, or
P); see
reference
46 and work described in this study]. In these
cases, the introduction of hydrophobic amino acid residues into
an
otherwise hydrophilic region of the E protein appears to disrupt
the
function of the protein at a stage following adsorption and
penetration
of the host cell. Interestingly, the introduction
of a strongly
hydrophobic Ile residue into hinge 4 abolishes both
HA activity and NI;
however, the introduction of a Val residue
(which is also strongly
hydrophobic but has a smaller side chain)
or a Pro residue (weakly
hydrophobic) reduces but does not abolish
HA activity and NI. Given
that adsorption and penetration rates
for all hinge mutant viruses were
unperturbed and that inhibition
of these viruses by soluble heparin was
similar to that observed
for CDV-1-51, evidence from this and other
studies suggests that
disruption of E protein function may be caused
not by altered
binding but by structural instability of the E protein.
This instability
appears to occur at a point after viral entry and is
exacerbated
by an increase in the hydrophobicity of the region, as
evidenced
by the phenotypes of the hinge mutant viruses described
above.
CDV-1-51v1 (S
I), the most attenuated of the four hinge
mutant
viruses constructed, contained the most hydrophobic of the
substituted
amino acids and displayed a complete loss of NI in mice,
coupled
with reduced replication kinetics in both Vero cells and
infected
mouse brain. Similarly, CDV-1-51v3 (S
V) and CDV-1-51v4
(S
P)
contained substituted amino acids of intermediate
hydrophobicity,
correlating with intermediate levels of both NI and
replication
in vitro and in vivo. Concomitantly, the substitution of an
Asn
residue at E-277 [(CDV-1-51v2 (S
N)], which has little
effect
on the hydrophobicity of the region, correspondingly had no
effect
on the phenotype of the virus (high NI and normal growth
kinetics
in vitro and in
vivo).
E-277 forms part of a Ser-Ser-Ser-Thr (SSST) motif at positions E-275
to E-278 of the MVE E protein, a strongly hydrophilic
-turn (linking
-sheets k and l) which is relatively well conserved
throughout the
JEV serocomplex of flaviviruses. Interestingly,
both WNV and KUN
contain a hydrophilic Asn residue at E-277, making
the resultant SSNT
motif identical to that present in CDV-1-51v2
(S
N). All of these
viruses are of high NI and NV in mice. It
therefore appears likely that
the functional basis for attenuation
caused by mutations at E-277 is
disruption of the
-turn, which
on the basis of the three-dimensional
structure of the protein
appears to form an integral part of the hinge
region.
Despite the inferences drawn above, the specific effect of
mutations at E-277 is difficult to ascertain. The inability of
CDV-1-51v1 (S
I) to hemagglutinate RBCs at low pH and the
observed
reduction of this ability in both CDV-1-51v3 (S
V) and
CDV-1-51v4
(S
P) suggests that a defect in fusion activity
suggested by McMinn
et al. (
47) is responsible for the
observed phenotypes. In the
aforementioned study, BHv1 [E-277
(S
I)] was also observed to
be less efficient in
fusion-from-within assays, lending further
support for reduced fusion
activity in viruses with hydrophobic
amino acid substitutions at E-277.
With the three-dimensional structure of the TBE E protein now known
(
60), it is possible to use this structure as a basis
for
modeling similar proteins from related flaviviruses, such
as MVE.
Threading of the MVE E protein sequence onto the known
structure of the
TBE E protein and subsequent energy minimization
of this basic model
shows a very high degree of structural similarity
between the two
proteins. This structural homology is likely to
be paralleled by a
functional homology which includes a substantial
rearrangement of the
protein during its transition from dimer
to trimer at low pH. In the
mechanism proposed by Rey et al. (
60),
mobility in the
hinge region is likely to project the conserved
hydrophobic fusion
peptide upwards for participation in fusion
with the endosomal
membrane, a mechanism analogous to that proposed
for members of other
virus families, including paramyxoviruses
(
3),
orthomyxoviruses (
5), and filoviruses (
71).
Similarities between the model proposed by Rey et al. (
60)
for TBE and that observed for other viruses are evident. Viral
fusion
proteins appear to undergo significant structural rearrangement
at low
pH, including a shift from dimer to trimer. In addition,
mutations in
regions which are reorganized during the dimer-to-trimer
transition can
increase the optimal pH for fusion in both viruses,
reflecting a
disruption of normal fusion activity. For example,
substitutions at
positions 55 or 71 of the HA
2 subunit of the
influenza A virus HA protein are sufficient to shift the optimal
pH for
fusion 0.6 point up from 5.1 to 5.7 (
58,
66). Similarly,
mutations at E-153 of DEN-2 have been shown to elevate the pH
threshold
of fusion (
22), as did mutations at E-277 of MVE
(S
V
and S
P) in this study. It is possible that these
substitutions
disrupt the stability of the protein, lowering the energy
barrier
between the metastable native state and the more stable final
conformation and allowing conformational change to occur at higher
pH.
