The association of positive transcription elongation factor b (PTEFb) with Dengue virus core protein stimulates the induction of
interleukin-8 (IL-8)
Li-li Li1, Hsing-Hui Lee2, Shao-Hung Wang1, Shiau-Ting Hu1, and YuehHsin Ping2,3, 
1
Institute of microbiology and immunology, 2Department and Institute of
Pharmacology, National Yang-Ming University, Taipei, Taiwan, 3Department of
Education and Research, Taipei City Hospital, Taipei, Taiwan
Corresponding author. Mailing address for Dr. Yueh-Hsin Ping: Department and
Institute of Pharmacology, National Yang-Ming University, Shih-Pai, Taipei, Taiwan
112.
Phone:
(886-2)-28267326;
yhping@ym.edu.tw
Fax:
(886-2)-28264372;
E-mail
address:
Abstract
Positive transcription elongation factor b (P-TEFb), a newly identified cellular
transcription complex consisting of CDK9 and cyclin T1, has been shown to be
crucial for enhancing RNA polymerase II (RNA pol II) processivity. P-TEFb is
participated in several transcriptional activations including NF-B. Dengue virus
(Den) infection causes several complications from a relative benign dengue fever (DF)
to the lethal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).
One of characterizations of Den infection is the induction of immuno-response
mediators such as interleukin 8 (IL-8). The core protein, the building block of the
nucleocapsid of Den, may serve as a transcription modulator by associating with
cellular proteins to affect and modulate a number of viral and cellular promoter
activities. We proposed here a hypothesis that the induction of IL-8 by Den infection
is caused by Den core protein and is mediated by P-TEFb. We have utilized a series of
approaches to test this hypothesis. Reporter assay results showed that Den core
protein could specifically stimulate the activity of NF-B as well as IL-8 promoter
activity. This activation is inhibited by either DRB, a pharmacological inhibitor of PTEFb or siRNA targeting to cyclin T1, indicating that P-TEFb is essential for the
transcription activation of Den core protein. In addition, results from coimmunoprecipitation and immunofluorescence staining assays elucidated that Den
core protein complexes with P-TEFb and alters the localization of P-TEFb in nuclear
region. These results suggest that P-TEFb interacts with Den core protein in vivo.
Moreover, chromatin immunoprecipiation (ChIP) results revealed that Den core
recruited P-TEFb to IL-8 promoter, resulting in the induction of IL-8 promoter
activity. Taken together, these studies provide new insights into the mechanisms of the
Den-induced IL-8 expression and the role of Den core protein in the regulation of IL8 gene expression. Importantly, our study is the first report demonstrating that P-TEFb
is directly involved in virus-induced host gene expression by interacting with the viral
protein.
Introduction:
The process of RNA Polymerase II (RNA pol II) transcription can be dissected
into number of stages, designed preinitiation, initiation, promoter clearance,
elongation and termination (48). Proper manipulation of eukaryotic transcription
initiation is dependent on the assembly of multi-protein regulatory complexes at
transactivation response elements located within gene promoters. Shortly after
promoter clearance, RNA pol II encounters the barrier of negative transcription
elongation factors (N-TEFs) and causes abortive elongation that could lead to
premature termination of transcription. The action of positive transcription elongation
factors (P-TEFs) is able to lower the barrier of N-TEFs and facilitates RNA pol II to
re-enter the elongation phase (38, 41). Over the past decade, the identification of
positive transcription elongation factor-b (P-TEFb) was uncovered. P-TEFb,
composed of two subunits including the catalytic subunit cyclic-dependent kinase
CDK9 (previously named PITALRE) and the regulatory subunit cyclin T1, was
originally recognized as a positive transcription factor by using a nucleoside analogue,
5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) (30, 31, 37, 53, 55). P-TEFb
was primarily known to release RNA polymerase II (pol II) from an elongation pause
in a DRB-sensitive manner and was proposed to assist the transition from abortive to
productive elongation by phosphorylation the C-terminal domain (CTD) of the largest
subunit of RNA pol II (30, 31). Although P-TEFb functions by phosphorylating the
CTD of RNA pol II during elongation steps, it has been demonstrated that P-TEFb is
recruited into preinitiation transcription complexes at initiation step (39, 40). Recently,
there are several strong evidences suggesting that P-TEFb is essential and limiting for
HIV-1 gene expression (39, 41). Formation of P-TEFb-Tat-TAR tertiary complex
during transcriptional elongation resulting in hyperphosphorylation of CTD of RNA
pol II is the critical step for the production of full length of HIV-1 mRNA (43). PTEFb also phosphorylates Spt5, a subunit of DRB sensitivity inducing factor (DSIF)
identified as a N-TEF, to enhance the processivity of RNA pol II during HIV-1 Tat
transactivation (39). In addition to being essential for HIV-1 gene expression, there
are a number of evidences showing that P-TEFb also participated in many various
cellular processes, such as differentiation, heat shock response, apoptosis, and
proliferation, by interacting with different cellular proteins (38). For example, P-TEFb
interacts with Rel A, a subunit of NF-B, to stimulate the elongation by RNA pol II
and the kinase activity of P-TEFb is critical for NF-B transactivation (2, 4).
