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Journal of Virology, November 2001, p. 10787-10799, Vol. 75, No. 22
Clinical Medical Virology Centre, University
of Queensland, St. Lucia, Queensland 4072, and Sir Albert Sakzewski
Virus Research Centre, Royal Children's Hospital, Herston, Brisbane,
Queensland 4029, Australia
Received 18 May 2001/Accepted 6 August 2001
The intracellular assembly site for flaviviruses in currently not
known but is presumed to be located within the lumen of the rough
endoplasmic reticulum (RER). Building on previous studies involving
immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin
virus (KUN)-infected cells, we sought to identify the steps involved in
the assembly and maturation of KUN. Thus, using antibodies directed
against envelope protein E in IF analysis, we found the accumulation of
E within regions coincident with the RER and endosomal compartments.
Immunogold labeling of cryosections of infected cells indicated that E
and minor envelope protein prM were localized to reticulum membranes
continuous with KUN-induced convoluted membranes (CM) or
paracrystalline arrays (PC) and that sometimes the RER contained
immunogold-labeled virus particles. Both proteins were also observed to
be labeled in membranes at the periphery of the induced CM or PC
structures, but the latter were very seldom labeled internally.
Utilizing drugs that inhibit protein and/or membrane traffic throughout
the cell, we found that the secretion of KUN particles late in
infection was significantly affected in the presence of brefeldin A and
that the infectivity of secreted particles was severely affected in the
presence of monensin and N-nonyl-deoxynojirimycin.
Nocodazole did not appear to affect maturation, suggesting that
microtubules play no role in assembly or maturation processes.
Subsequently, we showed that the exit of intact virions from the RER
involves the transport of individual virions within individual vesicles
en route to the Golgi apparatus. The results suggest that the assembly
of virions occurs within the lumen of the RER and that subsequent
maturation occurs via the secretory pathway.
The formation of flavivirus infectious particles
(~50 nm in diameter) involves the assembly of the nucleocapsid or
core particles (~30 nm in diameter) comprising the positive-sense RNA
genome and core protein C, all enclosed in a lipid envelope containing envelope protein E and premembrane prM (which is proteolytically cleaved late during maturation to yield protein M). The core particle has never been isolated in its free state from infected cells or
positively identified free within the cytoplasm by electron microscopy.
Virions accumulate as large cytoplasmic aggregates, but with rare
exceptions, the normal process of budding through membranes that is
associated with the envelopment of viruses has not been observed with
Flavivirus species (15, 37). Using Kunjin virus
(KUN) as a model to study the events of flavivirus replication, we have
characterized unique sets of induced membranes and their apparent
replication functions by cryoimmunoelectron microscopy (CIEM). Thus, we
have identified the intracellular site of KUN RNA synthesis within
packets of vesicles (VP) that associate closely with another
virus-induced membrane structure, termed convoluted membranes (CM)
interconvertible with paracrystalline array (PC) structures (33,
59). CM or PC is presumed to be the site of proteolytic
processing by the viral serine protease (NS3 and cofactor NS2B) located
therein (59). The consensus composition of the KUN
replicase complex in the VP comprises NS1, NS2A, NS3, NS4A, NS5, and
the template double-stranded RNA or replicative form, all identified
with monospecific antibodies by CIEM and radioimmunoprecipitation and
by biochemical analyses of infected cells (31, 33, 59). We
have proposed that this collection of membranes, along with associated
virions and proliferating endoplasmic reticulum (ER), comprises
virus factories in which compartmentalization within the induced
membranes ensures a more efficient replication process.
While it appears that virus particles are closely associated with the
KUN factories, the exact intracellular site of flavivirus assembly
remains obscure. Ultrastructural studies of flavivirus-infected cells
have consistently revealed apparent mature virions within distended ER,
large cytoplasmic vesicles, and vacuoles (37). In
addition, occasional sections have shown individual particles within
Golgi cisternae, suggesting that flavivirus maturation proceeds through
the Golgi apparatus (6, 14-16, 26, 32). After translation
and translocation of the KUN polyprotein into the ER lumen, processing
of prM and E occurs via host signal peptidase, whereas the core protein
remains within the cytosol and is cleaved at a dibasic site preceding
its carboxy-terminal hydrophobic domain by the KUN protease.
Immunolocalization studies have indicated that the core protein
translocates to the nucleus, associates in the cytosol with
virus-induced CM or PC, and is incorporated into virions
(58). In vitro studies with expressed recombinant proteins
of other flavivirus species have shown that a heterodimeric association
between prM and E is formed during the assembly and maturation
processes (3, 57), and domains required for this interaction have been identified within both E and prM (3, 4). However, the interaction that occurs between the core
protein and either prM or E to promote the assembly process is still
unknown. Several studies have indicated that prM facilitates the
correct folding of E (3, 17, 24) or, alternatively, masks
a retention signal within E, although no such signal has thus far been
identified. During maturation, prM appears to prevent E undergoing an
acid-catalyzed conformational change required for fusion within the
endosome formed during the entry of the virus (3, 12, 13,
18). During the final stages of maturation before virus release,
this preventive mechanism is overcome by cleavage of the pr
moiety from prM by the host protease furin (52).
Due to the orientation and topology of the structural proteins within
the lipid bilayer, it is presumed that flavivirus assembly must occur
via the core particle budding into the ER lumen (46). Although this process has not been convincingly shown, recent developments in cryofixation and CIEM have revealed budding of West
Nile virus strain Sarafend (glycosylated in both prM and E) at the
plasma and intracellular membranes (38). However, as noted
above, this process does not appear to be the general mechanism for
Flavivirus species. In this study, we have addressed the
ultrastructural location of flavivirus assembly and the subsequent maturation pathway by using CIEM in conjunction with various inhibitors of secretion.
Cells and virus.
Vero cells were grown and maintained in
Dulbecco modified Eagle medium (DMEM) (Gibco BRL) supplemented with 5%
fetal calf serum and penicillin-streptomycin. Cells were infected with
KUN strain MRM61C at a multiplicity of infection (MOI) of 3 as
previously described (59), and infected cells were
maintained in DMEM containing 0.1% bovine serum albumin.
Reagents.
