Review of Literature
1. 1teview Of fiterature
1.1 History of dengue and its current epidemic patterns
1.2 Dengue disease syndromes
1.2.1 Dengue fever
1.2.2 Dengue haemorrhagic fever
1.2.3 Dengue shock syndrome
1.3 Dengue viruses
1.3.1 Structure and genome organization
1.3.2 Life cycle
1.3.3 Transmission
1.4 Dengue pathogenesis
1.4.1 Antibody dependent enhancement
1.4.2 Structural correlations with pathogenesis
1.5 Dengue Non-translated regions
1.5.1 Sizes and structures of DEN NTRs
1.5.2 Roles ofNTRs in DEN life cycle
1.5.3 Significance ofNTR differences in DF vs. DHF
1.6 Host cell proteins that interact with viral NTRs
1.6.1 RNA viruses
1.6.2 Flaviviruses
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1.1 History of dengue and its current epidemic patterns
The origin of word 'dengue' is based on a Swahili phrase 'ki denga pepo', which
means a disease characterized by a sudden cramp like attack, caused by an evil spirit.
Dengue viruses cause dengue infection, which ranges from mild febrile illness to fatal
haemorrhagic manifestation leading to shock syndrome. Dengue virus is a mosquito borne
virus belonging to the genus Flaviviridae and is found in four closely related yet
antigenically distinct serotypes (DEN-I, DEN-2, DEN-3 and DEN-4), which are equally
capable of causing infection [1,2,3,41.
In recent years, dengue has become the most widespread vector-borne viral disease
of humans. It is currently estimated that there are 50-100 million cases of dengue fever (DF)
per annum worldwide, about half of which result in the severe forms of the disease, dengue
haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The main mosquito vector,
Aedes aegypti, is present in nearly every tropical country and consequently a third of the
world's human population is at risk of infection [5,6,71.
Dengue fever was a major cause of morbidity during World War II. Both US and
Japanese military established a commission to study dengue fever and were successful in
isolating the virus in 1944. However due to economic disruption and human population
migration during and immediately after the World War II, the disease spread beyond its
usual geographical locations and resulted in its reintroduction into some areas. It became an
epidemic in Asia-Pacific regions and has intensified during the last two decades. This is
mainly because the viruses and their mosquito vectors could easily be transported between
population centres by sailing vessels.
During the second half of the twentieth century, a rapid increase in the numbers of
air travelers, further population dislocations and poor public health measures worsened the
situation. Media and epidemiological reports revealed that 2001 saw the highest level of
dengue activity ever recorded and that dengue has a global distribution similar to that of
malaria. Epidemics occurred in Brazil, Cambodia, Columbia, Cuba, Ecuador, the Lao
People's Democratic Republic, Malaysia, Myanmar, Peru, Thailand, Venezuela and Viet
Nam. The outbreak in Cuba was the first and lasted for 4 years and that in Hawaii the first
since the World War II. Furthermore, in recent years, DHF has intensified as major
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epidemics on several islands. Initially, only DEN-2 was present in the Americas, however,
post 1970s, DEN-I, DEN-3 and DEN-4 were introduced causing major epidemics over a 16-
year period. A new DEN-2 strain from Southeast Asia caused a major DHF epidemic in
Cuba [81• The same strain has spread rapidly and caused major DHF epidemics in 14
countries in the American region.
In Southeast Asia, an epidemic of DHF first appeared in the 1950s, but by 1975 it
became a primary cause of hospitalization and death among children in many countries. In
the 1980s, DHF began a second expansion into Asia, when Sri Lanka, India and the
Maldives Islands had major DHF epidemics; Pakistan first reported an epidemic of DF in
1994. The recent epidemics in Sri Lanka were associated with multiple dengue virus
serotypes, but DEN-3 was predominant and was genetically distinct from the DEN-3 viruses
previously isolated from infected persons in the country [91. The People's Republic of China
had a series of epidemics caused by all four serotypes; its first major epidemic of DHF,
caused by DEN-2, was reported in Hainan Island in 1985[101• Singapore also had a
resurgence of denguelDHF from 1990 to 1994 after a successful control program had
prevented significant transmission for over 20 years. In other countries of Asia where DHF
is endemic, the epidemics have become progressively larger in the last two decades [ll].
During the last 25 years, major dengue epidemics have been reported for the first time from
other parts of the world such as the Kenya (1982, DEN-2), Mozambique (1985, DEN-3),
Djibouti (1991-92, DEN-2), Somalia (1982, 1993, DEN-2) and Saudi Arabia
(1994, DEN-2).
In India, DHF is the main cause of hospitalization and death among children. Dengue
virus was first isolated in 1945. Since then, all four serotypes have been demonstrated to
circulate and cause epidemics in the country. The number of dengue cases range from 7 to
16 thousand per year. However, only occasional cases of DHF/DSS have been reported in
India [121. Delhi had outbreaks of dengue virus infection in 1967 (circulating virus was DEN-
2), 1970 (DEN-l and 3) and 1982 (DEN-l and 2), but no signs ofDHFIDSS were reported
during these epidemics [131. In 1988 and 1996 (circulating virus was DEN-2), some cases of
DHF were reported. The virus was confirmed as DEN-2 by cell culture and indirect
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immunofluorescence with serotype-specific monoclonal antibodies. In this largest out-break,
a total of 8,900 cases were reported with 4.2% death rate.
The reasoris for this dramatic global emergence of denguelDHF are lack of an
effective mosquito control program in most dengue-endemic countries, major global
demographic changes like uncontrolled urbanization and concurrent population growth,
increased air travel which facilitated the movement of different serotypes and even
genotypes from one region to another. There may be other additional factors, such as
climatic changes and virus evolution that could influence the emergence of this disease (1-'1.
A licensed vaccine against dengue is not yet available, although vaccines against diseases
caused by related viruses such as yellow fever, Japanese encephalitis and tick-borne
encephalitis are available. Moreover, vaccine development has been complicated by the
apparent involvement of the immune system in disease pathogenesis.
In summary, dengue is the most widespread mosquito-borne human viral disease.
The disease is now endemic in more than 100 countries in Africa, the Americas, the Eastern
Mediterranean, Southeast Asia and the Western Pacific. An estimated 2.5 billion people are
living in areas at risk for epidemic transmission (WHO report, 2000) Figure 1.1 depicts the
global distribution of dengue and its vector.
908
~ 14001------.§ i i ;JUU I-----
c: li <!UU'I------
~i ~l
to .I ~
~!:'-",./ iJ <., . c: ., = Areas infesteo with Aedes aegypli V j. _ Areas with A.aegypti aM oengue apidemic activity c:?' Source: WHO
Figure 1.1. World distribution of dengue and its vector
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1.2 Dengue disease syndromes
Dengue virus infections range from a mild undifferentiated fever to classical dengue
fever (DF) and dengue haemorrhagic fever (DHF) or dengue shock syndrome (DSS) [3,4, 151.