Dengue virus (Den), a member of the flaviviridae family that comprises a
large genus of arthropod-transmitted, enveloped virus including yellow fever, West
Nile, tick-borne encephalitis (TBEV), and Japanese encephalitis virus, is one of the
most significant human viral pathogens transmitted by mosquitoes that cause more
than 50 million cases of infection and result in around 24000 deaths worldwide per
year. As there is currently no effective antiviral agent or vaccine against Den, the
spread of Den infection has become a major health issue around the world. DENs can
be classified into four different serotypes: Den-1, Den-2, Den-3, and Den-4. The
infection with any of these four serotypes will result in dengue fever (DF), dengue
haemorrhagic fever (DHF), or dengue shock syndrome (DSS) with a mortality rate of
approximately 5 % (15, 44). Particularly, DHF and DSS are of grave concerns during
heterologous secondary Den virus infections in patients with preexisting crossreactive antibodies and memory T lymphocytes from the primary Den virus infection
(45). It has been postulated that DHF or DSS is the result of sequential infection with
multiple serotypes. However, the molecular mechanism of Den pathogenesis is not yet
fully understood at present.
A three-dimensional image reconstruction shows that the virion comprises a
well-organized outer protein shell, a lipid bilayer membrane, and an inner
nucleocapsid core (23). The genome of Den is a positive-sense, 10.2 kb RNA
encoding a single, long open reading frame that is translated into a polyprotein
containing three structural proteins, including the core (C), membrane (M), and
envelope (E), and seven non-structural (NS) proteins. Signals sequences direct the
translocation of the polyprotein across the endoplasmic reticulum membrane to be
subsequently cleaved by both cellular and viral proteinases. The order of genes in the
polyprotein is C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (8). The core
protein can associate with viral RNA genome to form the building block of dengue
nucleocapsid. The mature form of Den core protein containing 100 amino acids is
generated after removal of the C-terminal hydrophobic signal sequence by the virally
encoded NS2B-NS3 protease (8). According to far-UV circular dichroism (CD), coimmunoprecipitation, and NMR analysis, Den core protein possesses four -helices
and forms homodimers by the homotypic interaction domain encompassing  -helices
II and III (19, 25, 50). Moreover, the existence of nuclear localization signal (NLS)
motifs within amino acid sequence of Den core protein suggest that the core protein
can enter the nucleus, though the replication cycle of Den virus occurs in the
cytoplasm of host cells (7). As a matter of fact, Den core protein is found to enter
nucleus and interacts with cellular protein hnRNP and potentially regulate the activity
of a transcriptional activator, C/EBP (9).
Chemokines, a family of small, structurally related chemoattractant cytokines,
have been shown to play an essential role in viral pathogenesis and immunity. Virus
have developed number of ways to either sabotage or exploit the chemokine system to
enhance viral replication (21, 26). There are several lines of evidence showing that
Den infection can induce the productions of interleukin-6 (IL-6) and interleukin-8
(IL-8) in cell culture system (6, 34, 52). Moreover, Den nonstructural protein NS5
induces IL-8 transcription in HEK293A cells through unknown mechanisms (32). In
clinic, the elevated level of IL-6 and IL-8 in blood or pleural fluid and the chemokine
gene expression in peripheral blood mononuclear cells (PBMC) are observed as well
in patients with Den infection (20, 36, 42, 49). Although chemokine production is
thought to be important in Den pathogenesis, however, the molecular mechanism of
Den-induced chemokine production has still not been defined.