Brefeldin A (BFA), monensin (MON), and nocadazole
(NOZ) were obtained from Sigma and used at concentrations of 5 µg/ml,
10 µM, and 2 µg/ml, respectively. Imino sugars, deoxynojirimycin (DNJ), and N-nonyl-DNJ (NN-DNJ) were obtained from Toronto
Research Chemicals (Toronto, Ontario, Canada) and used at a
concentration of 100 µM. Antibodies used were mouse monoclonal
anti-KUN E antibodies (1), rabbit polyclonal anti-Murray
Valley encephalitis virus E antibodies (cross-reactive with KUN E), and
mouse monoclonal antibodies to KUN prM glycoprotein (1E7), all provided
by R. Hall (University of Queensland, Brisbane, Queensland, Australia);
monoclonal antibodies to ERGIC53 (48) and to giantin
(28), provided by H.-P. Hauri (University of Basel, Basel,
Switzerland), and to protein disulfide isomerase (PD1) (ID3)
(55), provided by S. Fuller European Molecular Biology
Laboratory, Heidelberg, Germany); rabbit polyclonal antibodies to human
Lamp1 (93/B) (11), provided by M. Fukuda (La Jolla Cancer
Research Foundation, La Jolla, Calif.); goat polyclonal antibodies to
mannose-6-phosphate receptor (M6PR) (Zi I-2), provided by A. Hille
(Department of Biochemistry, Göttingen, Germany); and donkey
antibodies specific for rabbit or goat immunoglobulin G and conjugated
to fluorescein isothiocyanate (FITC) or Texas Red, purchased from
Edward Keller (Hallam, Victoria, Australia).
Radiolabeling.
Subconfluent monolayers of Vero cells were
infected with KUN at an MOI of 3. At 17 h postinfection (p.i.), cells
were incubated in methionine- and cysteine-deficient medium for 1 h. Cells were then pulse-labeled with 50 µCi of
[35S]methionine-cysteine (Trans-label; ICN) per ml for
120 min in the presence of 3 µg of actinomycin D per ml. When chase
experiments were performed, the labeling medium was removed and the
cells were washed twice with phosphate-buffered saline (PBS) before incubation with DMEM supplemented with 0.1% bovine serum albumin and a
10× excess of unlabeled methionine and cysteine (150 and 32 µg/ml,
respectively). After either labeling or chase periods (4 h), the tissue
culture fluid was collected and clarified by centrifugation at low
speed; the cell monolayer was harvested in coimmunoprecipitation buffer
(10 mM Tris [pH 8.0], 150 mM NaCl, 5 mM EDTA, 1% Triton X-100)
containing protease inhibitors and incubated on ice for 20 min before
clarification by centrifugation. Both the tissue culture fluid and
lysates were used for radioimmunoprecipitation (RIP) experiments with
mouse anti-E antibodies as previously described (22).
Immunofluorescence (IF).
Vero cell monolayers on coverslips
were infected with KUN at an MOI of approximately 3 to 5 and incubated
at 37°C for 24 h. The cells were subsequently washed with PBS,
fixed with 4% paraformaldehyde for 10 min at 20°C, and permeabilized
with 0.2% Triton X-100 in 4% paraformaldehyde for 10 min at 20°C.
The cells were washed with PBS, and aldehyde groups were quenched with
0.5 M NH4Cl for 7 min at 20°C. The cells were
subsequently washed twice with PBS before incubation with antibodies.
Electron microscopy.
Methods for resin embedding,
cryofixation, preparation of cryosections, and immunolabeling have been
described elsewhere (31, 32, 59). However, for the current
experiments, the cell blocks used for sectioning were embedded in 10%
gelatin and postfixed with 1% paraformaldehyde. Sections were cut with
a Diatome Cryo-P diamond knife and retrieved from the cryochamber with
a droplet of 14:1 2.3 M sucrose-2% methylcellulose.
IF analysis of the subcellular localization of KUN E protein.
To monitor the distribution of the KUN E protein in relation to
cellular organelles possibly involved in assembly or maturation of
virions, we examined the immunolocalization of E with antibodies to
various cell markers. The KUN strain (MRM61C) used for these experiments encodes a nonglycosylated E protein (60). At
24 h postinfection (p.i.), KUN-infected cells were fixed and
immunostained. The overall distribution of KUN E consisted of diffuse
perinuclear staining that often included several large densely staining
foci and small isolated foci that were widespread throughout the
cytoplasm. Dual-labeling experiments revealed extensive colocalization
of KUN E with the marker for the rough ER (RER) (anti-PDI) in the perinuclear region (Fig. 1A to C),
suggesting the accumulation of KUN E in this region. The perinuclear
staining pattern of E also overlapped that of the Golgi apparatus
(detected with antigiantin) (Fig. 1G to I), but precise colocalization
within this organelle could not be determined by light microscopy.
However, some costained cells displayed quite distinct staining
patterns with anti-E and antigiantin antibodies (results not shown).
Some anti-E foci associated with the perinuclear region were colabeled
with a marker for the intermediate compartment (IC) (anti-ERGIC53)
(Fig. 1D to F). A similar IF staining pattern with anti-ERGIC53
antibodies and anti-KUN NS3 antibodies was previously found
(30), suggesting that the anti-E-labeled foci may be
associated with KUN CM or PC. In contrast, the smaller anti-E-stained
foci distributed within the cytoplasm were strongly colabeled with a
marker for endosomes (anti-M6PR) (Fig. 1J to L). This coincidental
labeling of E within endosomes is unfortunately unable to differentiate
between incoming or outgoing E-labeled virions. E appeared to also show
some coincidental labeling with lysosomes in the perinuclear region
(Fig. 1M to O).
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.22.10787-10799.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Assembly and Maturation of the Flavivirus Kunjin
Virus Appear To Occur in the Rough Endoplasmic Reticulum and along the
Secretory Pathway, Respectively
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Markers for the RER, IC, and endosomal region are
associated by IF with the KUN E protein. KUN-infected Vero cells were
fixed at 24 h p.i. and processed for IF with anti-E antibodies
conjugated to Texas Red (B, E, H, K, and N) for comparisons
with FITC-labeled of antibodies to various cellular
compartments (A, D, G, J, and M) (same cells). Apparent partial
coincidence in the dual labels was observed as a yellow hue in panels
C, F, and L. No coincidence with KUN E was apparent for the marker for
lysosomes (Lamp1), but some partial overlap was observed with the
marker for the Golgi apparatus (giantin). (P and Q) Mock-infected Vero
cells incubated with mouse and rabbit anti-E
antibodies, respectively.