Primary infection, generally leads to DF and secondary infection may lead to DHF or DSS.
1.2.1 Dengue fever
Infection with each of four DEN serotypes (DEN-I, 2, 3 and 4) can cause dengue
fever, which may vary in severity. Dengue fever is a mild febrile non-fatal illness and its
clinical features depend on the age of the patient. It is mild in infants and young children and
severe in older children and adults. The fever with temperatures 102-1 OSoF lasting 2-7
days [41, myalgias, frontal headache, retro-orbital pain, nausea, vomiting, anorexia, altered
taste and olfactory perception and malaise, leucopenia and thrombocytopenia in some cases,
are observed in the patients. In some cases bleeding complications such as gingival bleeding,
gastrointestinal bleeding, heamaturia and menorrhagia are observed [161•
1.2.2 Dengue haemorrhagic fever
This is the severe form of the dengue fever with additional symptoms of high fever,
haemorrhagic phenomena, hepatomegaly and circulatory failure. The major physiological
change in DHF from DF is leakage of plasma, resulting in an elevated haematocrit.
Abdominal pain, epigastric discomfort and tenderness at right costal margin are common.
Positive tournique test, platelet counts less than 100,000 mm-3 and elevated haematocrit are
good indications of DHF. After 2-7 days of fever, the serious stage of the disease begins
with a sudden fall in body temperature and circulatory disturbances. At this stage the patient
may sweat and because of loss of plasma, may suffer from high pulse rate and low blood
pressure. Treatment for DHF patients includes injecting fluids to counteract plasma leakage.
Ifthe fluid loss is not corrected immediately, it may prove to be lethal for the patient [16,171.
1.2.3 Dengue shock syndrome
This is the severe form of DHF, which includes rapid fall in pulse pressure
«20mmHg), clammy skin and restlessness. The serious stage of the disease starts between
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the third and seventh day of the fever, with a sudden fall in temperature and signs of
circulatory failures such as skin blotches, congested, circumoral cyanosis and low pulse
pressure. If proper care is not taken promptly, the patient may pass into a stage of profound
shock, with the blood pressure or pulse becoming hardly noticeable. The duration of the
shock lasts for about 12-24 hours, before the terminal stage begins. At this stage, the patient
may die or may recover rapidly by appropriate volume replacement therapy (18).
1.3 Dengue viruses
It has been proposed that dengue viruses have originated from lower primates and
canopy-dwelling mosquitoes in a forest cycle of Malay Peninsula. Before being adapted to
lower primates and humans, the virus might have evolved as viruses of mosquitoes. The
causative agent of dengue was identified in the early part of the twentieth century. After
World War II, dengue became epidemic in Asia-Pacific region (19). Sabin isolated dengue
viruses, Dengue virus type-l (Hawaii) and Dengue virus type-2 (New Guinea C) in 1944(20).
Later in 1956, Gubler isolated Dengue virus type-3 and 4[211.
1.3.1 Structure and genome organization
Dengue viruses are classified into four groups, which share the same structure and
genome organization. The virus appears as a spherical particle of 40-50 nm in diameter, with
a lipid envelope and membrane, enclosing a nucleocapsid core of 30 nm in diameter (Figure
1.2)[22). The dengue virus genome is a ~ 11 kb single-stranded RNA of positive polarity
(mRNA sense) and contains a single ORF flanked by the non-translated regions at either
sides of the ORF. A type 1 cap structure (mGpppAmp) is present at the 5' NTR, but the 3'
NTR lacks a poly(A) tail and terminates with CUOH. Flavivirus genomes are the only
mammalian plus-stranded RNA virus genomes that do not have a 3' poly(A) tract. The 5'
NTR region of the genomic RNA in dengue virus ranges from 96-108 nucleotides in length,
while the 3' NTR ranges from 380-450 nucleotides in length. Ten mature viral proteins are
produced via proteolytic processing of the single polyprotein by the viral and the various
cellular proteases. The three viral structural proteins, capsid (C), membrane (prMl M) and
envelope (E) are encoded within the 5' portion of the genomic RNA, while the seven
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nonstructural proteins (NSl , NS2A, NS2B, NS3, NS4A, NS4B and NS5) are encoded within
the 3' portion (Figure 1.3 and Table 1.1) [23, 24,25,261•
A B c Figure 1.2. Structure of dengue virus. A. Electron microscopic structures of dengue virions. B. Schematic representation of dengue virus particle, C: capsid; M: membrane; E: envelope; RNA: ribonucleic acid. C. Cryo EM map of whole virus (fit of envelope dimers into density) showing envelope monomer with domains I, II and III (red, yellow and blue, respectively) and the fusion peptide is shown in green.
NTR
5'
NS2a
NSla NS2b
t-----.~ 7 non-structural 3 structural .... ---1
genes/proteins genes/proteins
NS4a NTR
3'
NS4b
Figure 1.3. Schematic representation of dengue virus genome. Dengue virus encodes three structural (C: capsid; prM: premembrane; E: envelope) and seven nonstructural proteins (NSl , NSla, NS2b, NS3, NS4a, NS4b and NS5) . NTR denotes non-translated regions.
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Table 1. 1. Roles of various dengue encoded proteins during its life cycle.
• , ,,;::.t, "''' ,,<;: ~ ..
Function
C Capsid protein, has signal sequence for M protein
PrMlM Membrane protein, believed to prevent conformational changes in E
E Envelope protein, host cell surface receptor, membrane fusion, major target for neutralizing antibodies
NSI Role suggested in replication
NS2a NS 1 processing
NS2b Required for NS3 mediated cleavage
NS3 Serine proteaselNTPase
NS4a Unknown, might be the cofactor along with the putative viral RNA dependent RNA polymerase, NS5
NS4b Unknown, might be the cofactor along with the putative viral RNA dependent RNA polymerase, NS5
NS5 RNA dependent RNA polymerase
1.3.2 Life cycle
12
19/9
60
48
20
14.5
70
16
27
105
, .J;' "~ f>" NO ~ ,,>'
Antibodies in patient's sera
Yes
Yes
Yes
Yes
No
No
Yes
Yes
No
Yes
Dengue virus replicates in a wide variety of cell cultures, including continuous cell
lines from monkeys, humans and insects, but does not cause obvious cytopathology in many
cell lines [271 . Glycosaminaglycans playa role in flavivirus entry [28,29
1. However, as in case
of another flavivirus, Murray Valley virus, mutants with substitutions in the hydrophilic
region (FG loop) of the E protein that resulted in an increased dependence on
glycosaminoglycans during entry of cultured cells showed decreased neurovirulence in
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mice. The identification of two glycoproteins as putative receptors of Dengue-4 virus
suggests that additional host cell surface molecules are necessary for flavivirus entry [301 .
Because flaviviruses are transmitted between insect and vertebrate hosts during their natural
transmission cycle, it is likely that the cell receptor(s) they utilize is a highly conserved
protein.
