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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2008.00382.x |
Biological characteristics of
dengue virus and potential targets for drug design
Rui-feng Qi1, Ling
Zhang1,
and Cheng-wu Chi1,2*
1 Institute of Protein Research, Tongji
University, Shanghai 200092, China
2 Institute of Biochemistry and Cell Biology,
Chinese Academy of Sciences, Shanghai 200031, China
Received: July 3,
2007
Accepted: November
20, 2007
This work was supported by a grant from the National Natural Science Foundation
of China (No. U0632001)
*Corresponding
author: Tel, 86-21-54921165; Fax, 86-21-54921011; E-mail, [email protected] or
[email protected]
Dengue
infection is a major cause of morbidity in tropical and subtropical regions,
bringing nearly 40% of the world population at risk and causing more than
20,000 deaths per year. But there is neither a vaccine for dengue disease nor
antiviral drugs to treat the infection. In recent years, dengue infection has
been particularly prevalent in India, Southeast Asia, Brazil, and Guangdong
Province, China. In this article, we present a brief summary of the biological
characteristics of dengue virus and associated flaviviruses, and outline the
progress on studies of vaccines and drugs based on potential targets of the
dengue virus.
Keywords dengue virus; NS3 protease; polyprotein processing; drug
target
Introduction
The family Flaviviridae is a large group of viral pathogens responsible
for causing severe disease and mortality in humans and animals. The family
consists of three genera, Flavivirus, Pestivirus, and Hepacivirus.
The flaviviruses (Latin “lavus” meaning yellow, because of the
jaundice induced by yellow fever virus) comprise a large genus of medically
important, arthropod-transmitted, enveloped viruses with more than 70 members
that include dengue virus, Japanese encephalitis virus (JEV), tick-borne
encephalitis virus (TBEV), West Nile virus (WNV), and yellow fever virus
(YFV). Symptoms of flavivirus infection can range from mild fever and malaise
to fatal encephalitis and haemorrhagic fever [1,2].
Dengue virus is responsible for the highest rate of disease and
mortality among members of the Flavivirus genus. Global epidemics of
dengue virus have occurred over the past few years. Dengue virus infects 50 to
100 million people each year, with 500,000 patients developing the more severe
disease, namely, dengue hemorrhagic fever (DHF), leading to hospitalizations
and resulting in approximately 20,000 deaths, mainly in children [3–5]. Dengue
viruses are transmitted to humans by the bite of infective female mosquitoes of
the genus Aedes. Throughout tropical and subtropical regions around the world,
over 2.5 billion people live in areas where dengue virus and its mosquito
vectors, the Aedes aegypti and Aedes albopictus, are endemic. The
most efficient epidemic vector is A. aegypti, although A. albopictus
and A. polynesiensis are also involved in dengue outbreaks [6]. Several
factors have been implicated in the global resurgence of dengue: failure to
control the Aedes population; increased airplane travel to dengue
endemic areas; uncontrolled urbanization; unprecedented population growth;
and global climate warming [7,8].
Infection of dengue virus is usually characterized by fever and
severe joint pain, but more serious syndromes, DHF or dengue shock syndrome,
sometimes occur following dengue infection. DHF was mostly confined to
Southeast Asia until the 1960s, then it also became endemic in Central
America, and more recently in South America. There are four antigenically
related but distinct serotypes of dengue virus, designated DEN-1, DEN-2, DEN-3,
and DEN-4, and infection by any one serotype does not protect the individual
from infection by the remaining three serotypes [9,10]. It has been postulated
that hemorrhagic fever or shock syndrome is usually the result of sequential
infection with multiple serotypes. Although vaccines have been developed for
several flaviviruses, control of dengue virus through the use of vaccination
has proven to be elusive [3].
The dengue viruses share many characteristics with other
flaviviruses, such as a single-stranded RNA genome that is packaged by the virus
capsid protein in a host-derived lipid bilayer, and surrounded by 180 copies of
two glycoproteins. The complete virion is approximately 50 nm in diameter and
contains an approximately 10.7 kb positive-sensed RNA genome that has one open
reading frame encoding a single polyprotein [11]. The 5‘-end of the genomic RNA has a type 1 cap, and the 3‘-end is devoid of a poly(A) tail. The
5‘-end encodes three structural
proteins: capsid (C); membrane precursor protein (prM) proteolytically cleaved
by the host protease furin to form the membrane protein in mature virions; and
envelope (E) constituting the enveloped virus particle [11,12]. Seven
non-structural (NS) proteins essential for viral replication are encoded by the
remainder of the genome. The order of proteins encoded is 5‘-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3‘ [12] [Fig. 1(A)].