Ultrastructural analysis shows that KUN E and prM are primarily
associated with reticular membranes and the KUN-induced CM or PC
structures within infected cells.
To further define the
intracellular localization of KUN during its assembly and maturation,
we immunolabeled cryosections from KUN-infected cells at 24 h p.i.
with monoclonal antibodies raised against the E and prM proteins. As
shown in Fig. 2, gold particles were
primarily associated with reticular membranes present within the
cytoplasm of infected cells. Some of these reticular membranes appeared
to show direct connections with KUN-induced CM or PC structures (Fig.
2D and E). In most sections, labeling with anti-E and/or anti-prM
antibodies within the reticular membranes was associated with
accumulated virus particles (Fig. 2A, B, C, and E); however, in some
sections labeling did not appear to be associated with any particular
structure other than the ER or virus particles (Fig. 2D) and therefore
may have represented either the accumulation of E at assembly sites or
the detection of E translocated into the ER during its synthesis. Both
KUN E and prM were also immunolabeled at similar sites on reticular
membranes, at or around the periphery of induced CM or PC (Fig. 2E) and
on virus particles that were either extracellular (Fig. 2F) or
intracellular, within a vacuole (Fig. 2G). Gold particles appeared to
sparsely label the inner components of some CM or PC structures (Fig.
2B and E), but gold particles were never observed to label VP
structures. Interestingly, previous immunoelectron microscopy
observations with anticore antibodies showed a labeling pattern (of the
periphery of induced membranes and virions [58]) very
similar to that of anti-E and anti-prM antibodies presented here (Fig.
2A, B, C, and E). Notably, gold particles within the Golgi apparatus were always associated with virions (Fig. 2C), implying that the E
protein alone does not traverse this organelle.
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Intracellular distribution of the KUN E protein after treatment of
infected cells with metabolic inhibitors.
As part of the studies
on the intracellular location of flavivirus assembly, we sought to
determine the effects of metabolic inhibitors on this process late in
infection. Our initial screening was performed by dual IF with
antibodies to KUN E and the Golgi apparatus (antigiantin). Antigiantin
antibodies were used to determine the effects of the inhibitors on
cellular morphology, as most of them have an impact on the Golgi
apparatus. BFA affects many cellular functions, but its primary
target is the G-coupled ADP-ribosylation factors (10, 19).
The effect of BFA on ADP-ribosylation factors leads to an interruption
in the vesicular flow from the IC to the Golgi apparatus, resulting in
Golgi apparatus disassembly (9, 49). MON arrests protein
movement within the medial Golgi compartment (44), whereas
NOZ disrupts microtubules and thus microtubule-dependent traffic. DNJ
and NN-DNJ are inhibitors of the 
-glucosidases, involved in the
removal of terminal glucose residues from the glycans of nascent
glycoproteins in the RER, and thus cause some viral glycoproteins to be
misfolded and retained within the ER (8, 36, 62).
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Effects of metabolic inhibitors on the assembly, secretion, and maturation of KUN. To extend the above study, we used radiolabeling in pulse-chase experiments to evaluate both assembly and secretion of KUN in the presence of inhibitors. For the assembly studies, we reasoned that the coprecipitation of core protein from treated lysates or infected culture fluid during RIP with anti-E antibodies would suggest that the formation of virus particles had occurred in the presence of inhibitors. This presumption stems from the observations of Khromykh et al. (22), who found that core protein interacted with the other structural proteins only in the presence of replicating KUN RNA. Our aims were to attempt inhibition of assembly at specific cellular sites, namely, RER (with DNJ or NN-DNJ), IC (with BFA), and medial Golgi compartment (with MON), and to look for the presence of pulse-labeled core in anti-E immunoprecipitates. The effects of the addition of inhibitors from 18 h p.i. on virus secretion when approaching the maximum period of virus release were analyzed by collecting radiolabeled culture fluids for RIP and plaque assay analyses.
In all lysates, incorporation of the radiolabel in E, C, and prM from 18 to 20 h p.i. appeared efficient and did not appear to be inhibited by the presence of inhibitors (Fig. 4A); however, in lysates of MON-treated cells, the immature precursor Ci of virion C appeared to be inefficiently cleaved because normal product C (61) was only weakly labeled (Fig. 4A, lane 9). Control (untreated) samples revealed that the KUN virion was efficiently secreted into the culture fluid during the chase period of 4 h, and most of the labeled prM glycoprotein in lysates was proteolytically processed to pr and M in the tcf (Fig. 4A, compare lanes 1 and 9). In stark contrast, incubation of infected cells in the presence of BFA drastically reduced the amounts of all secreted structural proteins to undetectable amounts, yet the level of incorporation of radiolabel in BFA-treated cells was only slightly reduced (Fig. 4A, lanes 2 and 8) compared to the results obtained for other lysates associated with each inhibitor treatment. Treatment of cells late in infection with MON allowed efficient secretion of particles comprising E, prM, and C; notably, however, prM glycoprotein was not further processed (Fig. 4A, lanes 3 and 9), suggesting that either these particles did not pass through the trans-Golgi compartment or treatment with MON affected the pH-dependent cleavage of prM to M. Treatment of cells with either NOZ, DNJ, or NN-DNJ did not appear to have a significant influence on the secretion of virus particles or on the normal processing of prM, although the amounts of immunoprecipitated secreted structural proteins were reduced in each of these samples compared with MON-treated cells (Fig. 4A, lanes 3 to 6). In all samples other than the BFA culture fluid, core protein appeared to be coprecipitated with anti-E antibodies, indicating that particle formation had occurred.
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-glucosidase activity by NN-DNJ was unique in that it had a profound
effect on the infectivity of secreted virus, even though prM appeared
to be cleaved.