After binding to an unidentified cell receptor(s), virions enter the cell via receptor
mediated endocytosis followed by low-pH fusion of the viral membrane with the endosomal
vesicle membrane releasing the nucleocapsid into the cytoplasm [311. The genomic RNA is
released and translated into a single polyprotein. The viral serine protease, NS2B-NS3 and
several cell proteases then cleave the polyprotein at multiple sites to generate the mature
viral proteins. The viral RNA dependent RNA polymerase (RdRp), in conjunction with
other vital nonstructural proteins and possibly cell proteins, copies complementary minus
strands from the genomic RNA template and these minus strand RNAs in turn serve as
templates for the synthesis of new genomic RNAs. Flaviviral RNA synthesis is semi
conservative and asymmetric. Genomic RNA synthesis is about 10 times more efficient than
minus-strand RNA synthesis [32, 331 . Data obtained by Chu and Westaway in 1985 with a
Kunjin virus suggested that only a single, nascent (-) RNA is copied from a plus strand
template at a time (replicative form, RF) while the minus strand template is efficiently
reinitiated so that multiple, nascent plus strands are simultaneously copied from a single
minus strand template (replicative intermediate, RI). Once established, both plus and minus
strand viral RNA synthesis can continue even in the absence of protein synthesis indicating
that transient viral polyprotein precursors are not required [32, 33,341. Extensive reorganization
and proliferation of cytoplasmic, perinuclear endoplasmic reticular (ER) membranes are
observed in infected cells [351• Nascent genomic RNAs could function as templates for
translation and transcription and as substrates for encapsidation. Data obtained with Kunjin
replicon suggests that translation is a prerequisite for replication of nascent RNAs and that
replication is a prerequisite of encapsidation [361• At the beginning of the replication cycle,
nascent genomic RNAs may alternate between replication and translation because of
insufficient pool of structural proteins.
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Virion assembly occurs in association with rough ER membranes. Little is known
about this process because budding intermediates and free nucleocapsids in the cytoplasm
have rarely been observed by electron microscopy. The E and prM proteins are inserted into
rough ER membranes during protein synthesis. Rapid assembly and maturity of virions
occurs within the intracellular vesicles. Finally, the virions are released by exocytosis via
host cell secretory pathway (Figure 1.4) (371.
1.3.3 Transmission
• • • New Genomes~ __ _
~ "'. -~ . , ... -.-' Transport and Assembly
Figure 1. 4. Dengue virus life cycle.
The principal vector for dengue transmission is the female Aedes mosquito. The
transmission cycle of dengue virus by the Aedes mosquito begins with a dengue-infected
person, who has the virus circulating in the blood that lasts for about 4-5 days (38 ,391. At this
stage, an uninfected female Aedes mosquito bites the person and ingests blood that contains
dengue virus. Then within the mosquito, the virus replicates during an extrinsic incubation
period of eight to twelve days (6, 40,41 1. The mosquito transmits the virus to every other
susceptible person that it bites for the rest of its lifetime. The infected mosquito may also
transmit the virus to the next generation of mosquitoes by transovarial transmission. The
virus then replicates in the newly infected person and produces symptoms. In the mosquito,
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the ViruS replicates within the midgut epithelium brain and salivary glands and also
replicates in the females genital track and may infect her progeny. The Figure 1.5 depicts the
transmission of dengue virus by the mosquito vector.
Humans provide the major amplifying reservoir for the virus and the symptoms
begin to appear an average of four to seven days after the mosquito bites. This is the
intrinsic incubation period, within humans. While the intrinsic incubation period averages
from four to seven days, it can range from three to 14 days. The viremia begins slightly
before the onset of symptoms. Symptoms caused by dengue infection may last three to 10
days, with an average of five days, after the onset of symptoms (Figure 1.5).
Mosquito feeds / Mosqu ito refeeds / acq uires virus transmits virus
1 Extrinsic Intrinsic incubation incubation period period
/Viremi~ "'iremi~
12 16 DAYS
20 24 28
" Illness .J Human #1 Human #2
Figure 1.5. Transmission of dengue virus by Aedes aegypti
1.4 Dengue pathogenesis
The pathogenesis of DHF and OSS is still controversial and the understanding has
been hampered by the lack of in vitro and in vivo models of disease . The studies of viral
factors involved in the production of severe dengue haemorrhagic fever (OHF), versus the
more common dengue fever (OF) have been limited to indirect clinical and epidemiologic
associations. Two theories, which are not mutually exclusive, are frequently cited to explain
the pathogenetic changes that occur in OHF and DSS. These are antibody dependent
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enhancement (ADE) and structural basis for pathogenesis [7,42,43 441 . There is epidemiologic
and laboratory evidence to support both of these hypotheses; however, neither has been
confirmed due to presence of limited data along with lack of suitable animal model. But
what can be summarized conclusively is that both viral (structural basis for pathogenesis)
and host immunologic factors (antibody dependent enhancement) are involved in the
pathogenesis of severe dengue disease.
1.4.1 Antibody dependent enhancement
This is the most commonly accepted theory and is known as the secondary-infection
or immune enhancement hypothesis. According to this school of thought, patients
experiencing a second infection with a heterologous dengue virus serotype have a
significantly higher risk for developing DHF and DSS. Pre-existing heterologous dengue
antibody recognizes the infecting virus and forms an antigen-antibody complex, which is
then bound to and internalized by immunoglobulin Fc receptors on the cell membrane of
leukocytes, especially macrophages. Because the antibody is heterologous, however, the
virus is not neutralized and is free to replicate once inside the macrophage. Thus, it is
hypothesized that prior infection enhances the infection and replication of dengue virus in
cells of the mononuclear cell lineage through a process known as antibody-dependent
enhancement (ADE) [45, 461. It is thought that these cells produce and secrete vasoactive
mediators in response to dengue infection, which causes increased vascular permeability
leading to hypovolemia and shock.
The proposed role of antibody in DHF and DSS has stimulated much research into
the mechanism of antibody enhancement of infection (ADE). Although in vitro ADE was
first reported in the 1930s, Hawkes performed the first definitive studies in 1964 [471. A
critical component in this phenomenon is the presence of Fcy receptors on the surface of a
permissive cell (usually a member of the mononuclear phagocytic lineage), enhancing
immunoglobulin G (IgG) antibodies bound to virus that attach to the cell surface, bringing
the infectious virion into close proximity to the normal virus receptor. Although detailed
studies have been performed on only a few viruses, the virus does not appear to enter cells
through binding to Fcy receptors alone, but requires the normal virus receptor. Thus virus-
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specific antibody and the Fc receptor appear to act together as co-receptors, enhancing the
efficiency of virus binding and increasing the number of infected cells /481. Enhancement via
IgM and complement C3 receptors has also been reported. In vitro, ADE has been observed
with many viruses, including both the mosquito-borne and tick-borne flaviviruses. Thus in
an infected patient, pre-existing antibody could result in increased viral load, shortened
incubation times and increased disease severity. Moreover, as many components of the cell
mediated immune system (CM!) display Fey receptors on their cell surface, ADE could act
by destroying these cells and further compromising recovery from disease.