Structure
The dengue virus surface is composed of 180 copies of the envelope
glycoprotein and the membrane protein. The E protein of dengue virus contains a
class II fusion peptide sequence that is important for viral invasion of a
host cell. There are remarkable structural deviations between the immature and
mature dengue envelopes as revealed by elegant cryo-electron microscopy
studies [11,13]. The immature dengue virus particle is covered with 60
asymmetric trimers of prM-E heterodimers that stick out like spikes from its
surface [Fig. 2(A)]. The prM protein protects E from premature fusion
while passing through the acidic environment of the trans-Golgi network (TGN)
during morphogenesis [13]. During maturation, the N-terminal part of the prM
protein is released by the host cell furin that induces a rearrangement of the
E proteins essential for fusion. In the mature virus, the E proteins exist as
homodimers that lie on the viral membrane in the form of 30 so-called
“rafts”. Each raft contains three parallel dimers arranged in
icosahedral symmetry and organized into a herringbone pattern [11] [Fig.
2(B)].
Life Cycle
Virions attach to the surface of a host cell and subsequently enter
the cell by receptor-mediated endocytosis. Acidification of the endosomal
vesicle triggers an irreversible trimerization of the E protein in the virion
that results in fusion of the viral and cell membranes [14]. After fusion has
occurred, the nucleocapsid (NC) is released into the cytoplasm, leading to the
dissociation of the C protein and RNA. Once the genome is released into the
cytoplasm, the positive-sense RNA is translated into a single polyprotein that
is processed cotranslationally and post-translationally by viral and host
proteases. Genome replication occurs on intracellular membranes. Assembly and
formation of immature virus particles occur on the surface of the endoplasmic
reticulum (ER) when the structural proteins and newly synthesized RNA bud into
the lumen of ER [14–16]. Although these particles contain E and prM, lipid membrane and
NC, they cannot induce host-cell fusion, remaining non-infectious, because the
prM protein is needed to be further processed [17,18]. Subviral particles are
also produced in ER, but only contain the glycoproteins and membrane, and lack
the C protein and genomic RNA, making them also non-infectious [19]. The
resultant non-infectious, immature viral and subviral particles are transported
through the TGN. The immature virion particles are then cleaved by the host
protease furin, resulting in mature, infectious particles. Subviral particles
are also cleaved by furin. The mature virions and subviral particles are
subsequently released from the host cell by exocytosis (Fig. 3) [20].
Entry, Fusion, and Infection
The structure of soluble E protein elucidated by X-ray
crystallography consists of three domains: domain I, the N-terminal part structurally
located in the central part; domain II, the fusion domain containing a
hydrophobic fusion peptide; domain III, the putative receptor binding domain
[21,22]. Cryo-electron microscopy revealed the presence of a C-terminal
“stem” and two transmembrane sequences through which the E protein is
anchored to the viral surface [23]. During endocytosis, under the acid
condition in endosome, the E proteins undergo a dramatic structural change from
dimer into trimer. These trimers cluster on the viral surface and induce
curvature that might promote fusion. In the E trimer, the fusion peptide is
exposed at the tip of the trimer, leading the virus and endosomal membranes to
merge [24–26].
Dengue virus is known to enter cells through receptor-mediated
endocytosis [14,27–37]. Several primary cellular receptors and low-affinity
coreceptors for flaviviruses have been identified. Dendritic cell-specific
ICAM-grabbing non-integrin and CD-14-associated molecules have been suggested
as the primary receptors for dengue virus [28,31,36]. Heparin and other
glycosaminoglycans act as low-affinity coreceptors for several flaviviruses
[27,29,30,32–35,37].
Enzymatic Activities and
Processing
Once dengue virus enters cells, the viral genome consisting of a
single positive-strand RNA is liberated into the cytoplasm, and is used as a
template for translation into a large polyprotein precursor. The
cotranslational and post-translational processing of the polyprotein precursor
by the host cell proteases (e.g. signalase, furin) within the ER and by the
viral protease (NS3pro) in cytoplasm gives rise to three structural proteins of
the enveloped virus particle (C-prM-E) and seven nonstructural proteins
(NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5), most of which are thought to be required for
assembling together with yet poorly-defined host proteins to form a replication
machine in the cytoplasm of the infected cells that catalyze copying of the
viral RNA [38] [Fig. 1(B)]. The newly-generated RNAs are then used for
translation to produce more viral proteins and for copying more viral RNAs of
virus particles.