By combining the model for dimer-to-trimer transition outlined by
Allison et al. (
1) with that of defective fusion in the
endosome at low pH described by McMinn (
45), hypothetical
models
for the basis of attenuation caused by mutations in the hinge
region can be postulated. For example, limited mobility in the
hinge
region (E-277) may prevent correct presentation of the buried
fusion
peptide on the tip of each protein in the homotrimer. Alternatively,
undue stress may be placed on the receptor binding site, causing
premature release of the virion from the receptor just prior to
fusion.
Receptor binding is an important part of the viral entry
process and is
likely to be essential for holding the viral and
cellular membranes in
close proximity for initiation of the fusion
reaction. In human
parainfluenza virus type 3, the binding of
hemagglutinin-neuraminidase to its sialic acid receptor is essential
for the fusion process (
51). Furthermore, increased
avidity
of the receptor-ligand interaction correlates with increased
fusogenicity,
as evidenced by the introduction of site-directed
mutations into
the sialic acid binding site of
hemagglutinin-neuraminidase (
52).
In contrast, cleavage of
the hemagglutinin-esterase protein of
influenza C virus from its
receptor is a prerequisite for the
low pH-triggered conformational
change required for fusion (
67).
Whether this is the case
in flaviviruses or whether ongoing receptor
binding in the endosome is
required for fusion to occur remains
to be determined. McMinn
(
45) and Corver et al. (
16) have,
however,
shown that the presence of a receptor is not required
for fusion of MVE
or TBE with target liposomes. Furthermore, the
attenuated BHv1 strain
of MVE was able to fuse to liposomes with
equal efficiency to wild-type
virus, suggesting that the defective
fusion phenotype of BHv1 may be
influenced by a dynamic interaction
between the E protein, the
receptor, and the target membrane (
45).
Given that
preexposure of TBE to acidic pH results in a loss of
infectivity and
fusogenicity (presumably due to an irreversible
structural
rearrangement of the E protein) (
16), evidence suggests
that the fusion active state of the E protein is transient and
that the
receptor, which is dispensable for the fusion process
itself, may be
required to orient the endosomal membrane with
a transitional form of
the E protein. If this were the case, premature
release of the virion
from its receptor would allow the E protein
to assume a final
"fusion-inactive" conformation before it had
a chance to interact
with the endosomal
membrane.
Mutations in the RGD motif.
Mutations were introduced into the
RGD motif at position E-390 of MVE (on the lateral face of domain III
of the E protein) to gain further insight into the role of this region
in receptor binding. Evidence for the involvement of the lateral face
of domain III in receptor binding is compelling but for the most part
is based solely on the observed structure of the region
(60). Direct experimental evidence is limited to the
observation that partial-length C-terminal E protein constructs of
DEN-2 (including residues 281 to 423) have potent cell binding
activity, while N-terminal constructs do not (12). Whether
the RGD motif in mosquito-borne flaviviruses plays an important role in
binding to host cell GAGs or integrins and whether these serve as
receptors for virus entry is difficult to ascertain. The RGD motif is
not present in any of the DEN viruses, and these viruses are able to
bind heparin with greater affinity than MVE (see reference
39 and work presented in this study). Integrin binding is
further complicated by the fact that motifs other than RGD have been
implicated in the process. Motifs such as EILDV (29) and
IDAPS (70) are involved in the binding of fibronectin to
41 integrins.
Similarly, DGEA-containing peptides are capable of inhibiting the
binding of collagen to
21 integrins, suggesting that this motif may also play a role (65).
Regardless of whether integrins are involved in flavivirus entry, it is
clear that RGD-dependent binding to host cell integrins
is not an
important determinant of virulence in flaviviruses.
While the D
N
and D
Y substitutions at E-390 of MVE caused a complete
loss of
NI in the mouse model, coupled with attenuated replication
kinetics in
vitro and in vivo, the D
E substitution had no effect
on viral
phenotype. Integrin binding should be severely affected
by such a
mutation, as evidenced by studies of FMDV where disruption
of the RGD
motif by R
K and/or D
E substitutions has been shown
to
disrupt RGD-dependent binding to host cell integrins (
44).
The presence of an RGE motif in WNV (
72) and KUN
(
15), both
of which are highly virulent in mice, plus the
observation that
all of the E-390 mutants derived in this study were
able to adsorb
and penetrate host cells with equal efficiency to
wild-type virus,
suggests that the RGD motif is not essential for virus
binding.
This does not rule out integrin binding as a means of host
cell
entry by flaviviruses; rather, it suggests that RGD-dependent
binding to host cell integrins is not a major pathway for flavivirus
entry.
In contrast to the hinge region of the E protein, the lateral face of
domain III is strongly hydrophilic. As such, it is possible
that
the introduction of a large hydrophobic Tyr residue [CDV-1-51v7
(D
Y)] may destabilize the FG loop (nomenclature of Rey et al.
