Journal of General Virology (2008), 89, 3047–3051
Short
Communication
DOI 10.1099/vir.0.2008/002535-0
Functional importance of dengue virus maturation:
infectious properties of immature virions
Izabela A. Zybert, Heidi van der Ende-Metselaar, Jan Wilschut
and Jolanda M. Smit
Correspondence
Jolanda M. Smit
Department of Medical Microbiology, Molecular Virology Section, University Medical Center
Groningen, University of Groningen, 9700 RB Groningen, The Netherlands
[email protected]
Received 3 April 2008
Accepted 30 July 2008
Prior to the release of flavivirus particles from infected cells, the viral surface protein prM is
cleaved to M by the cellular enzyme furin. For dengue virus (DENV), this maturation process
appears to be very inefficient since a high proportion of progeny virions contain uncleaved prM.
Furthermore, it has been reported that prM-containing DENV particles are infectious. These
observations contradict the general assumption that prM processing is required to render virus
particles infectious. Therefore, in this study, we reinvestigated the infectious properties of
immature DENV virions. DENV particles were produced in furin-deficient LoVo cells. We observed
that DENV-infected LoVo cells secrete high numbers of prM-containing particles. Subsequent
analysis of the infectious titre revealed that immature particles lack the ability to infect cells, the
infectious unit to particle ratio being 10 000-fold reduced compared with that of wild-type virus.
Our results indicate that cleavage of prM to M is required for DENV infectivity.
Dengue virus (DENV) is a mosquito-transmitted pathogen, which together with other arthropod-borne viruses
like yellow fever virus, West Nile virus and tick-borne
encephalitis virus (TBEV) are members of the genus
Flavivirus. Flaviviruses are small, enveloped, icosahedral
viruses with a single copy of a positive-strand RNA
genome. The viral envelope anchors two transmembrane
proteins: the small (8 kDa) membrane protein M and the
major (51–60 kDa) envelope glycoprotein E (Lindenbach
& Rice, 2001). The E glycoproteins are organized in 90
homodimers, which lie flat on the viral surface (Kuhn et al.,
2002; Kuhn & Rossmann, 2005).
prM-containing TBEV virions revealed that these particles
cannot undergo the structural rearrangements required for
membrane fusion (Guirakhoo et al., 1991; Heinz et al.,
1994a; Stadler et al., 1997). Consequently, immature
particles are considered to be non-infectious (Elshuber et
al., 2003; Elshuber & Mandl, 2005; Heinz et al., 1994b;
Stadler et al., 1997). These results led to the widely accepted
hypothesis that prM protects the E protein from undergoing premature conformational changes during transit
through the acidic TGN and that cleavage of prM to M is
required to render the virus particle infectious (Guirakhoo
et al., 1992; Heinz et al., 1994a).
Assembly of virus particles is initiated in the endoplasmic
reticulum by formation of an immature virion. In
immature particles, the E protein forms a heterodimeric
association with prM, the precursor protein of M. Each
particle contains 60 trimers of E–prM heterodimers and
thus differs structurally from a mature virion (Kuhn et al.,
2002; Lorenz et al., 2002; Zhang et al., 2003b, 2004).
Maturation of flavivirus particles occurs during transport
through the exocytic pathway. The viral envelope proteins
are believed to undergo conformational changes triggered
by the low pH in the lumen of the trans-Golgi network
(TGN). Shortly before or during the final release of the
virions, prM is cleaved by the host cell endoprotease furin
into virion-associated M and a soluble peptide (Mackenzie
& Westaway, 2001; Wengler & Wengler, 1989).
Interestingly, the DENV cleavage of prM to M is not very
efficient. Several studies have shown that both mammalian
cells (BHK-21 or Vero) and insect cells (C6/36) infected
with DENV type 2 (16681, NGC and PR159 S1) release
high numbers of particles containing unprocessed prM
(Anderson et al., 1997; He et al., 1995; Henchal et al., 1985;
Murray et al., 1993; Putnak et al., 1996; Randolph et al.,
1990; van der Schaar et al., 2007; Wang et al., 1999). These
recurring in vitro observations have their authentication in
vivo, since anti-prM antibodies are commonly found in
sera of DENV-infected patients (Bray & Lai, 1991; Cardosa
et al., 2002; Se-Thoe et al., 1999). Furthermore, it has been
shown that the specific infectivity of prM-containing
DENV-2 particles produced on chloroquine-treated
BHK-21 cells is only reduced by six- to eightfold compared
with that of a wild-type virus preparation, which suggests
that immature DENV particles may retain considerable
levels of infectivity (Randolph et al., 1990). Remarkably,
the authors also observed that viral infectivity could be
The biological significance of the maturation process has
been investigated in considerable detail, particularly for
TBEV. Characterization of the infectious properties of
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3047
I. A. Zybert and others
restored to wild-type levels upon addition of prM
antibodies to the titration reaction. The high proportion
of immature particles released from DENV-infected cells
and the only limited reduction of the specific infectivity of
immature virus particles suggest that DENV may have
evolved such that cleavage of prM to M is not required for
infectivity per se. In an attempt to resolve this question, we
reinvestigated the infectious properties of immature DENV
particles. Unlike the approach used in the above study
(Randolph et al., 1990), we prepared immature DENV
particles in LoVo cells, which lack functional furin.