During translation of the polyprotein, the structural proteins are
translocated and anchored in ER by various signal sequences and membrane anchor
domains (Fig. 4). And the C-terminal region of the C protein, serving as
a hydrophobic signal sequence, anchors the C protein into the ER membrane, and
thus translocates prM into the lumen of ER. Subsequently, this signal sequence
is cleaved off by the host cell signalase, liberating the N-terminus of prM,
whereas the C protein remains closely associated with the ER membrane [39].
This association is present in all flaviviruses, and promotes viral assembly
[40]. The prM protein has two transmembrane-spanning domains, a stop transfer
sequence and a signal sequence (Fig. 4), and the sequentially linked E
protein is then also translocated into the lumen of ER. After the appropriate
proteolytic cleavages, the C protein remains associated with the ER membrane,
whereas the viral RNA is released into the cytoplasm after replication. On the
lumenal side of ER, the prM and E proteins form a stable heterodimer within a
few minutes of translation [41–43].
NS3 is a multifunctional protein. Based on sequence comparison with
known proteases, a classic trypsin-like serine protease with a catalytic triad
(His51, Asp75, and Ser135) was identified in the N-terminal 180 amino acid
residues of NS3 [38,44–48]. The enzyme requires NS2B as a cofactor for activation of
protease activity [46,49]. The minimum sequence for protease activity was
mapped to the first 167 residues of NS3 [50]. The C-terminal part of NS3
carries three other enzymatic activities: an RNA-stimulated nucleoside
triphosphatase (NTPase); an RNA helicase; and an RNA 5‘-triphosphatase (RTPase) [45,47,50–55] [Fig. 5(A)]. The
latter is most likely required for removal of the terminal phosphate group from
the newly-synthesized RNA, and for formation of the viral cap structure at
the 5‘-end of the virus RNA genome
[51,54,55]. The helicase functions to unwind the double-stranded nucleic acids
during viral RNA replication [45,50]. This activity is energy-dependent and is
carried out by its NTPase activity that hydrolyses ATP to generate the
necessary energy [50,52,53]. The minimal domain for helicase and NTPase
activities was reported to comprise the full C-terminal part of NS3 [45,51,56].
It is most likely that the two different enzymatic activities (RTPase and
NTPase) are exerted by one active site in the same protein, and are strictly Mg2+-dependent [51,54].
Unlike trypsin, the NS3 protease has a marked preference for
dibasic residues (e.g. Arg and Lys at positions P1 and P2 [57]) and requires a cofactor supplied by the non-structural
protein NS2B for efficient cleavage of the dengue virus polyprotein [46]. The
NS2B-NS3 protease catalyzes the cleavage of the viral polyprotein precursor in
the non-structural region at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5
sites [58,59]. Additional proteolytic cleavages are within the viral C
protein, NS2A, and NS4A, and at the C-terminal part of NS3 [49,60–62], whereas the
host cell proteases (such as signalase and furin) act on the remaining cleavage
sites [63–66] [Fig. 1(B)]. Deletion studies have further shown that a
central 40-amino acid conserved hydrophilic domain within NS2B is sufficient
for the cofactor activity [67]. The flanking hydrophobic residues of NS2B are
likely to function to associate the protease complex and the infected cell
membranes [68] [Fig. 5(A)]. The residues within the core hydrophilic
segment of the cofactor NS2B responsible for binding the NS3 protease domain
have been further identified [69].
The NS5 proteins of all flaviviruses consist of at least three very
important enzymes that are essential for viral propagation [70,71]. Located at
the NS5 N-terminal part, approximately 320 residues comprise the S-adenosylmethionine-dependent
methyltransferase (MTase), possessing the MTase and guanylyltransferase
activities responsible for capping and methylation of the capped positive-strand
genomic RNA at the 5‘-end [Fig.
5(A)]. The structure of MTase of DEN-2 and its complex with relevant small
molecules has been determined by X-ray crystallography [72]. As the RNA capping
is an essential viral function, it provides a structural basis for the rational
design of drugs against flaviviruses. The C-terminal part of NS5 is the
RNA-dependent RNA polymerase (RdRp) at residue position 420–900, responsible
for synthesis of the intermediate RNA template for further replication of the
positive-strand genomic RNA [73,74] [Fig. 5(A)]. The RdRp activity of
dengue virus has been shown for several other flaviviruses, including West Nile
virus and Kunjin virus [71,75–77]. In all flavivirus RdRp, there is an essential and classical amino
acid sequence signature, the Gly-Asp-Asp motif [71].