NF-B is a ubiquitous transcriptional regulator and promotes the expression of
multitude of target genes, including a number of cytokines, receptors for immune
recognition, proteins for antigen presentation, and adhesion receptors involved in
migration across blood vessel walls, the majority of which participate in human
immune response (54). The activation of NF-B by various virus/viral products
including HIV-1, HBV, EBV, and HTLV-1 has been reported (1). Den virus infection
is also capable of activating NF-B in human endothelial cells and hepatoma cells (3,
29). Accordingly, Den core protein is detected in the nucleus in early stage of viral
infection and interacted with cellular proteins (9, 51). We reasoned that Den core
protein is highly possible to induce the chemokine systhesis through binding with
cellular transcriptional factors. In this paper, we demonstrate that Den core protein
can specifically activate both NF-B and IL-8 promoter activity and this activation is
dependent on the activity of P-TEFb. Our results also show that the activity of Den
core protein is due to its interaction with P-TEFb. Furthermore, the recruitment of
Den core-P-TEFb complex onto IL-8 promoter results in the production of IL-8.
These results provide a novel molecular mechanism by which Den core protein
induces the production of chemokine. This is also the first evidence, to our knowledge,
that P-TEFb is adapted by a viral protein to alter host gene expression.
Material and methods:
Plasmids
Construction of pFC1-100, pFC1-84 and pFC1-72 were described earlier (Wang, Syu
et al., 2004), they express the core protein with a Flag tag at the N-terminus. pNF-kBluc, pAP-1-Luc, pCRE-Luc, p53-Luc, pNFAT-Luc and pSRE-Luc were purchased
from Stratagene, La Jolla, CA, and the lucifersase expression vector containing the 5’
flanking region of IL-8 gene (-133 to -50) was kindly provided by Dr. N. Mukaida
(Ishikawa, Japan). (Ref)
Antibodies
Antibodies used were goat anti-cyclin T1 antibody (T-18; Santa Cruz), rabbit anticdk9 antibody (C-20; Santa Cruz) and anti-Flag mouse monoclonal antibody (M2,
Sigma).
siRNA preparation
Twenty-one-nucleotide dsRNAs were synthesized as 2' bis(acetoxyethoxy)-methyl
ether-protected
oligonucleotides
by
Dharmacon
(Lafayette,
Colo.).
The
oligonucleotides were deprotected, annealed, and purified according to the
manufacturer's recommendations. Duplex formation was confirmed by 8%
nondenaturing polyacrylamide gel electrophoresis (PAGE). All siRNAs were stored in
0.1% diethyl pyrocarbonate-treated water at -80°C. Sequences of siRNA duplexes is
hCycT1 ds, 5'-UCCCUUCCUGAUACUAGAAdTdT-3' (12).
Cell culture and transfection
Human cervical carcinoma HeLa cells were cultured at 37ºC, 5% CO2 in Delbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 60μ
g/ml penicillin and 100μg/ml streptomycin. For transcient transfection, cells seeded
onto 10-cm2 or six-well dishes were incubated for 24 hours and then transfected with
appropriate amounts of plasmid DNA by Lipofectamine 2000 (Invitrogen) as
described by the manufacturer for adherent cell lines. For co-immunoprecipitation and
EMSA, cells were transfected with 20 μg of pFC1-100 or empty plasmid vector. For
luciferase assay, 0.1μg of different reporter vector encoding the luciferase gene, 0.1
μ g of pEGFP (for normalization) and increasing amount of pFC1-100 were
transfected. All transfections were balanced for a total of 1.0 μg of DNA with the
empty plasmid vector.
Luciferase assay
Cells were extracted with the use of 100 μl of luciferase cell culture lysis reagent
(Promega) 48 h post-transfection. The luciferase assay was performed with the
Luciferase Assay System (Promega, Madison, WI) using the standard protocol
provided by the manufacturer. In brief, cell lysate were centrifuged at 12,000 x g for 2
minutes (at 4ºC) to remove the cell debris. 20μl of the supernatant were dispensed
into a 96-well ELISA plate for the detection of fluorescence with microreader Wallac
Victor 2 (Perkin Elmer), and then 100μl of luciferase substrate (Luciferase Assay
System, Promega, Madison, WI) were added to the supernatant and quantitated.