Despite several clinical studies, evidence for the role of ADE in human disease
remains circumstantial. High levels of viremia are correlated with the incidence of DHF and
DSS, as are secondary infections with heterotypic virus. Low levels of viremia and high
levels of pre-existing homotypic neutralizing antibodies in secondary infections are
associated with mild disease. A detailed analysis of the outbreaks in Cuba in 1977-79 and in
1997 supported the role of ADE and indicated that it could affect disease severity as long as
20 years after the primary infection [491• The careful cohort studies support these general
conclusions, but emphasize that dengue pathogenesis is a multifactorial process and
exceptions frequently occur [501. In addition, children in the first year of life who have
acquired maternal antibodies with ADE characteristics experience DHF with a primary
immune response. This observation is consistent with the role of ADE in DHF, but it is
difficult to assign a role for T cells in such young children. It has also been suggested that
the fact that different dengue serotypes can coexist in the same human population is
consistent with an enhancement phenomenon. However, direct evidence for the role of
virus-specific antibody in DHF or DSS is still lacking.
1.4.2 Structural correlations with pathogenesis
Despite the biological plausibility of the "antibody enhancement" hypothesis, it does
not adequately explain all the clinical and epidemiological observations. It is important
therefore to understand the molecular basis of dengue pathogenesis to aid diagnosis and
treatment and facilitate the design of vaccines, which will be protective, but not enhance
disease. Thus, the other school of thought maintains that the mutation of the viruses could
have produced viruses with greater virulence and therefore greater epidemic potential. This
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hypothesis assumes that dengue viruses, like all animal viruses, vary and change genetically
as a result of selection pressures as they replicate in humans and/or mosquitoes and that there
are some virus strains with greater epidemic potential [51, 521. Phenotypic expression of
genetic changes in the virus genome may include increased virus replication and viremia,
severity of disease (virulence) and epidemic potential.
Studies of wild and attenuated dengue viruses have suggested that genetic
differences among strains of the four serotypes can be associated with attenuation, virulence
and/or epidemic potential. Despite the fact that a number of nucleotide and amino acid
differences were found in coding or noncoding regions of the genome, no specific site( s)
could be correlated with attenuation or severe disease in humans. In vitro (e.g., plaque size)
and in vivo (e.g., mouse neurovirulence and monkey viremia) markers have been used to
pinpoint probable virulence determinants and have shown to be imperfect models of human
disease. The most consistent argument for a strain basis to DHF and DSS has been put
forward by Rico-Hesse and colleagues [511. Briefly, they proposed that a low-virulence strain
of DEN-2 virus circulated in Latin America since the late 1960s and that epidemics of DHF
and DSS did not occur in this region until the arrival of a strain of higher pathogenicity
originating from South-East Asia, where serious dengue disease is more common; this
suggestion is supported by epidemiological study which shows that the native American
genotype seemed to have been displaced by the imported South-east Asian genotype since
there have been no isolations of the former in the areas where latter used to be circulating.
This study indicated a direct association between the introduction of the imported strain
(DEN-2 As) and the disease severity based on intensified surveillance in Peru and Mexico.
Further, Leitmeyer et al. have done a significant study of genetic differences between two
DEN-2 genotypes that have been associated with distinct clinical presentations in humans:
the Southeast Asian genotype with DF and DHF and the American genotype with DF only
[421. They adopted an approach of full genome sequencing directly from patient plasma to
avoid any selection of virus variants by cultivation or cloning methods. Therefore, the
resulting nucleotide and/or amino acid differences that they have reported reflect the virus
population in the host and permit association with pathogenesis in a better way.
Their observation supports the conclusion that all viruses belonging to the Southeast
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Asian genotype have the potential to cause severe disease ; only epidemiologic associations
provided the first indication that transmission of this genotype is directly associated with the
occurrence of severe dengue. Ongoing studies in Peru reveal that no cases of DHF have
occurred in a population with high secondary infection rates and this is probably explained
by the fact that only the American genotype ofDEN-2 is being transmitted in this region [53J.
They have shown that a comparison of viruses from genetic groups with distinct clinical and
epidemiologic associations can better identify structural differences that correlate with
pathogenesis potential. A total of six encoded amino acid charge differences- prM (Glu to
Lys at position 28, Val to Thr at position 31), envelope (Asn to Asp at position 390), NS4b
(Ser to His at position 17) and NS5 (Asn to Asp at position 645 and Ser to Arg at position
676) were seen to occur consistently in DF vs. DHF causing DEN-2 strains. The sequence
differences observed consistently within the NTRs are tabulated in Table 1.2, which
compares the mutations/substitutions between the DF and DHF causing DEN-2 strains and
the region of the dengue virus genome they belong to.
Table 1.2. Comparison of the mutations/substitutions between the DF and DHF DEN-2 strains.
S.No NTR Positioll 011 gellome Mutatioll FUllctiOllll1 relevallce
1. 5' 69 A toT 4-nucleotide bulge at the 5'
terminus with reduced stem
2. 5' 77 AtoG length and longer 3' terminal
loop of 12 nucleotides.
3. 3' 10297 GtoA
4. 3' 10331 A toG Compact structure with
5. 3' 10388 A toT possibility of more tertiary
6. 3' 10390 A to G interactions
7. 3' 10527 A toG
In summary, available evidence suggests that both viral and host immune factors are
involved in the pathogenesis of severe dengue disease. Unfortunately, the role of each is not
fully understood and the lack of an animal model makes this a difficult area to study. It
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would appear that different clinical pathologic manifestations of the disease might be caused
by different pathogenetic mechanisms [21. For example, it has been suggested that hepatic
injury may relate more to viral factors whereas vascular permeability may be mediated
predominantly by the immune response. Clearly, the strain of virus is important since ADE
apparently occurs only with selected virus strains when tested in vitro. Also, the rate of virus
replication and infectivity in various tissues varies with the strain of virus. Collectively, the
data suggest that only certain strains of dengue virus are associated with major epidemics
and severe disease and it is most likely that these are the viruses that infect cells of the
monocytic line via ADE [541 .
1.5 Dengue Non-translated regions (NTRs)
1.5.1 Sizes and structures of DEN NTRs
The genome of the dengue virus is -11 kb single-stranded RNA of positive polarity
and serves as the viral mRNA. It contains a single open reading frame (ORF), flanked at its
5' and 3' ends by non-translated regions (5' and 3' NTRs) . It carries a type I cap structure at
its 5' end, but lacks a poly(A) tract at its 3' end [23, 24, 25,261. Replication proceeds through a
complementary minus sense genomic RNA intermediate [32, 56, 571. Systematic studies on the
roles of the nontranslated regions (NTRs) of the viral RNA in the gene expression of DEN
or other flaviviruses are lacking. The size and sequence of the NTRs vary among different
flaviviruses, but their secondary structure comprising stable stem-loop (SL) structures
appears to be highly conserved (Figure 1.6 and Table 1.3) [35,58, 59, 60, 611. Thus, NTRs are
expected to play an important role earlier during viral infection in coordinating viral gene
expression and the onset of RNA replication.