First, we assessed the growth of DENV-2 in LoVo cells
(human adenocarcinoma cells), and compared it to virus
production in C6/36 mosquito cells, a cell line commonly
used for DENV production. LoVo cells have been used since
Takahashi and co-workers demonstrated that these cells lack
the endoprotease furin, which suggested that these cells
should secrete fully immature DENV particles (Takahashi et
al., 1993). LoVo cells were cultured in Ham’s medium
(Invitrogen) supplemented with 20 % fetal bovine serum
(FBS) at 37 uC and 5 % CO2. Aedes albopictus C6/36 cells
(CRL-1660; ATCC) were maintained in minimal essential
medium (Life Technologies) supplemented with 10 % FBS,
25 mM HEPES, 7.5 % sodium bicarbonate, penicillin
(100 U ml21), streptomycin (100 mg ml21), 200 mM glutamine and 100 mM non-essential amino acids at 28 uC and
5 % CO2. Cells were infected with DENV-2 strain 16681 at an
m.o.i. of 10, to ensure initial infection of all cells, for 1.5 h at
37 uC. Following infection, viral inoculum was removed and
the cells were washed twice with PBS. Subsequently, fresh
medium was added and incubation was continued. The
number of genome-containing particles (GCP) was determined in samples collected at 24 h time intervals, as
described previously (van der Schaar et al., 2007). Briefly,
viral RNA was extracted from the samples by use of QIAamp
Viral RNA mini kit (Qiagen). Next, cDNA was synthesized
from the RNA by RT-PCR, copies of which were quantified
using quantitative-PCR.
Fig. 1(a) shows that comparable numbers of GCP were
secreted from LoVo and C6/36 cells at each time point
examined. This indicates that the release of viral particles,
based on quantification of RNA copies, is not impaired in
LoVo cells and that these cells are at least as permissive to
DENV infection as mosquito cells. In both cell lines,
maximal virus particle release was observed at 48 h postinfection (p.i.).
Subsequently, we analysed the protein composition of the
virus particles produced in LoVo and C6/36 cells by SDSPAGE. To allow accurate visualization and quantification
of the protein bands, [35S]methionine-labelled virus was
prepared. Briefly, 2 h after infection of both cell lines at an
m.o.i. of 10, 400 mCi (14.8 MBq) of [35S]methionine
(Amersham Biosciences) was added to 20 ml medium
and incubation was continued overnight. At 23 h p.i., the
medium was supplemented with an additional 200 mCi
(7.4 MBq) of the radioactive label. At 72 h p.i., the
3048
Fig. 1. (a) Growth kinetics of GCP of DENV-2 in C6/36 and LoVo
cells. Samples were analysed in duplicate; each data point
represents the average of two independent cultures. (b) Protein
composition of DENV-2 particles harvested from C6/36 (lane 1)
and LoVo (lane 2) cells. Approximately 109 GCP of purified
[35S]methionine-labelled DENV were loaded per lane. Positions of
the structural proteins are indicated on the right, based on their
molecular mass. (c) Identification of the prM protein band by
Western blot using 2H2 antibody. DENV-2 particles harvested
from C6/36 (lane 1) and LoVo (lane 2) are shown next to the
protein marker (M).
supernatant containing the viral particles was cleared from
cell debris by low-speed centrifugation and the virions were
pelleted by ultracentrifugation at 4 uC in a Beckman type
SW41 rotor for 2.5 h at 35 000 g. Subsequently, virus
pellets were resuspended in HNE buffer (5 mM HEPES,
150 mM NaCl, 0.1 mM EDTA, pH 7.4) and further
purified on a discontinuous (20 and 55 % w/v) Optiprep
gradient (Axis-Shield) ultracentrifugation in a Beckman
type SW41 rotor, 4 uC at 210 000 g for 2 h. Viruses were
harvested from the gradient interface and subjected to
SDS-PAGE analysis. The results showed that virus particles
secreted from LoVo cells do not contain the M protein,
since only E, prM and C protein bands were detected (Fig.