Assembly, Maturation, and
Release
During the assembly and maturation of viral particles, the C protein
of dengue virus is crucial. The C proteins of DEN-2 readily form dimers in
solution, and can be regarded as building blocks for NC assembly [78–80]. The
secondary structure of the C protein from residue 21 to 100 is composed of
four a-helices: helix I, the N-terminal part; helix II, hydrophobic and
essential for the ER membrane association; and helices III and IV, the
C-terminal part containing the signal sequence for anchoring the ER membrane.
The N-terminal 20 residue fragment is flexible [78,81]. The 3D picture of the
dengue C protein shows a dimeric structure maintained by the homotypic binding
domain, facilitating the interaction between RNA and the C protein by an
asymmetric charge distribution, suggesting the membrane-associated C protein
mediates viral assembly by a highly coordinated interaction with the prM-E
heterodimer in ER [40,81]. Several copies of the C protein and one copy of the
genomic RNA form the NC that finally buds into the lumen of ER and produces
immature viral particles. It was shown that the C protein can also be found in
the nucleus, and can possibly interact with heterogeneous nuclear
ribonucleoprotein K, suggesting a role in regulation of the dengue life cycle
probably by controlling apoptosis [82].
Virus maturation is a two-step process. First, during maturation in
the TGN, under low pH conditions, the prM proteins are conformationally changed
and cleaved by the host cell furin. As a result, the 60 “spikes”
composed of the three prM-E heterodimers that project from the immature virus
surface are dissociated, consequently forming a smooth surface of mature virus
composed of 90 E homodimers (Fig. 2) [66,83,84]. Second, during
exocytosis, a major rearrangement of the E protein occurs. The anti-parallel E
homodimers dissociate into monomers, that then re-associate into parallel
homotrimers [14,85,86]. The mature viral particles are then eventually released
from the host cell by exocytosis.
Potential Targets and Progress
in Study of Vaccines and Drugs
The re-emerged dengue fever/DHF has becomes a global threat and
endemic in more than 100 countries throughout the Americas, Southeast Asia, and
western Pacific islands. It is an increasingly important public health concern,
and challenges scientists to discover new vaccines and antiviral drugs. There
are three strategies for the control of dengue virus disease: the control and
elimination of the mosquito vector; the development of safe vaccines to prevent
infection; and the search for specific antidengue drugs for treatment of
disease. So far, the only way to prevent dengue transmission is to control the
principal vector mosquito and reduce human-vector contact, because there is no
approved vaccine or effective antiviral drug for dengue disease.
In the absence of effective antiviral drugs, vaccination offers a
good option for decreasing the incidence of these diseases. An ideal dengue
vaccine must be effective for all four virus serotypes, be safe in 9–12-month-old
children, and provide a long-lasting protective immunity. Various strategies
have been used to develop dengue vaccines [87–89]. Using primates for
preclinical evaluation, chimeric tetravalent vaccines show a high level of
neutralizing antibody against all serotypes, and clinical trials are in
progress [88–90]. Another type of dengue vaccine is the DNA vaccine [91,92].
Recently, a new dengue tetravalent DNA vaccine against DEN-3 and DEN-4, based
on prM/E and combined with two previously constructed DNA vaccines against
DEN-1 and DEN-2, has been constructed [93]. Molecular biology techniques have
facilitated the development of the recombinant subunit vaccines. The
structural proteins (E and prM) and non-structural proteins (NS1 and NS3) are
the dominant sources of cross-reactive CD4+ and CD8+
cytotoxic T-lymphocyte epitopes [94–97]. Some immunization studies show that these
proteins are important for inducing protective immunity [98–105]. The
combined DNA and protein vaccines have a synergistic effect on the antibody
titers [106–109].
A tetravalent live attenuated vaccine was developed at the Walter
Reed Army Institute of Research (Silver Spring, USA) and licensed to
GlaxoSmithKline [87]. However, there are still several issues that make the
live-attenuated vaccines problematic, including the phenomenon of antibody-dependent
enhancement [87,110].
There are four stages of the viral life cycle, and each stage can be
considered for the development of drugs. In stage 1, prevent viral entry or
infection of the host cell, or inhibit fusion of the viral envelope with the
host vesicles. The E protein can be taken as an ideal target. In stage 2,
prevent maturation processing of the individual viral protein. The well-studied
viral protease is considered a good target. In stage 3, prevent viral RNA
synthesis by inhibiting the viral helicase and RdRp. Finally, in stage 4,
target the host proteins such as furin and signalase that help the maturation
and release of infectious viral particles.