Table 1.3. Sizes and related secondary structures (of those known in literature) of NTRs of various dengue viruses.
DEN NTR Si:;e Structure NTR Si:e Structure
1 5' 110 -- 3' 418 --2Am 5' 96 A 3' 444 D 2As 5' 95 B 3' 455 E 3 5' 124 -- 3' 416 --4 5' 89 C 3' 369 F
18
(A)DEN2Am
(D)DEN2Am
;~~;(~; :"J
,~ ()~.~ . . ,," . .: );:f'~ .. "!G
. "" .-<~.!. ;,. .. ,..
:.-~.\'.:\:;>:: .. '._/ :"'.'
(B) DEN 2 As
(E) DEN 2 As
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(C) DEN 4
(F) DEN 4
Figure 1.6.Secondary-structure of NTRs of various dengue viruses. Details of (A), (B), (C), (D), (E) and (F) are given in Table 1.3.
1.5.2 Roles of NTRs in dengue virus life cycle
The flavivirus RNA replication strategy has been established with Kunjin virus
(KUN) and the terminal nucleotides of the flavivirus genome have been shown to play a
significant roles in the replication [32, 56, 62, 63l • Upon infection, the first step during the life
cycle of the virus is translation of the genomic or positive-strand RNA. Genomes of
positive-strand RNA viruses often lack a 5' cap and/or a poly(A) tail and hence must
compete with cellular mRNAs for ribosomes to support robust viral gene expression. Viral
RNAs which lack a 5' cap often possess an internal ribosome entry site in the 5' NTR that
directs interaction with the small ribosomal subunit directly or through the binding of an
initiation factor, such as eIF4G [64,65). This can lead to initiation at an AUG triplet that is not
5' proximal, as occurs with the picornaviruses [66) and the members of two genera
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(Hepacivirus and Pestivirus) of the family Flaviviridae [351. Since the genomes of DEN and
other members of the genus Flavivirus have a 5' cap and initiate translation at the 5'-most
AUG triplet, standard cap-dependent initiation rather than internal ribosome entry has been
considered to be the gene expression strategy used by these viruses [35).
The 3' NTRs of some viral RNAs that possess a 5' cap but lack a poly(A) tail,
promote translation to an extent similar to that of NTRs with a poly(A) tail and through a
similar synergistic interaction with the cap. This is true for rotavirus mRNA [671, whose 3'
terminal GACC sequence acts as a translational enhancer [681 by binding the viral protein
NSP3, which competes with poly(A) binding protein for interaction with eIF4G [671. It is also
true for some plant viral RNAs whose 3' NTRs terminate in a tRNA-like structure, like those
of Tobacco Mosaic virus, Brame Mosaic virus and Turnip Yellow Mosaic virus [701 although
in these cases, the molecular interactions have not been identified. In case of flavivirus such
as HCV and dengue virus, cellular proteins like La auto antigen and PTB bind to both the 5'
and the 3' NTRs and might function to bring them together to carry on efficient translation [71,72,731
Once the genome is translated, a replication complex (RC) consisting of the virus
encoded NS proteins is formed co-translationally on the 3' non-translated region (NTR) of
the template RNA and transcribes a negative strand RNA that remains base paired with the
positive strand RNA as a replicative form (RF). The RF functions as a recycling template
(shown by the kinetics of pulse-chase labeling) for asymmetric and semi-conservative
replication of progeny RNA positive strands by the viral replicase [32). Thus the 3' NTR of the
negative RNA serves as the promoter for the synthesis of the genomic RNA similar to the
role played by the 3' NTR of genomic RNA in the synthesis of negative RNA. It is well
documented that the RNA synthetic machinery of the flaviviruses, like most other
eukaryotic cytoplasmic plus-sense RNA viruses, is associated with host cell membrane
components. Mackenzie et al. in 1996 [741, observed for the first time, unique cytoplasmic
entities known as vesicle packets (VPs), in DEN-2 virus-infected cells [75, 76, 771.
Subsequently, it was shown that NS1, NS3, NS2a and NS4a (all components of the viral
RNA replicase) as well as dsRNA (the putative template for viral RNA synthesis) co
localized to the VPs in Kunjin virus-infected Vero cells [34, 78,791.
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Consistent with the roles played by the various NTRs during the infection cycle of
dengue virus, mutations and deletions in these regions appear to reduce the infectivity and
virulence of the virus. Figure 1.7 highlights the roles played by the NTR in the flavivirallife
cycle.
1.5.3 Significance of NTR differences in DF vs. DHF
The NTRs of several positive-strand RNA viruses are predicted to fold into stem
loop structures that interact with viral or cellular proteins. At the 5' NTR, ribosomes bind to
the 5' terminus of the positive strand to initiate translation and replicase binds to the 3'
terminus of the plus and minus strand to initiate transcription of negative and positive
strands, respectively, thus regulating replication. Mutations that modify these structures and
thereby alter the RNA-protein interactions, have been shown to affect virulence or cause
attenuation. A single-nucleotide mutation in the 5' NTR of poliovirus is associated with
neurovirulence and attenuation was correlated with disruption of its secondary structure [801•
Engineered mutations and deletions in the 5' NTR of a full-length DEN-4 cDNA clone were
shown to restrict DEN-4 virus growth in cell culture and in inoculated mosquitoes; most
mutations within the long stem structure were lethal, but RNA transcripts containing
-;r deletions in the loop or short stem regions were usually infectious [811. The results shown by
f- Leitmeyer et al. indicate that an A-to-U mutation that distinguishes Southeast Asian from
American genotype viruses at position 69 was predicted to change the secondary structure of
the viral RNA: a 4-nt-long bulge was formed at the 5' terminus of the American genotype,
which reduced the length of the stem, but increased the length of the 3 '-terminal loop [411.
Whether the presence of this small bulge could reduce translation efficiency remains
unclear; although a bulge as small as 1 nucleotide has been shown to reduce RNA-protein
interactions in other viral systems.
The 3' NTR contains sequences essential for virus replication and growth, serving as
signals for the initiation of minus-strand synthesis and possibly packaging as known for
yellow fever and DEN-4 viruses [821. A stable secondary structure motif, formed by the 3'
terminal 100 nucleotides, was described for all mosquito-borne flaviviruses studied to date.