When BFA was added as early as 1.5 h p.i., viral protein synthesis
and release of infectious virus late in infection were drastically
impaired (Fig. 5), compared with the
small effects when BFA was added late (Fig. 4). These results indicate
that the early addition of BFA severely restricts expression, probably by inhibiting the membrane induction (involving the
trans-Golgi membranes) required for optimal RNA synthesis
(30), as suggested for the inhibitory effects of BFA on
poliovirus replication (35). The effects of the other
inhibitors were similar when they were added either early or late
(compare Fig. 4 and 5). Notably, the early disruption of microtubules
by NOZ had no effect on virus replication and release.
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Exit of KUN virions from the RER occurs via transport
vesicles.
The observations presented in this article and those of
others (46) suggest that the assembly of the flavivirus
virion occurs within the RER. No direct evidence of budding of
nucleocapsids into the lumen of the RER has been published, indicating
that this event either is extremely rapid or occurs infrequently. We therefore carefully scanned cryosections for any evidence of a maturation pathway for KUN virions involving transport from the RER via
individual vesicles to the Golgi apparatus. Figure
6A shows quite clearly KUN virions
apparently exiting the RER (gold labeled with antibodies against PDI)
via individual protuberant vesicles emerging from the RER membrane.
It is apparent that each virion is being transported to the
Golgi apparatus via a separate vesicle carrier (Fig. 6B and C). These
vesicles most likely represent transport vesicles associated with the
IC, as tubular-vesicular clusters (tvc) synonymous with the IC
(29, 54) were frequently observed associated with
virus-carrier vesicles (Fig. 6C). We propose that subsequent transport
proceeds through the Golgi apparatus (Fig. 6) and that the accumulation
of "mature" virions occurs in the trans-Golgi region or
endosomal region. KUN virions do not appear to accumulate within the
Golgi organelle, indicating that transit through the stacks must be a
rapid event.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The experiments described in this paper have attempted to define the intracellular site of assembly and the maturation pathway for the flavivirus KUN. For these analyses, we used antibody markers to cellular compartments and to the KUN structural proteins E and prM for both dual IF (Fig. 1 and 3) and immunolabeling of cryosections (Fig. 2, 5, and 6). We showed that KUN virions appear to accumulate within the RER and endosomal vesicles (Fig. 1 and 6), that the E and prM proteins localize to distended ER and also to the periphery of the induced CM or PC structures (sometimes associated with virions) (Fig. 2), and that the KUN virion appears to initiate maturation by exiting the RER via individual transport vesicles (Fig. 6) and probably fuses with the cis-Golgi compartment. Although no direct evidence for virus assembly was observed, the accumulation of apparently intact virions within the RER and the assembly of infectious virions in the presence of BFA (Fig. 4) suggest that this event occurs pre-Golgi apparatus, presumably within the RER.
IF analyses revealed that KUN E was distributed within the perinuclear region in a diffuse manner and in large densely staining foci and accumulated throughout the cytoplasm as small isolated foci (Fig. 1). When compared to locations of cell markers, the staining pattern of KUN E indicated accumulation largely within the RER and endosomal compartments, plus some coincidence with the IC marker. Interestingly, a minor overlap with the Golgi apparatus was observed (Fig. 1G to I) and persisted in the presence of the inhibitor MON, thought to arrest transport within this organelle (Fig. 3D to F). CIEM results suggested that E transversed the Golgi apparatus only when incorporated in a virus particle (Fig. 2C), suggesting that assembly occurs pre-Golgi apparatus. Virus particles were not frequently observed within the Golgi apparatus, implying that transport through the individual stacks occurs quite rapidly.
The use of metabolic inhibitors to study aspects of flavivirus assembly
has not been investigated extensively. Sreenivasan et al.
(51) added BFA at 1 h p.i. to investigate its effects on West Nile virus strain Sarafend replication in Vero cells. The
results showed some reduction in protein synthesis and severe impairment of [3H] mannose incorporation in viral
glycoproteins E and prM in the presence of 1 µg of BFA per ml, and
these effects were associated with a reduction of 3 log units in the
release of infectious extracellular virus at 20 h p.i. We
performed similar experiments and found that the presence of 5 µg of
BFA per ml early in infection severely hampered the metabolic
incorporation of radiolabel into KUN structural proteins (Fig. 5), most
likely by restricting membrane induction and thus KUN replication
(30). Courageot et al. (8) incubated dengue 1 virus-infected cells in the presence of 30 µM DNJ and 300 µM
castanospermine (both -glucosidase inhibitors) from 19 to 25 h
p.i. and found a 50 to 80% reduction in the titer of extracellular virus. In contrast, our similar experiments with KUN and 100 µM DNJ
showed no effect on infectious virus release. However, our observed
effect of NN-DNJ on KUN infectivity is in agreement with the results of
Zitzmann et al. (62), who found a similar effect of NN-DNJ
on the infectivity of bovine viral diarrhea virus (also a member of the
Flaviviridae). Currently, the only explanation we have for
the discrepancy in the lack of effect of DNJ on infectious KUN release
is that the strain of KUN that we are currently using encodes an
unglycosylated E protein. In fact, preliminary experiments using a
mutated strain of KUN with glycosylated E (1) suggest this
may be the case, as in these experiments DNJ reduced the release of
infectious virus by 50% whereas NN-DNJ surprisingly had no effect on
the infectious virus titer (unpublished results).
The results obtained with MON are intriguing. First, incubation of cells with MON is thought to arrest viral glycoprotein movement within the Golgi apparatus (20, 21, 42, 44), yet our RIP analysis after pulse-chase experiments (Fig. 4A) indicated that KUN structural proteins are still secreted under these conditions. Second, during treatment with MON, a large amount of prM associated with E and C was secreted into the medium (Fig. 4A), suggesting that movement of virus particles within the medial Golgi compartment was not arrested and that subsequent furin cleavage of prM was either not occurring or was very inefficient. This deficiency in cleavage is primarily responsible for the reduction in infectious virus titer by not allowing the conformational change in the E protein (of immature particles containing prM) that is required for uncoating and fusion with endosomal membranes after infection (2, 18, 53), rather than arrest of particles within the Golgi apparatus. Similar results were observed when acidotropic amines (such as NH4Cl, chloroquine, or methylamine) were added to the culture fluid of flavivirus-infected cells (45, 47). Based on these earlier results and our present observations, it would appear that the ionophore MON causes an increase in the pH of endosomal compartments, disabling the low-pH-dependent action of furin that normally occurs just prior to virus secretion (52). In addition, anchored or immature core protein (Ci) appeared to accumulate in lysates (Fig. 4A, lane 9) in lieu of normal end-product virion C and was not secreted. This accumulation of nonassembled Ci may have also enhanced the formation and release of noninfectious slowly sedimenting hemagglutinin particles, comprising predominantly E and prM (50), into the tissue culture fluid, and this factor would also contribute to the observed reduction in infectivity. Thus, in addition to the obvious inhibition of cleavage of prM, MON also appears in some unknown way to inhibit the cleavage of Ci that generates virion C.