It is well known that dengue virus NS3 is a multifunctional protein
with an N-terminal protease domain (NS3pro), RTPase, an RNA helicase, and an
RNA-stimulated NTPase domain in the C-terminal region [45,46] [Fig. 5(A)].
Thus, the dengue virus NS3 plays a crucial role in viral replication and
represents an interesting target for the development of specific antiviral
inhibitors/drugs.
NS3pro is required to process the polyprotein precursor into the
individual functional proteins that are essential for viral replication, thus
NS3pro is a promising drug target [111]. The 3D structure of the protease
domain of NS3 was solved [112]. Several inhibitors targeting hepatitis C virus
(HCV) NS3pro are now in different stages of clinical trial [113]. A
recombinant NS2B-NS3 fusion protein has been engineered in which a 40 residue
cofactor corresponding to the core part of NS2B is covalently connected through
a flexible glycine-rich linker to DEN-2. NS3 protease has been successfully
expressed in Escherichia coli, and the purified protein was found to be
highly active on peptide substrates designed on the base of the polyprotein
cleavage sites [114] [Fig. 5(B)]. The cofactor NS2B, which has three
hydrophobic regions flanking a conserved hydrophilic domain of approximately 40
amino acid residues, revealed that this hydrophilic region is necessary and
sufficient for activation of the NS3 protease domain in vivo and in
vitro [67,68]. The substrate-based inhibitors with a natural dengue
recognition sequence can inhibit the DEN-2 protease in a competitive manner
[114–116]. Similar to the HCV NS3 protease, the small molecule
inhibitors of NS2B-NS3pro, based on the peptide substrates, have been
synthesized [114,115,117–119]. However, in contrast to HCV NS3 protease, some synthetic
peptides representing the polyprotein cleavages sites do not show an
appreciable inhibition on this protease [115] and some other small inhibitors
(molecules) based peptide substrate have an apparent Ki value at mM and nM
[114,117–119]. According to the crystal structure of the NS3 protease
complexed with the mung bean Bowman-Birk inhibitor [120], several
non-substrate-based compounds were developed [121]. The crystal structures of a
dengue NS2B-NS3pro complex, and of a West Nile virus NS2B-NS3pro complex, with
a substrate-based inhibitor Bz-Nle-Lys-Arg-Arg-H have also been solved [122].
The structures identify the key residues for NS3pro substrate recognition and
clarify the mechanism of NS3pro activation [122].
The NS3 helicase essential for in viral replication also makes it an
attractive target for the design of antiviral compounds [123,124]. The 3D
structure of dengue virus helicase/NTPase shows that there are three domains:
domains I and II situated at the N-terminal (the NTPase site resides between
these two domains); and the C-terminal domain III bound to NS5 [125]. A tunnel
that runs across the interface between domain III and the tip of domains I and
II can accommodate a single-stranded nucleic acid tail along which the enzyme
can translocate. This motion is triggered by NTP hydrolysis to provide the
energy [125,126]. There is no drug to target NS3 helicase. Several low
molecular weight compounds that inhibit the NS3 helicase from the West Nile
virus or the Japanese encephalitis virus have been described [127]. The
regions crucial for the ATPase or nucleic acid duplex unwinding activity have
been identified by mutagenesis that might be suitable for the design of
allosteric inhibitors [124,128].
The viral polymerase NS5 (RdRp) is also a potential target for drug
design [73,74]. The crystallographic structure of an active fragment of the
dengue virus NS5 RdRp has been refined at 1.85 Å resolution [129]. This
structural information of NS5 RdRp will facilitate the design of antiviral
compounds because the host cells are devoid of this enzymatic activity. In
fact, the selective inhibitors against HIV-1 reverse transcriptase, and the
inhibitors against hepatitis B virus, cytomegalovirus, and herpes simplex
virus polymerases have been approved as drugs for treatment of the associated
viral infections [130]. In addition, the interaction between viral NS5 RdRp and
the NS3 helicase also offers a possible target for drug design [131].
In conclusion, although there are no ideal
vaccines or therapy for the prevention and treatment of DHF, the understanding
of the life cycle of dengue virus has made great progress over the past few
years, and all the life cycle stages can represent potential targets for
antiviral drug discovery.
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