[
60]). Alternatively, the replacement of a negatively
charged
Asp residue with an uncharged Tyr or Asn residue could affect
the formation of a functionally important salt bridge. Formation
of
such a bridge has recently been shown to be an important determinant
of
virulence in TBE. Based on the three-dimensional structure
of the TBE E
protein, an Asp residue at E-308 (Asp-308) is believed
to form a salt
bridge with a Lys residue at E-311 (
60). Replacement
of
Asp-308 with a Lys residue resulted in a virus which was highly
unstable, and passage of this virus in baby mouse brain
resulted
in spontaneous reversion of the replacement Lys to a
Glu residue
(
42). Interestingly, reversion to a negatively
charged Glu residue
required only a single nucleotide change, while
reversion to the
wild-type Asp would have required two such changes. It
therefore
seems evident that the virus took the shortest route possible
to restore the salt bridge between residues E-308 and E-311.
Confirmation
of whether a salt bridge involving E-390 (in MVE) exists
will
require elucidation of the three-dimensional structure of the
MVE
E protein; however, at least three positively charged His
residues are
in close proximity to E-390: His-395, His-396, and
His-398.
Furthermore, the Arg residue which forms part of the
RGD motif is also
positively charged. It is entirely possible
that one of these four
residues could participate in the formation
of a functionally important
salt bridge in the MVE E protein.
The instability of the Asn
substitution in CDV-1-51v5 (D
N), as
evidenced by its reversion
to a wild-type Asp residue after passage
in Vero cells, is reminiscent
of the reversion observed in the
Mandl et al. study (
42).
Furthermore, if a functionally important
salt bridge were playing a
role in this region of the MVE E protein,
a Glu residue at E-390 would
be expected to functionally substitute
for an Asp residue, as
demonstrated for CDV-1-51v6 (D
E), which
had an almost identical
phenotype to that of clone-derived wild-type
virus.
Since mutations in the RGD motif of MVE do not appear to affect the
ability of virus to bind host cells, it seems likely that
this motif is
not functionally important in receptor binding.
Disruption of the motif
had little to no effect on the heparin
binding ability of the virus, as
evidenced by heparin inhibition
assays which showed approximately 40%
inhibition for all viruses
produced during the study. Incomplete
inhibition by heparin suggests
that receptors other than GAGs can serve
as mediators of viral
entry.
Variation in the amount of HS expressed on different cell types, as
well as the degree of sulfation of HS, may go some way
towards
explaining the different levels of inhibition by heparin
seen in
previous studies. Lee and Lobigs (
39) have shown that
inhibition of MVE binding by soluble heparin is low in Vero cells
and
high in BHK-21 cells. In addition, Chen et al. (
13) have
shown that highly sulfated HS is capable of significantly inhibiting
the binding of DEN-2, while low-sulfate HS is not. Similar work
on
respiratory syncytial virus has shown that the interaction
of virus
with cell surface GAGs is not based on a simple charge
interaction but
rather a specific GAG structural configuration
including
N-sulfation and iduronic acid (
25). Thus,
binding
of flaviviruses to host cells is likely to be a complicated
process
involving multiple receptors with various levels of affinity.
The use of such receptors may depend on their relative availability
on
the host cell, as well as whether these receptors have been
posttranslationally modified in a way amenable to virus
binding.
Aside from receptor binding, it is possible that mutations at E-390
could affect virulence by disrupting the conformation
of the E protein
in much the same way as that described for the
hinge region. Recent
work by van der Most et al. (
69), involving
SDM of
the RGD motif of YF, has shown that viruses containing
Arg
Thr,
Gly
Ala, or Asp
Glu/Ser substitutions at E-390 are
able
to bind to and infect both SW-13 and C6/36 cells. In addition,
RGD-containing peptides did not inhibit infection of primary chicken
embryo fibroblasts by YF-17D (Preugschat and Strauss, unpublished
results cited by van der Most et al. [
69]). In the van
der Most
et al. study (
69), the explanation for the
observed phenotypes
was instability in the E protein. Intracellular
levels of the
E protein were considerably lower in viruses with RGD
motif mutations;
however, this instability could be overcome by
incubation of infected
cell cultures at a suboptimal temperature
(30°C) (
69). This
prompted the authors to suggest that
the nature of attenuating
mutations in the FG loop of flaviviruses may
be to cause incorrect
folding of the protein at a posttranslational
stage. Incorrectly
folded E proteins may be defective in their ability
to interact
with prM or another chaperone protein, resulting in their
retention
in the endoplasmic reticulum and eventual degradation. Such
retention
has been observed in cells expressing misfolded HA of
influenza
A virus (
19). The suggestion that attenuation of
flaviviruses
caused by mutations in the RGD motif are caused not by
altered
receptor binding but by posttranslational instability of the E
protein is an interesting one and warrants further investigation.
It
may be that the attenuation of MVE mutants with substitutions
at E-390
has the same functional basis as that described for similar
mutants of
YF.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Virology
Division, TVW Telethon Institute of Child Health Research, Subiaco WA
6008, Australia. Phone: 61 8 9489 7896. Fax: 61 8 9489 7700. E-mail: robh{at}ichr.uwa.edu.au.
| |
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Journal of Virology, August 2001, p. 7692-7702, Vol. 75, No. 16
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.16.7692-7702.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