The results obtained with NOZ indicated that microtubules play no role in the release of KUN from infected cells (Fig. 4 and 5), similar to the findings of Ng et al. (39), who found reductions of about only 10-fold in extracellular virus titers after treatment of KUN-infected cells with the cytoskeletal disrupting agents vinblastine sulfate, colchicine, and cytochalasin B.
Previous localization data obtained with anticore antibodies
(58) revealed a distribution in the cytoplasm similar to
that obtained here with anti-E and anti-prM antibodies (Fig. 2). Thus, all three structural proteins appeared to be labeled on the periphery of the induced CM or PC structures in association with the RER, which
is involved in their sequential translocation and
posttranslational cleavage. We have suggested that the induced CM or PC
structures are the sites of viral protease cleavage (59);
thus, the posttranslational cleavage of C from prM by NS2B-NS3
(5, 27, 41, 61) may also occur at this location. In
addition, as VP are the intracellular sites of KUN RNA synthesis
(59) and are also closely associated with the CM or PC
structures, it is not surprising that the structural proteins would
also remain closely associated with these structures and package the
newly synthesized genomic RNA. One could speculate that assembly thus
occurs at or near the ER membranes continuous with the
virus-induced membrane structures. The subsequent steps involve
maturation and secretion of virions that exit the RER via individual
transport vesicles en route to the Golgi apparatus (Fig. 6). Once these
vesicles fuse with the Golgi apparatus, the virions appear to move
through the individual stacks into the trans-Golgi region,
where they appear to cluster and accumulate within large vesicles until
exocytosis occurs. This process is schematically depicted in Fig.
7.
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Questions arise as to how virions exit the RER via vesicles for transport to the Golgi apparatus and what signals allow interactions with the coatomer proteins (COP [34]) required for vesicle formation. Obviously, prM plays a role in this process, as the expression of Japanese encephalitis virus E alone led to the retention of the protein within the ER (24). It was additionally shown that the ectodomain of the E protein was responsible for the interaction with prM to allow assembly and secretion of subviral particles of tick-borne encephalitis and dengue viruses (3, 57). These results suggest either that a retention signal within the ectodomain of E is masked in the presence of prM or that prM is required for the correct folding of E. Generally, membrane proteins destined for secretion have a cytoplasmic tail with encoded motifs, such as DxE, that interact with coatomer components to facilitate vesicle formation (40). However, after virus assembly, this type of signal within either E or prM would necessarily interact with the nucleocapsid within the lumen of the ER. Martinez-Menarguez et al. (34) very elegantly showed that soluble secretory proteins were concentrated in noncoated tubular clusters associated with ER membranes containing COPI components, however, after further transport, enrichment of the soluble cargo was observed within COPII-bearing structures and the secretory proteins were then excluded from COPI-bearing structures. Unfortunately, the exact mechanism for this exclusion is still under investigation, but it would be interesting to investigate the distributions of COPI and COPII within flavivirus-infected cells. In addition, because KUN E is nonglycosylated, some as-yet-undefined peptide signal(s) within the protein may exist to target the virion to the trans-Golgi apparatus to allow furin cleavage of pr from prM Another aspect of viral exit is the observed budding of the E-glycosylated strain of West Nile virus (Sarafend) at the plasma membrane (38). Because West Nile virus strains and KUN share 93 to 98% amino acid identity (7, 25, 43), it is surprising that glycosylation of E in the Sarafend strain may induce a distinctly different maturation pathway. Budding of flavivirus nucleocapsids from intracellular membranes has been observed very infrequently and appears to be virus strain dependent and also cell type dependent (14, 15).
The results presented in this paper have highlighted the maturation pathway of the flavivirus KUN. Some limited evidence suggests that assembly occurs within the RER in close association with the KUN-induced CM or PC and VP structures. Molecular approaches, such as the use of the KUN replicon (23), with the opportunity to vary the addition of structural proteins, should enable further investigation of the interactions of structural proteins during the assembly process and aid in visualizing possible assembly sites within the RER.
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ACKNOWLEDGMENTS |
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We thank R. Hall, S. Fuller, H.-P. Hauri, A. Hille, and M. Fukuda for generously providing antibodies. We also thank A. Khromykh for critical reading of the manuscript and for helpful discussions.
Funding for this work was provided by the National Health and Medical Research Council of Australia (grant 102481).
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FOOTNOTES |
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* Corresponding author. Mailing address: Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston Rd., Herston, Brisbane, Queensland 4029, Australia. Phone: (617) 3636 1569. Fax: (617) 3636 1401. E-mail: j.mackenzie{at}mailbox.uq.edu.au.
SASVRC publication 134.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Adams, S. C., A. K. Broom, L. M. Sammels, A. C. Hartnett, M. J. Howard, R. J. Coelen, J. S. Mackenzie, and R. A. Hall. 1995. Glycosylation and antigenic variation among Kunjin virus isolates. Virology 206:49-56[CrossRef][Medline]. |
| 2. | Allison, S. L., J. Schalich, K. Stiasny, C. W. Mandl, C. Kunz, and F. X. Heinz. 1995. Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH. J. Virol. 69:695-700[Abstract]. |
| 3. | Allison, S. L., K. Stadler, C. W. Mandl, C. Kunz, and F. X. Heinz. 1995. Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate forms. J. Virol. 69:5816-5820[Abstract]. |
| 4. |
Allison, S. L.,
K. Stiasny,
K. Stadler,
C. W. Mandl, and F. X. Heinz.
1999.
Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E.
J. Virol.
73:5605-5612 |
| 5. |
Amberg, S. M., and C. M. Rice.
1999.
Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing.
J. Virol.
73:8083-8094 |
| 6. | Barth, O. M. 1999. Ultrastructural aspects of the dengue virus (flavivirus) particle morphogenesis. J. Submicrosc. Cytol. Pathol. 31:407-412[Medline]. |
| 7. |
Coia, G.,
M. D. Parker,
G. Speight,
M. E. Byrne, and E. G. Westaway.
1988.
Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virus-specified proteins.
J. Gen. Virol.
69:1-21 |
| 8. |
Courageot, M. P.,
M. P. Frenkiel,
C. D. Dos Santos,
V. Deubel, and P. Despres.
2000.
Alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum.
J. Virol.
74:564-572 |
| 9. |
Dascher, C., and W. E. Balch.
1994.
Dominant inhibitory mutants of ARFI block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus.
J. Biol. Chem.
269:1437-1448 |
| 10. | Donaldson, J. G., D. Finazzi, and R. D. Klausner. 1992. Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature 360:350-352[CrossRef][Medline]. |
| 11. |
Fukuda, M.,
J. Viitala,
J. Matteson, and S. R. Carlsson.
1988.
Cloning of cDNAs encoding human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Comparison of their deduced amino acid sequences.
J. Biol. Chem.
263:18920-18928 |
| 12. | Guirakhoo, F., R. A. Bolin, and J. T. Roehrig. 1992. The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology 191:921-931[CrossRef][Medline]. |
| 13. |
Guirakhoo, F.,
F. X. Heinz,
C. W. Mandl,
H. Holzmann, and C. Kunz.
1991.
Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions.
J. Gen. Virol.
72:1323-1329 |
| 14. | Hase, T., P. L. Summers, K. H. Eckels, and W. B. Baze. 1987. An electron and immunoelectron microscopic study of dengue-2 virus infection of cultured mosquito cells: maturation events. Arch. Virol. 92:273-291[CrossRef][Medline]. |
| 15. | Hase, T., P. L. Summers, K. H. Eckels, and W. B. Baze. 1987. Maturation process of Japanese encephalitis virus in cultured mosquito cells in vitro and mouse brain cells in vivo. Arch. Virol. 96:135-151[CrossRef][Medline]. |
| 16. | Hase, T., P. L. Summers, K. H. Eckels, and J. R. Putnak. 1989. Morphogenesis of flaviviruses. Subcell. Biochem. 15:275-305[Medline]. |
| 17. | Heinz, F. X., G. Auer, K. Stiasny, H. Holzmann, C. Mandl, F. Guirakhoo, and C. Kunz. 1994. The interactions of the flavivirus envelope proteins: implications for virus entry and release. Arch. Virol. Suppl. 9:339-348[Medline]. |
| 18. | Heinz, F. X., K. Stiasny, G. Puschner-Auer, H. Holzmann, S. L. Allison, C. W. Mandl, and C. Kunz. 1994. Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology 198:109-117[CrossRef][Medline]. |
| 19. | Helms, J. B., and J. E. Rothman. 1992. Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. Nature 360:352-354[CrossRef][Medline]. |
| 20. | Johnson, D. C., and M. J. Schlesinger. 1980. Vesicular stomatitis virus and sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores. Virology 103:407-424[CrossRef][Medline]. |
| 21. |
Kaariainen, L.,
K. Hashimoto,
J. Saraste,
I. Virtanen, and K. Penttinen.
1980.
Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins.
J. Cell Biol.
87:783-791 |
| 22. |
Khromykh, A. A.,
A. N. Varnavski, and E. G. Westaway.
1998.
Encapsidation of the flavivirus Kunjin virus replicon RNA by using a complementation system providing Kunjin virus structural proteins in trans.
J. Virol.
72:5967-5977 |
| 23. | Khromykh, A. A., and E. G. Westaway. 1997. Subgenomic replicons of the flavivirus Kunjin virus: construction and applications. J. Virol. 71:1497-1505[Abstract]. |
| 24. |
Konishi, E., and P. W. Mason.
1993.
Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein.
J. Virol.
67:1672-1675 |
| 25. |
Lanciotti, R. S.,
J. T. Roehrig,
V. Deubel,
J. Smith,
M. Parker,
K. Steele,
B. Crise,
K. E. Volpe,
M. B. Crabtree,
J. H. Scherret,
R. A. Hall,
J. S. MacKenzie,
C. B. Cropp,
B. Panigrahy,
E. Ostlund,
B. Schmitt,
M. Malkinson,
C. Banet,
J. Weissman,
N. Komar,
H. M. Savage,
W. Stone,
T. McNamara, and D. J. Gubler.
1999.
Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States.
Science
286:2333-2337 |
| 26. | Leary, K., and C. D. Blair. 1980. Sequential events in the morphogenesis of Japanese encephalitis virus. J. Ultrastruct. Res. 72:123-129[CrossRef][Medline]. |
| 27. |
Lee, E.,
C. E. Stocks,
S. M. Amberg,
C. M. Rice, and M. Lobigs.
2000.
Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage in vitro is lethal for virus production.
J. Virol.
74:24-32 |
| 28. |
Linstedt, A. D.,
M. Foguet,
M. Renz,
H. P. Seelig,
B. S. Glick, and H. P. Hauri.
1995.
A C-terminally-anchored Golgi protein is inserted into the endoplasmic reticulum and then transported to the Golgi apparatus.
Proc. Natl. Acad. Sci. USA
92:5102-5105 |
| 29. |
Lotti, L. V.,
M. R. Torrisi,
M. C. Pascale, and S. Bonatti.
1992.
Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein from the intermediate compartment to the Golgi complex.
J. Cell Biol.
118:43-50 |
| 30. |
Mackenzie, J. M.,
M. K. Jones, and E. G. Westaway.
1999.
Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells.