Revie}v (~( Literature
CS2 and CS3, are thought to be functionally important elements of the 3' NTR [821. While
conservation of the 3' terminal region is assumed to be essential for flaviviruses, there is
evidence that regions further upstream determine virus replication efficiency. Deletions
introduced into full-length DEN-4 cDNA clones that did not extend beyond the 3' terminal
113 nucleotides were viable when transfected into cells, but they exhibited a range of growth
restrictions. In the 19 viruses compared by Pandey and Igarashi [831, a conserved region of
110 nucleotides was found at the 3' terminus, which folded in a form observed in other
flaviviruses. However, a striking size and sequence heterogeneity was observed in the 300-
nucleotides upstream region that allowed distinguishing the genotypes, based on nucleotide
alignments. All American genotype samples revealed deletions in the region immediately
downstream of the stop codon as well as 5 interspersed nucleotide differences when
compared to the Southeast Asian genotype; secondary structure predictions yielded
drastically different conformations that were characteristic for each genotype [51, 53,841• The
importance of these structures in defining dengue virus pathogenicity is unknown, but 3'
NTR structure has also been shown to correlate with virulence in yellow fever and TBE
viruses.
Hence along with the amino acid substitutions within the coding region, which may
affect antigenicity or cell attachment, nucleotide changes within the NTRs, affecting
secondary structure and thereby replication, may be the viral determinants of severe dengue
in humans. Genomic differences in nonstructural genes or in the NTRs potentially altering
replication efficiency could possibly be measured most effectively in terms of human
viremias.
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Chaperonemediated
folding, assembly. or disassembly
Review of Literature
Dengue genomic RNA
STEPS IN VIRAL LIFE CYCLE
Translation of viral repl. factors R~uloted expression
Post-translational modification
lNVOLVEMENT OF I RNA localization? t 5'(+)13'(+) NTRs
NTRs
tt I I I I I I
Template RNA recognition 5'(+)13'(+) NTRs
O " , , R~ulated stability, interaction I I'
t I I Inhibition of translation 5'(+)13'(+) NTRs Intracellular localization ;IF > I
Replication complex assembly HF _"
Membrane rearrangements
Recruitment to replication complex 3'(+)13'(-) N TRs
Negative-strand RNA replication interm~iate
I Negative-strand RNA t initiation, synthesis 5'(+)13'(+) NTRs
••••••••••• I Positive-strand RNA t initiation, synthesis
_____ . 5. '(.-).13 .. '(-) NTRs
Progeny RNA
(-) RNA degradation ...... ------/ , 5'(-)13'(-) NTRs
Encapsidation 5'(+)13'(+) NTRs
Figure 1. 7. Highly simplified scheme of major steps in positive-strand RNA virus genome replication indicating the possible roles for the genomic as well as replication intermediate NTRs. Arrows 1 and 2 depict the sequential use of the infecting, dengue genomiC RNA as a template first for translation and then for RNA replication. As discussed in the text, precedents exist for the involvement of NTRs with host cellular factors (HF) in most steps shown.
Interestingly, although distinguishing mutations are scattered throughout the genome
of DEN-2 Am and As strains, some occur in regions known to affect pathogenicity such as
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Revie'w q( Literature
the 3'-NTR, which is fundamental to viral replication, translation and assembly, or in the
envelope protein, which is the site of attachment for cellular proteins and stimulates
neutralizing antibodies to induce cell-mediated immune responses. The 3' NTRs of the
American strains are characterized by a set of point mutations and a deletion and are
reported to alter the RNA secondary structure; additionally, an amino acid replacement at
position 390 in the envelope protein is suggested to alter the strength of virus binding to host
cells. This was supported by the observation that other amino acid changes at this residue
affect neurovirulence in mice [421. Unfortunately, the lack of a suitable animal model for
dengue makes it difficult to confirm whether these and other characteristic mutations really
determine virulence, either in isolation or synergistically, or whether they have accumulated
simply because of the phylogenetic isolation of the Latin American strains. Despite this
caveat, whole genome analysis clearly takes the study of dengue pathogenesis to a new level
and provides a valuable set of testable hypotheses.
1.6 Host cell proteins that interact with the viral NTRs
1.6.1 RNA viruses
Viruses are intracellular parasites that infect cells and use host machinery to
multiply. During multiplication, the viral genomes replicate and these progeny genomes,
together with newly synthesized viral proteins, are assembled into new virus particles.
Because viruses contain only limited genetic information, they must rely on existing or
modified cellular machineries for many steps of macromolecular synthesis. This is true not
only for protein translation, but also for the replication and transcription of viral genomes.
For example, gene expression of most DNA viruses is affected by cellular polymerases and
regulated largely by cellular transcription factors. In contrast, the participation of cellular
factors in the transcription and replication of viral RNA genomes is less obvious in RNA
viruses. At first glance, this uncertain role of cellular factors appears to be logical since most
RNA viruses replicate and transcribe their genomes by RNA-dependent RNA synthesis, a
process foreign to most eukaryotic cells. Thus, it is assumed that normal cells do not have
pre-existing machinery to support viral RNA-dependent RNA synthesis and that RNA
viruses are more self-reliant, depending on their own proteins for RNA replication and
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Review (?f Literature
transcription. This view is consistent with the fact that most RNA viruses shut off cellular
transcription and translation for the purpose of viral replication. However, it is paradoxical
because RNA viruses typically have a small genome; thus, we would intuitively assume that
they must rely more on the cell to assist in viral replication. Indeed, there is increasing
evidence that RNA viruses frequently subvert cellular factors for replication and
transcription of viral RNAs. Many of these factors are normal components of cellular ,RNA
processing or translation machineries, which are subverted to play an integral or regulatory
role in the replication and transcription of viral RNA. This revised view is consistent with
the long-recognized observation that replication of many RNA viruses and their mutants is
cell type-specific suggesting their dependence on cell-specific factors [85,86,871.
The most compelling evidence for the participation of cellular factors in viral RNA
synthesis came from a heterologous system that studied brome mosaic virus (BMV)
replication in yeast [881. Multiple yeast mutants that affect BMV RNA replication or
transcription have been isolated [891. Therefore, it now appears that cellular factors are
involved in the replication and transcription of viral RNAs, even-though cells are not
equipped to carry out RNA-dependent RNA synthesis on their own. Many cellular factors,
such as protein kinases or other protein-modifying enzymes' may affect viral RNA synthesis
indirectly by modulating the properties or biosynthesis of viral proteins.
Purified viral RNA polymerases alone are usually enzymatically inactive or
nonselective for template and often require cellular proteins for their normal functioning.