Coimmunoprecipitation
Cells transfected with pFC1-100 or pFCMV2 vector only were washed twice with
cold PBS and lysed with 300μl lysis buffer (150 mM NaCl, 1 mM EDTA, 50 mM
Tris-Cl pH 7.5 and 10 mM PMSF). The cell lysate were then snap freeze with liquid
nitrogen and thaw at 37ºC three times followed by centrifugation. Cell extracts were
incubated with 50 l anti-Flag antibody-conjugated agarose beads (Sigma-Aldrich)
for 4 h at 4ºC. The immunoprecipitated complexes were extensively washed to reduce
nonspecific contaminants, subjected to SDS-PAGE and transferred onto a
polyvinylidene difluoride membrane (PVDF; BioRad), followed by immunoblotting
using antibodies against Cyclin T1 and Flag.
Chromatin Immunoprecipitation (ChIP)
Cells transfected with FC1-100 or vector only were treated 24 h after transfection by
adding formaldehyde directly to tissue culture medium to a final concentration of 1%
and incubated for 10 min at room temperature. Approximately 2 x 106 cells were used
for each immunoprecipitation. Cross-linking reactions were stopped by the addition of
phosphate-buffered saline-glycine to a final concentration of 0.125 M. Cells were
washed twice with ice-cold phosphate-buffered saline, scraped, and centrifuged at
2000 rpm for 2 min. Cells were then resuspended in cell lysis buffer (150 mM NaCl, 1
mM EDTA, 50 mM Tris-Cl pH 7.5 and 10 mM PMSF) containing protease inhibitors
(Complete, Roche) and kept on ice for 15 min. Cells were sonicated on ice to an
average chromatin length of 200-1000 bp and then centrifuged at 12,000 rpm for 30
min at 4 °C. After centrifugation, 1% of the extract was aliquoted and used for the
total input control. The remaining extracts were precleared by adding Protein G
Sepharose (Amersham Bioscience) incubated for 2 h at 4 °C and was aliquoted and
incubated with the anti-Flag antibody or rabbit IgG and 1 g/l Herring sperm DNA
(Promega) overnight at 4 °C on a rotator. Immunoprecipitated material was washed 7
times with wash buffer. Cross-links were reversed by incubating samples for 5 h at 65
°C in 200 mM NaCl and 10 g of RNase A to eliminate RNA. Recovered material
was
treated
with
proteinase
K
for
1
h
at
45
ºC,
extracted
with
phenol/chloroform/isoamyl alcohol (25:24:1), and precipitated. The pellets were
resuspended in 50 l of H2O and analyzed by PCR with Taq Polymerase (Bioman)
and the following primers: IL-8 promoter (accession number M28130 [GenBank] )
sense (nucleotides 1303-1325), 5'-aagaaaactttcgtcatactccg-3'; antisense (nucleotides
1450-1473), 5'-tggctttttatatcatcaccctac-3', GADPH For – 5’-ccccacacacatgcacttacc-3’,
GADPH Rev – ‘5-cctagtcccaggctttgatt-3’.
Immunofluorescence
For immunofluorescence microscopy, HeLa cells were seeded onto coverslips and
transfected with pFC1-100. After 24 hours cells were washed twice with phosphatebuffered saline (PBS) and fixed with 4% p-formaldehyde/PBS. The fixed cells were
then permeabilized for 5 min with 0.2% Triton X-100 in PBS. Coverslips were
washed with PBS and incubated for 60 min in 1% bovine serum albumin-PBS at 37℃.
Antibodies were diluted in 5% bovine serum albumin-PBS. Coverslip were incubated
with primary antibody for 60 mins at 37℃and then wash thrice with PBS followed by
incubation with secondary antibody for 60 mins at 37℃. After washing with PBS, the
specimens were observed by laser-scanning confocal microscopy.