J. Virol.
73:9555-9567 |
| 31. | Mackenzie, J. M., M. K. Jones, and P. R. Young. 1996. Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology 220:232-240[CrossRef][Medline]. |
| 32. | Mackenzie, J. M., M. K. Jones, and P. R. Young. 1996. Improved membrane preservation of flavivirus-infected cells with cryosectioning. J. Virol. Methods 56:67-75[CrossRef][Medline]. |
| 33. | Mackenzie, J. M., A. A. Khromykh, M. K. Jones, and E. G. Westaway. 1998. Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A. Virology 245:203-215[CrossRef][Medline]. |
| 34. | Martinez-Menarguez, J. A., H. J. Geuze, J. W. Slot, and J. Klumperman. 1999. Vesicular tubular clusters between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles. Cell 98:81-90[CrossRef][Medline]. |
| 35. |
Maynell, L. A.,
K. Kirkegaard, and M. W. Klymkowsky.
1992.
Inhibition of poliovirus RNA synthesis by brefeldin A.
J. Virol.
66:1985-1994 |
| 36. | Mehta, A., N. Zitzmann, P. M. Rudd, T. M. Block, and R. A. Dwek. 1998. Alpha-glucosidase inhibitors as potential broad based anti-viral agents. FEBS Lett. 430:17-22[CrossRef][Medline]. |
| 37. | Murphy, F. A. 1980. Morphology and morphogenesis, p. 65-103. In T. P. Monath (ed.), St. Louis encephalitis. American Public Health Association Inc, Washington, D.C. |
| 38. | Ng, M. L., J. Howe, V. Sreenivasan, and J. J. Mulders. 1994. Flavivirus West Nile (Sarafend) egress at the plasma membrane. Arch. Virol. 137:303-313[CrossRef][Medline]. |
| 39. | Ng, M. L., J. S. Pedersen, B. H. Toh, and E. G. Westaway. 1983. Immunofluorescent sites in Vero cells infected with the flavivirus Kunjin. Arch. Virol. 78:177-190[CrossRef][Medline]. |
| 40. |
Nishimura, N.,
S. Bannykh,
S. Slabough,
J. Matteson,
Y. Altschuler,
K. Hahn, and W. E. Balch.
1999.
A di-acidic (DXE) code directs concentration of cargo during export from the endoplasmic reticulum.
J. Biol. Chem.
274:15937-15946 |
| 41. | Nowak, T., P. M. Farber, and G. Wengler. 1989. Analyses of the terminal sequences of West Nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic cleavages involved in their synthesis. Virology 169:365-376[CrossRef][Medline]. |
| 42. | Pesonen, M., and L. Kaariainen. 1982. Incomplete complex oligosaccharides in semliki forest virus envelope proteins arrested within the cell in the presence of monensin. J. Mol. Biol. 158:213-230[CrossRef][Medline]. |
| 43. | Poidinger, M., R. A. Hall, and J. S. Mackenzie. 1996. Molecular characterization of the Japanese encephalitis serocomplex of the flavivirus genus. Virology 218:417-421[CrossRef][Medline]. |
| 44. |
Qiu, Z.,
F. Tufaro, and S. Gillam.
1995.
Brefeldin A and monensin arrest cell surface expression of membrane glycoproteins and release of rubella virus.
J. Gen. Virol.
76:855-863 |
| 45. | Randolph, V. B., G. Winkler, and V. Stollar. 1990. Acidotropic amines inhibit proteolytic processing of flavivirus prM protein. Virology 174:450-458[CrossRef][Medline]. |
| 46. | Rice, C. M. 1996. Flaviviridae: the viruses and their replication, p. 931-959. In B. N. Fields, D. N. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, and T. P. Monath (ed.), Virology, 3rd ed. Lippincott-Raven, Philadelphia, Pa. |
| 47. | Schalich, J., S. L. Allison, K. Stiasny, C. W. Mandl, C. Kunz, and F. X. Heinz. 1996. Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions. J. Virol. 70:4549-4557[Abstract]. |
| 48. |
Schweizer, A.,
J. A. Fransen,
T. Bachi,
L. Ginsel, and H. P. Hauri.
1988.
Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus.
J. Cell Biol.
107:1643-1653 |
| 49. |
Sciaky, N.,
J. Presley,
C. Smith,
K. J. Zaal,
N. Cole,
J. E. Moreira,
M. Terasaki,
E. Siggia, and J. Lippincott-Schwartz.
1997.
Golgi tubule traffic and the effects of brefeldin A visualized in living cells.
J. Cell Biol.
139:1137-1155 |
| 50. | Shapiro, D., W. E. Brandt, R. D. Cardiff, and P. K. Russell. 1971. The proteins of Japanese encephalitis virus. Virology 44:108-124[CrossRef][Medline]. |
| 51. | Sreenivasan, V., K. L. Ng, and M. L. Ng. 1993. Brefeldin A affects West Nile virus replication in Vero cells but not C6/36 cells. J. Virol. Methods 45:1-17[CrossRef][Medline]. |
| 52. | Stadler, K., S. L. Allison, J. Schalich, and F. X. Heinz. 1997. Proteolytic activation of tick-borne encephalitis virus by furin. J. Virol. 71:8475-8481[Abstract]. |
| 53. | Stiasny, K., S. L. Allison, A. Marchler-Bauer, C. Kunz, and F. X. Heinz. 1996. Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus. J. Virol. 70:8142-8147[Abstract]. |
| 54. |
Stinchcombe, J. C.,
H. Nomoto,
D. F. Cutler, and C. R. Hopkins.
1995.
Anterograde and retrograde traffic between the rough endoplasmic reticulum and the Golgi complex.
J. Cell Biol.
131:1387-1401 |
| 55. | Vaux, D., J. Tooze, and S. Fuller. 1990. Identification by anti-idiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal. Nature 345:495-502[CrossRef][Medline]. |
| 56. |
Wang, S.,
R. He, and R. Anderson.
1999.
PrM- and cell-binding domains of the dengue virus E protein.
J. Virol.
73:2547-2551 |
| 57. |
Wengler, G., and G. Wengler.
1989.
Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release.
J. Virol.
63:2521-2526 |
| 58. | Westaway, E. G., A. A. Khromykh, M. T. Kenney, J. M. Mackenzie, and M. K. Jones. 1997. Proteins C and NS4B of the flavivirus Kunjin translocate independently into the nucleus. Virology 234:31-41[CrossRef][Medline]. |
| 59. | Westaway, E. G., J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh. 1997. Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA and of NS2B with NS3 in virus-induced membrane structures. J. Virol. 71:6650-6661[Abstract]. |
| 60. |
Wright, P. J.