1b, lane 2). In contrast, virus preparations harvested from
C6/36 mosquito cells did show the presence of the M
protein (lane 1). Next, we quantified the prM and M
contents of these virus preparations by phosphorimaging
analysis using ImageQuant TL software (Amersham
Biosciences). The percentage of prM and M in virions
was determined by relating the intensity of prM and M
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Journal of General Virology 89
Infectious properties of immature dengue virus
bands to that of E, on the basis of the relative numbers of
methionine residues in the distinct viral proteins and with
the assumption of uniform labelling. The number of
methionine residues in the E, prM and M protein of
DENV-2 16681 are 19, 11 and 5, respectively. Three
independent SDS-PAGE analyses revealed that DENV
particles released from LoVo cells contain on average
94±9 % prM, this number representing the observed
amount of prM relative to the theoretical prM content of a
completely immature virus preparation calculated on the
basis of the intensity of E. Conversely, DENV-infected
mosquito cells yielded primarily mature virions with an
average M content of 89±13.4 %. In agreement with other
studies, we also detected a substantial amount of prM in
C6/36-produced virus (Fig. 1b, lane 1), quantification of
which revealed an average content of 28.7±9.8 %. In both
DENV preparations, the identity of the prM protein was
ensured by Western blot analysis using the anti-prM
antibody 2H2 (Fig. 1c). Collectively, these results clearly
demonstrate that DENV-infected LoVo cells secreted fully
immature prM-containing viruses.
Next, we determined the infectious properties of the
distinct virus preparations using an infectious centre assay
(ICA) (van der Schaar et al., 2007). Briefly, serial dilutions
of virus samples harvested during the growth curve analysis
(Fig. 1) were used to infect BHK-15 cells. Cells were fixed
at 24–27 h p.i. and stained intracellularly with monoclonal
antibody (mAb) 3H5 (Chemicon International) against the
DENV E glycoprotein. Fig. 2(a) shows that the infectious
titre of immature DENV-2 was severely reduced compared
with that of virions produced in the insect cells at any given
time point. Indeed, very few DENV-infected cells were
observed (Fig. 2b). Viruses were also titrated on Vero and
C6/36 cells with very similar results (data not shown).
Subsequently, we calculated the ratio of GCP to infectious
units (IU) for each of the virus samples (Table 1). The
results show that the specific infectivity of immature
DENV was at least 10 000-fold lower than that of virus
generated in C6/36 cells.
Since insect cells are quite different from mammalian cells
and might produce virus with a high specific infectivity for
reasons other than the presence of functional furin, we also
determined the specific infectivities of DENV generated in
two mammalian cell lines, BHK-15 and Vero cells (p149).
These cells were maintained at 37 uC, 5 % CO2 in C6/36
medium without glutamine and non-essential amino acids
and infected at an m.o.i. of 10 as described above. Samples
were collected at different time points p.i. for analysis. As
shown in Table 1, at all time points the GCP : IU ratio of virus
produced in these cells was very similar to that of virus
generated in C6/36 cells, thus confirming that immature
DENV generated in LoVo cells is at least 10 000-fold less
infectious due to the absence of functional furin.
Fig. 2. (a) Growth kinetics of infectious units of DENV-2 in C6/36
and LoVo cells. Samples from each time point were analysed in
triplicate for IU content ml”1. Each data point represents the
average of two independent cultures. (b) Representative read out
of the ICA. Infected BHK-15 cells stained intracellularly with antidengue E mAb 3H5 (right panels) and 4,6-diamidino-2-phenylindole (DAPI) nuclei staining (left panels). Samples of DENV-2 in
C6/36 and LoVo cells were diluted 500-fold and twofold,
respectively. Magnification ¾400. (c) ICA of immature DENV-2treated cells in vitro with furin (diluted 20-fold) and uncleaved
control (diluted twofold). Magnification ¾100.
To further substantiate that cleavage of prM to M by furin
is essential for DENV infectivity, we investigated whether
treatment of immature DENV with exogenous furin could
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I. A. Zybert and others
Table 1. Specific infectivities of DENV-2 16681 in time.
DENV-2
production
GCP : IU ratio*
24 h p.i.