Results:
Effect of DEN2 core protein on different transcription factors
It is unclear whether Den core protein can enhance the activities of
transcription activators in nucleus. To address this speculation, we used the
PathDetect® in Vivo Signal Transduction Pathway cis-Reporting system to determine
the effect of DEN2 core protein on the trans-activation activities of five different
transcription factors including the activator protein 1 (AP-1), cyclic AMP response
element (CRE), the nuclear factor of activated T cells (NFAT), the nuclear factorκB
(NF-κB) and the serum response element (SRE). HeLa cells were co-transfected Den
core protein with reporter plasmids encoding a luciferase gene driven by various
transactivator as described above. In figure 1, the results of luciferase assay showed
apparently that DEN2 core protein had no effect on NFAT, SRE, CRE and AP-1. In
contrast, the activity of NF-B was indeed enhanced by Den core protein in a doesdependent manner (Figure 1, black bar). These results indicated that DEN2 core
protein could specifically induce the activity of NF-B.
The activation domain of DEN2 core protein for NF-kB activity
Next, we would like to further characterize the activation domain of DEN2
core protein that is essential for NF-B transactivation. DEN2 core proteins harboring
N-terminal fragment variants were co-transfected with luciferase reporter plasmid.
Compared to wild type DEN2 core protein (FC1-100), FC1-72 mutant core protein
could not induce the activity of NF-B (Figure 2, black and white bars). Deletion of
73-100 amino acids of DEN2 core protein disrupted the effect of core protein on NFB transactivation, indicating that this region is required for the function of DEN2
core protein to modulate gene expression. In addition, FC1-84 mutant was sufficient
to increase NF-B transactivation with less activity (Figure 2, gray bar). Taken
together, amino acids from 73-83 of DEN2 core protein may be the activation domain
of DEN2 core protein.
P-TEFb is required for DEN2 core protein-mediated NF-kB transactivation
Accordingly, we had shown that DEN2 core protein could induce the activity
of NF-B. DEN2 core protein had been suggested to function as a transcription
regulator (9); however, the molecular mechanism of trans-activation of DEN2 core
protein is still unclear. Previous studies reported that NF-B activation is required PTEFb to stimulate transcriptional elongation (4). In addition, P-TEFb is also required
for viral gene expression, such as HIV-1 and EBV (5, 56). Since DEN2 core protein
affected the activity of NF-kB, we hypothesized that P-TEFb might participate in
DEN2 core protein-mediated NF-B transactivation. To test this possibility, luciferase
reporter assay was performed by co-transfecting pNF-B-luc, pFC1-100 and pEGFP
into HeLa cells and adding 30μM of DRB, a pharmacological inhibitor of CDK9, 6
hours post-transfection. Luciferase activity was determined 48 hours post-transfection.
DEN2 core protein failed to activate NF-B in the presence of DRB (Figure 3, white
bar), whereas in the absence of DRB, it activated NF-B in a dose-dependent manner
which is consistent with our previous observation (Figure 3, black bar). These results
suggest that P-TEFb is required for DEN2 core protein-mediated NF-B
transactivation.
The interaction of DEN2 core protein with P-TEFb
P-TEFb affects viral gene expression by interacting with viral regulatory
proteins (5, 56). Next, co-immunoprecipitation was perfomed to investigate whether
P-TEFb can associate with DEN2 core protein. The FLAG-tagged core protein was
expressed in cells and immunoprecipitated with anti-FLAG monoclonal antibody M2.
The protein content in immunoprecipitated pellet was analyzed by western blot assay.
In Figure 4A, compared to control, cyclin T1 was detected while only FLAG-tagged
core protein was expressed (Lanes 1 and 3). In addition, cyclin T1 was also
precipitated with FC1-84 mutant core protein (Figure 4A, lane 2) that could explain
the reason why this mutant still could induced NF-B transactivation (Figure 2, gray
bar). To further confirm this result, we examine whether these protein display the
same cellular localization in nucleus. Immunofluorescence staining assay results
revealed that DEN2 core protein was detected in both cytoplasm and nucleus,
consistent with previous reports (Figure 4B-I). In contrast, the distribution of cyclin
T1 appeared in nucleus, nucleuos in particular (Figure 4B-II). This observation is
conflicting with early studies that both cyclin T1 and CDK9 were found in the nucleus
in a speckled pattern (16, 27, 35). However, the merging image of the distribution of
DEN2 core protein and cyclin T1 showed that they overlapped inside the nucleus
(Figure 4B-III). These results strongly suggest that DEN2 core protein is associated
with P-TEFb in nucleus.