Prokaryotic and eukaryotic DNA-dependent RNA polymerases usually consist of multiple
subunits. Typically the core enzymes do not confer template specificity. The specificity of
RNA synthesis is usually determined by other factors, such as bacterial or bacteriophage
sigma factors or mammalian transcription factors, which are either tightly associated with
the polymerase holoenzyme or exist independently of the polymerases but interact with the
template. Similarly, relatively pure preparations of viral RNA-dependent RNA polymerase
from several viruses, such as poliovirus [901, hepatitis C virus [911, or dengue virus [921, can
replicate most natural or synthetic RNA or even DNA templates in vitro with very little
template specificity (which usually requires a primer or is self-priming, in contrast to viral
RNA synthesis in vivo, which is usually primer-free). Only a few viral RdRps, e.g., those of
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cucumber mosaic virus (CMV) (93), brome mosaic virus (BMV) [94,95) and turnip crinkle
virus (TCV) (96) have been shown to replicate or transcribe viral RNAs specifically;
however, all of the RdRp preparations purified from virus-infected cells (for positive-strand
RNA viruses) contained some cellular proteins. Removal of the cellular factors invariably
resulted in the loss of RdRp activity or template specificity. Thus, it appears that the core
viral polymerase proteins, at best, carry only the basic RdRp activity but not the determinant
of their template specificity. In negative-strand RNA viruses, RdRps are typically present in
the virion; disruption of the virion leads to transcription of viral RNA under in vitro reaction
conditions. Even under these conditions, viral replicase and its associated viral proteins
(typically nucleocapsid protein and some other proteins, eg., phosphoprotein) are not
sufficient to replicate or transcribe virion RNA. Instead, cellular factors, such as tubulin,
actin and heat-shock are required for RdRp reactions [97, 98, 99, 100, 101). In the poliovirus
translation-replication-coupled system, initiation of RNA synthesis from a pre-initiation
complex also requires a cellular factor (102). All these observations suggest that cellular
factors are necessary for template-specific RNA-dependent RNA synthesis, which is
undoubtedly a pre-requisite for successful viral replication, considering the fact that viral
RdRps have to replicate only viral RNA but not numerous cellular RNAs present in the
infected cells.
The participation of cellular factors in viral RNA-dependent RNA synthesis follows
two modes (103). In the first, cellular proteins are present as part of the RdRp holoenzyme; in
the second, they bind directly to the RNA template, thereby directing RdRp to the template.
However, the two modes are not mutually exclusive. In fact, some factors may possess both
functions and thus serve as a bridge between viral RdRps and viral RNA template. The
classification of these two modes of action may merely reflect the degree of difference in the
affinity of a factor for either RdRp or RNA and the method of detection used by the
investigators.
Another mechanism by which cellular proteins participate in viral RNA synthesis is
by binding to viral RNA. An increasing number of cellular proteins have been shown to bind
60 discrete viral RNA regions that regulate RNA synthesis of various viruses. These regions
include the 5' and 3' NTRs and internal promoters of the various viral genomes and
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antigenomes. The proteins are typically detected by in vitro UV crosslinking or gel
retardation experiments using viral RNA and uninfected cell lysates. In some cases, these
protein-RNA interactions were also demonstrated in virus-infected cells and their formations
correlated with viral RNA synthesis [29, 104, 105]. These viral RNA-protein complexes
frequently contain both viral (polymerase) and cellular proteins. It should be noted that,
although some purified viral polymerase proteins alone (polio virus [106] and
encephalomyocarditis virus (EMCV)[107]) can preferentially bind homologous viral RNA at
the regions required for viral RNA synthesis (e.g., 5' and 3' ends), their binding specificity
and affinity is probably not sufficient to account for the specificity of viral RNA synthesis.
In fact, many other viral RdRps do not bind to viral RNA specifically at all; thus, their
binding to template RNA must be mediated by cellular proteins that bind specifically to viral
RNA. Table 1.4 summarizes the various cellular proteins that have been found to be capable
of binding to viral RNAs. Cellular proteins implicated in more than one virus system include
the polypyrimidine tract binding protein, the protein hnRNP Al and the autoantigen La. La
associates with the RNAs of many viruses, but it is a known RNA-binding protein; it has
been difficult to show that the binding is functional. There is evidence that La binding to
poliovirus RNA and HIV RNA is required for optimal efficiency and fidelity of their
translation, but in other viruses it may be involved in RNA replication.
Abundant genetic evidences suggest that cellular proteins are components of the
replicases of plant and animal RNA viruses, but the identification of such proteins has been
slow. Purification of active replicases has been difficult, still a few studies with purified
rep Ii cases have been reported. As an alternative, many laboratories have turned to the
identification of cellular proteins that bind to the viral RNA (with mobility shift assays) or to
virally encoded replicase components (with cross linking or two-hybrid system assays) to
identify participating host proteins. Cell proteins identified by these various means comprise
a diverse lot. Most exciting has been the identification of plant and animal elongation factors
EF -1 a, 13 and y as potential components of replicases. In 1998, Das et al. provided the
strongest evidence for the participation of EF -1. They have shown that the polymerase of
vesicular stomatitis virus, which replicates in both insects and mammals, binds EF-la very
tightly and that the resulting complex binds EF-la and 13 [108]. All three EF-I proteins are
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required for replicase activity in vitro. EF-Ia is functionally homologous to bacterial Tu and
EF -1 a and y to Ts. It is not known whether these proteins are true homo logs that have
descended from common ancestors (although the sequences are known, no sequence
identities between the bacterial and animal proteins are apparent), but if they are it would
suggest that the participation of these translation factors as components of viral RNA
replicases is ancient and has co evolved with the viruses and their hosts.
Apart from being a part of the viral replicase complex, the cellular proteins that
bind to viral RNA may serve to bring various regions of a viral RNA template together to
form transcription or replication complexes. Such RNA-protein complexes may help to
recruit and stabilize the RdRp to the initiation sites for viral RNA synthesis. Increasing
evidences suggest that the cis-acting signals for viral RNA replication or transcription often
consist of multiple discontinuous sequences on the viral RNA. Particularly, in many cases,
there appears to be crosstalk between the 5' and 3' NTRs of viral RNAs, so that the 3' NTR
sequence often can regulate RNA synthesis or translation initiated from the 5' NTR of the
RNA[l09, 1101. The interactions between 5' and 3' NTRs and other internal viral RNA
sequences may also involve complementary nucleotides of RNA, as do the 5' and 3' ends of
VSV, influenza virus and flavivirus [36, 111, 112, 113, 1141. Influenza viral RdRp can then bind
directly to the duplex structure formed between the two ends of viral RNA; even in these
cases, the participation of cellular factors has not been ruled out [1151. Alternatively, where no
apparent sequence complementarity exists between the 5' and 3' NTRs, RNA-protein and
protein-protein interactions must be involved. The cellular proteins may fulfill such
function. Indeed, several cellular proteins shown to bind different regions of a viral RNA
have the ability to interact with each other (e.g., between PTB and hnRNP Al for MHV),
thus allowing different RNA regions to interact.
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Table 1.4. Various cellular factors involved in viral RNA life cycle.