C6/36
Vero
BHK-15
LoVo
139, 32
29, 22
4, 4
26106, 76105
48 h p.i.
108,
28,
33,
36105,
192
65
18
16106
72 h p.i.
82, 289
18, 20
13, 7
56105, 56105
96 h p.i.
34,
19,
47,
46105,
48
26
16
76105
*For all cell lines, two independent cultures were carried out. GCP : IU ratio for each of the cultures was
determined at least twice.
restore viral infectivity. To this end, immature particles
were incubated with furin (New England BioLabs) for 16 h
at pH 6.0, after which the infectious properties of the
particles were measured by ICA. Fig. 2(c) shows that the
infectivity of the virus preparation was dramatically
increased (approx. by a factor of 1000) upon in vitro
treatment with furin. This observation indicates that
immature DENV is actually non-infectious due to a
complete lack of prM cleavage and hence demonstrates
that processing of prM to M is a prerequisite for DENV
infectivity.
Earlier studies have shown that immature DENV particles
produced on cells treated with chloroquine had a reduced
(factor six to eight), but still quite high, level of infectivity.
However, this study clearly demonstrates that immature
DENV particles are not infectious. This conclusion is in
agreement with previous results obtained with TBEV
(Elshuber et al., 2003; Elshuber & Mandl, 2005; Heinz et
al., 1994b). Immature TBEV virions produced in LoVo
cells, BHK-21 cells treated with furin inhibitor or particles
in which the prM cleavage motif was mutated showed a
significant drop (up to a factor of 10 000) in viral
infectivity. A less profound effect (factor 20–50) on viral
infectivity was seen when immature TBEV virions were
generated in chicken embryo cells treated with acidotropic
reagents (Heinz et al., 1994b). This may suggest that usage
of acidotropic reagents is not sufficient to completely
prevent viral maturation in the TGN and that the presence
of M-containing particles in the viral preparations
contributes to the residual infectious titre observed. The
results presented in this manuscript show that, once
cleavage of prM to M is completely blocked, DENV
infectivity is essentially abolished.
While the very large majority of virus particles in the
immature preparation were non-infectious, we did detect
some infectious virions in the titration assay. A possible
explanation for this finding may be that some prMcontaining particles mature during cell entry, since furin,
although predominantly localized in the TGN, also shuttles
between early endosomes and the cell surface (Molloy
et al., 1999; Shapiro et al., 1997). This notion has been
experimentally confirmed for Semliki Forest virus (SFV),
3050
an alphavirus (Zhang et al., 2003a). The authors observed
that immature SFV particles can be processed during
endocytic uptake in furin-containing cells, albeit with a low
efficiency since the infectious titre increased only by a
factor of 10. Envelope maturation during cell entry may
therefore explain the low but detectable number of
infectious particles in our immature DENV preparation.
Interestingly, antibodies against prM have been shown to
enhance DENV infection (Henchal et al., 1985; Randolph
et al., 1990; Huang et al., 2005, 2006). Enhancement of viral
infection by prM antibodies was observed with both wildtype virus particles, presumably due to the substantial
content of unprocessed prM in these preparations, and
with DENV particles containing high levels of prM
generated from cells in the presence of chloroquine. It is
not yet clear how prM antibodies stimulate the infectious
properties of these virions. As argued above, it is
conceivable that DENV virus particles generated in cells
in the presence of acidotropic agents exhibit a low residual
level of maturation and corresponding infectivity, which
might be enhanced in the presence of anti-prM antibodies.
Also, one could speculate that prM antibodies enhance
uptake of immature particles in cells, which might
consequently lead to increased processing of prM to M
and creation of potentially fusogenic virions within the
target cell. This report shows for the first time that entirely
immature and almost completely non-infectious DENV
particles can be generated in furin-negative LoVo cells, and
therefore may serve as a novel tool to elucidate the role of
prM antibodies in DENV infectivity. Using fully immature
DENV, we are currently investigating if prM antibodies
only facilitate viral entry or whether other processes are
involved that might render immature virions infectious.
Acknowledgements
We thank Hilde van der Schaar for stimulating scientific discussions.
We would also like to thank Tjarko Meijerhof for excellent assistance
with virus production. We are grateful to Claire Y.-H. Huang (Center
for Disease Control and Prevention, USA) for generously providing
us with DENV-2 strain 16681 and Shee Mei Lok (Purdue University,
USA) for kindly providing mAb 2H2. This work was supported by
Paediatric Dengue Vaccine Initiative.
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Journal of General Virology 89
Infectious properties of immature dengue virus
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