1982.
Envelope protein of the flavivirus Kunjin is apparently not glycosylated.
J. Gen. Virol.
59:29-38 |
| 61. |
Yamshchikov, V. F., and R. W. Compans.
1994.
Processing of the intracellular form of the West Nile virus capsid protein by the viral NS2B-NS3 protease: an in vitro study.
J. Virol.
68:5765-5771 |
| 62. |
Zitzmann, N.,
A. S. Mehta,
S. Carrouee,
T. D. Butters,
F. M. Platt,
J. McCauley,
B. S. Blumberg,
R. A. Dwek, and T. M. Block.
1999.
Imino sugars inhibit the formation and secretion of bovine viral diarrhea virus, a pestivirus model of hepatitis C virus: implications for the development of broad spectrum anti-hepatitis virus agents.
Proc. Natl. Acad. Sci. USA
96:11878-11882 |
Journal of Virology, November 2001, p. 10787-10799, Vol. 75, No. 22
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.22.10787-10799.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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[Abstract] [Full Text] -
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(2004). The Transmembrane Domains of the prM and E Proteins of Yellow Fever Virus Are Endoplasmic Reticulum Localization Signals. J. Virol.
78: 12591-12602
[Abstract] [Full Text] -
Scholle, F., Girard, Y. A., Zhao, Q., Higgs, S., Mason, P. W.
(2004). trans-Packaged West Nile Virus-Like Particles: Infectious Properties In Vitro and in Infected Mosquito Vectors. J. Virol.
78: 11605-11614
[Abstract] [Full Text] -
Yoshii, K., Konno, A., Goto, A., Nio, J., Obara, M., Ueki, T., Hayasaka, D., Mizutani, T., Kariwa, H., Takashima, I.
(2004). Single point mutation in tick-borne encephalitis virus prM protein induces a reduction of virus particle secretion. J. Gen. Virol.
85: 3049-3058
[Abstract] [Full Text] -
Lontok, E., Corse, E., Machamer, C. E.
(2004). Intracellular Targeting Signals Contribute to Localization of Coronavirus Spike Proteins near the Virus Assembly Site. J. Virol.
78: 5913-5922
[Abstract] [Full Text] -
Op De Beeck, A., Voisset, C., Bartosch, B., Ciczora, Y., Cocquerel, L., Keck, Z., Foung, S., Cosset, F.-L., Dubuisson, J.
(2004). Characterization of Functional Hepatitis C Virus Envelope Glycoproteins. J. Virol.
78: 2994-3002
[Abstract] [Full Text] -
Lobigs, M., Lee, E.
(2004). Inefficient Signalase Cleavage Promotes Efficient Nucleocapsid Incorporation into Budding Flavivirus Membranes. J. Virol.
78: 178-186
[Abstract] [Full Text] -
Allison, S. L., Tao, Y. J., O'Riordain, G., Mandl, C. W., Harrison, S. C., Heinz, F. X.
(2003). Two Distinct Size Classes of Immature and Mature Subviral Particles from Tick-Borne Encephalitis Virus. J. Virol.
77: 11357-11366
[Abstract] [Full Text] -
Chiou, C.-T., Hu, C.-C. A., Chen, P.-H., Liao, C.-L., Lin, Y.-L., Wang, J.-J.
(2003). Association of Japanese encephalitis virus NS3 protein with microtubules and tumour susceptibility gene 101 (TSG101) protein. J. Gen. Virol.
84: 2795-2805
[Abstract] [Full Text] -
Liu, W. J., Chen, H. B., Khromykh, A. A.
(2003). Molecular and Functional Analyses of Kunjin Virus Infectious cDNA Clones Demonstrate the Essential Roles for NS2A in Virus Assembly and for a Nonconservative Residue in NS3 in RNA Replication. J. Virol.
77: 7804-7813
[Abstract] [Full Text] -
Jones, C. T., Ma, L., Burgner, J. W., Groesch, T. D., Post, C. B., Kuhn, R. J.
(2003). Flavivirus Capsid Is a Dimeric Alpha-Helical Protein. J. Virol.
77: 7143-7149
[Abstract] [Full Text] -
Lorenz, I. C., Kartenbeck, J., Mezzacasa, A., Allison, S. L., Heinz, F. X., Helenius, A.
(2003). Intracellular Assembly and Secretion of Recombinant Subviral Particles from Tick-Borne Encephalitis Virus. J. Virol.
77: 4370-4382
[Abstract] [Full Text] -
Kien, F., Abraham, J.-D., Schuster, C., Kieny, M. P.
(2003). Analysis of the subcellular localization of hepatitis C virus E2 glycoprotein in live cells using EGFP fusion proteins. J. Gen. Virol.
84: 561-566
[Abstract] [Full Text] -
Op De Beeck, A., Molenkamp, R., Caron, M., Ben Younes, A., Bredenbeek, P., Dubuisson, J.
(2002). Role of the Transmembrane Domains of prM and E Proteins in the Formation of Yellow Fever Virus Envelope. J. Virol.
77: 813-820
[Abstract] [Full Text] -
Liu, W. J., Sedlak, P. L., Kondratieva, N., Khromykh, A. A.
(2002). Complementation Analysis of the Flavivirus Kunjin NS3 and NS5 Proteins Defines the Minimal Regions Essential for Formation of a Replication Complex and Shows a Requirement of NS3 in cis for Virus Assembly. J. Virol.
76: 10766-10775
[Abstract] [Full Text] -
Chu, J. J. H., Ng, M. L.
(2002). Infection of polarized epithelial cells with flavivirus West Nile: polarized entry and egress of virus occur through the apical surface. J. Gen. Virol.
83: 2427-2435
[Abstract] [Full Text] -
Carrere-Kremer, S., Montpellier-Pala, C., Cocquerel, L., Wychowski, C., Penin, F., Dubuisson, J.
(2002). Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus. J. Virol.
76: 3720-3730
[Abstract] [Full Text]
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