Heterogeneous nuclear ribonucleoprotein (hnRNP)
Protein Virus hnRNPAl MHV, HIV, HTLV-2 hnRNP 1 (PTB) PV, MHV, HCV, HA V, HIV, HTL V hnRNP E (poly rC-binding PV protein) hnRNP Cl Influenza Virus
Pol III transcript- binding proteins
Protein Virus La auto anti en Sindbis virus, VSV, HIV, HPIV-3, PV, RV
Translation factors
Protein Virus EF-la ~V,QP,VSV,PV,T~
EF-l~, Y QP, VSV eIF-3 BMV, TMV Ribosomal protein QP
Cytoskeletal or chaperone protein
Protein Virus Tubulin VSV, Sendai virus, RSV Actin HPIV-3 Heat shock protein CDV
Miscellaneous
Protein Virus GAPDH HAV, HPIV-3 Calreticullin RV Sam68 PV
One striking feature of these RNA-binding proteins is that many of them can bind
to several different, unrelated viral RNAs [1031. La antigen, PTB and EF-la all have this
ability. The binding specificity of each of these proteins for respective viral RNA has been
well established. Considering the fact that the RNAs in question do not share a primary
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sequence, the overlap among the RNA-binding protein profiles of the various viruses is
surprising. This observation has two implications: (1) The RNA-binding specificity of these
cellular proteins is more flexible than commonly realized and these RNAs may share
unrecognized structural features common among the viral RNA sequences that regulate
RNA synthesis; and (2) some of these proteins bind to viral RNA in one virus but associate
with viral polymerase in another. Thus, repeated detection of these proteins cannot be
attributed solely to nonspecific RNA-protein interactions in all cases, but most likely reflects
the possibility that these cellular proteins are associated with viral RNA synthetic machinery [103]
Several paramyxoviruses and rhabdoviruses require other types of cellular factors for
RNA synthesis in vitro. These include tubulin for VSV and Sendai viruses [981, measles virus
[991 and respiratory syncytial virus [1001; actin for human parainfluenza virus 3 [lOll; and heat
shock protein for canine distemper virus [971• Tubulin is tightly associated with the purified
RdRp complex of measles virus [991; the modes of action of the other proteins are not clear.
One possibility is that they serve a structural function, holding the various components of
RNA transcription or replication complex in correct topology. Many other viruses (other
than paramyxoviruses) also synthesize their RNAs on or in discrete subcellular
compartments, such as the endoplasmic reticulum (BMV) [1161 or membranous vesicles
(poliovirus) [117, 1181• Cellular RNAs may be excluded from such restricted compartments, thus
allowing viral RNA to be exclusively synthesized, providing an alternative mechanism to
confer template specificity for viral RdRp. Actin, tubulin, heat shock proteins and other
chaperone proteins may provide the means to transport or orient RNA and RdRps in proper
topology in these compartments. In this regard, it is significant to note that heat shock
proteins are required for the reverse transcriptase activity of hepatitis B virus [1191 and bind to
the capsid protein of poliovirus[120I . These arguments notwithstanding, it cannot be ruled out
that these cytoskeletal and chaperone proteins may also have a more direct role in viral RNA
synthesis; for example, tubulin can replace the acidic domain of the viral P protein of VSV
during transcription in vitro. These proteins are not normally thought of as RNA-binding
proteins. However, they have been shown to bind RNA under various conditions.
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One notable feature of these proteins, with the exception of the translation factors, is
that they are located predominantly in the nucleus of uninfected cells, whereas most RNA
viruses replicate exclusively in the cytoplasm. This apparent contradiction may cast doubt
on the biological significance of these cellular factors in viral RNA synthesis; however,
some of these proteins may be shuttled between cytoplasm and nucleus (e.g., hnRNPAl and
PTB) and, certainly, all are synthesized in the cytoplasm. Thus, the association of these
proteins with viral components in cytoplasm, should not pose a conceptual difficulty.
Indeed, the relocalization of these cellular proteins from nucleus to cytoplasm in virus
infected cells has been demonstrated for several proteins (La, hnRNP AI, PTB) [121,122,123).
1.6.2 Flaviviruses
Translation and replication in the flaviviruses too would require interaction between
cis-acting elements of the NTRs with viral as well as host proteins. As mentioned earlier,
while the 5' NTR of the plus sense genomic RNA [5'(+) NTR] is thought to be involved in
the initiation of translation, 3' NTR of the plus sense genomic RNA [3'(+) NTR] is
implicated to function as a promoter for the synthesis of the complementary minus sense
RNA, resulting in a double stranded (ds) replicative form (RF). A number of host factors
have been reported to associate with the different viruses (Table 1.5), for example, the
cellular proteins, La antigen and polypyrimidine tract binding protein (PTB) have been
shown to bind to the 5'(+) NTR of DEN-4, WNV and HCV and facilitate translational
initiation [72,73,124,1251•
The viral proteins, NS3 and NS5, which function as components of the RNA
replicase of flaviviruses, have been found to be associated with the 3 '( +) NTR in all the
members of the flavivirus group [29, 1261• PTB mentioned above also binds to the 3'(+) NTR of
HCV RNA and may thus affect transcription as well [71,1271• In case of West Nile virus, it has
been reported that the translation factor EF-la binds to the 3'(+) NTR [128,1291• Similarly,
Japanese Encephalitis virus 3'(+) NTR has been reported to interact with a cellular protein
known as Mov34 [1301. It has been speculated that the binding of cellular factors to the 3'(+)
NTRs may confer template-specificity. The 3' NTR of the minus strand [3'(-) NTR] is
believed to function as promoter for the synthesis of the genomic plus sense RNA from the
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dsRF molecule through an apparently asymmetric, semi-conservative mechanism. Host cell
RNA binding proteins TIAR and TIA-l involved in translation and splicing are known to
interact with 3'(-) NTR of the flavivirus, West Nile virus and facilitate plus sense RNA
synthesis.
Table 1.5. Various cellular factors interacting withjlaviviral NTRs.
Proteill . lIo I. mass .\TR FUllctioll (/iDa)
PTBI hnRNPAl 57 +/- Modulates secondary and tertiary RNA structures, facilitates recognition by replicase through protein-protein interactions.
La autoantigen 52 + Chaperone, helicase, protection from degradation
EF-la 50 + Role in replication
hnRNPC 35 + Modulates secondary and tertiary RNA structures, facilitates recognition by replicase through protein-protein interactions.
PCBP2 39 + mRNA stability, regulation of translation & replication
hnRNPK -do-
TWTIAR 42 - Stability, recognition by replicase, reduction in stress granule formation
CalreticullinIPDI 60/62 - Binding to ER membrane during assembly
MoV34 36 + Cell cycle regulation & translation
Interestingly, there is no information regarding host cellular proteins that may
interact with the 5'(-) NTR of the flaviviruses. The identification of host factors that
specifically interact with the 5'(+) NTR, 3'(+) NTR and 3'(-) NTR is consistent with the
proposed roles for these NTRs in translation, synthesis of the minus sense RNA intermediate
and the genomic plus sense RNA, respectively. Intuitively it is not clear what role the 5'(-)
NTR may have, if any, during viral life cycle. However, the conservation of the relative
location and shape of the SL structures in the flavivirus 3'(+) NTR, suggests that this would
be mirrored in its complement, the 5' (-) NTR. Therefore, it is likely that the 5'(-) NTR may
indeed also interact with host proteins through these putative conserved elements.
32