The activation of IL-8 promoter by DEN2 core protein is P-TEFb-dependent
Very high levels of circulating IL-8 were detected in all cases of dengue
hemorrhagic fever and dengue shock syndrome (DHF/DSS) (3). NF-B element is
one of transactivator binding sites within IL-8 promoter. Since DEN2 core protein
affected the activity of NF-kB (Figure 2), we examined whether DEN2 core protein
affected IL-8 promoter activity. Luciferase reporter assay was performed by
cotransfecting promoter/reporter hybrid plasmids pIL-8(-133 to -50)Luc with DEN2
core protein expressing construct (pFC1-100). DEN2 core protein clearly activated
the IL-8 promoter activity in a dose-dependent manner (Figure 5A). Next, to examine
whether P-TEFb is participated in the activation of IL-8 promoter by DEN2 core
protein, we performed the same luciferase reporter assays with two additional
treatments: addition of DRB or siRNA targeting to cyclin T1. In figure 5B, DEN2
core protein failed to activate IL-8 promoter activity in the presence of DRB, whereas
in the absence of DRB, it activated IL-8 promoter activity in a dose-dependent
manner that is consistent with results shown in figure 5A. Previous studies has
reported that cyclin T1 knockdown by siRNA specifically affected the protein stability
of CDK9, resulting in the reduction of P-TEFB activity (12). To further confirm the
role P-TEFb in DEN2 core protein-mediated NF-B transactivation, cells were treated
with siRNA targeting cyclin T1 prior to the luciferase reporter assay. In the presence
of cyclin T1 siRNA, DEN2 core protein was not able to activate IL-8 promoter
activity (Figure 5C). In contrast, DEN2 core protein activated IL-8 promoter activity
in a dose-dependent manner in the absence of siRNA (Figure 5C). Taken together,
DEN2 core protein activates IL-8 promoter activity and the activation is required PTEFb.
DEN2 core protein directly target to IL-8 promoter
We then addressed whether the recruitment of DEN2 core protein to the IL-8
gene resulted in the induction of IL-8 expression. Chromatin immunoprecipitation
(ChIP) assays were performed with an anti-FLAG monoclonal antibody M2, allowing
us to examine occupancy of the IL-8 gene by DEN2 core protein. The IL-8 fragment
was immunoprecipitated by M2 antibody in DEN2 core expressed cells (Figure 6A,
lane 3). No signal was detected in control or beads alone groups (Figure 6A, lanes 1
and 2). Immnoprecipitation with antibody specific for RNA pol II was conducted as
well. The IL-8 gene was only amplified in the presence of DEN2 core protein (Figure
6B). These finding suggest that DEN2 core protein was recruitment to IL-8 promoter
and associated with RNA pol II complex to induce IL-8 expression.
Discussion:
We have utilized a series of approaches to demonstrate that the P-TEFb is a
key coactivator that mediates the ability of Den core protein to activate the IL-8
promoter. Our results showed that Den core protein specifically stimulates the activity
of NF-B, but had no effect on AP-1, CRE, NFAT and SRE. In addition, results from
co-immunoprecipitation and immunofluorescence staining assays elucidated that Den
core protein complexes with P-TEFb and alters the localization of P-TEFb in nuclear
region. Finally, chromatin immunoprecipitation (CHIP) results showed the
recruitment of Den core protein to IL-8 promoter, resulting in the induction of IL-8
expression. These studies provide new insights into the mechanisms of the Dengueinduced IL-8 expression and the role of Den core protein in the regulation of IL-8
gene expression.
IL-8 and other cytokines have been reported as inducers of alternation in
endothelial function, because of elevated levels of cytokines in serum and pleural
fluid of patients with Dengue hemorrhagic fever. A number of studies have reported
that the infection of dengue virus induces secretion of IL-8 (6, 11, 32). The core of IL8 promoter located at -1 to -133 within the 5’ flanking region of IL-8 gene is essential
and sufficient for transcriptional regulation of the gene (18). Many transcriptional
activators, including NF-B, AP-1 and CAAT/enhancer-binding protein (C/EBP),
have been reported to present in this core region. By using luciferase assay, our results
showed that DEN2 core protein can specifically enhance NF-B promoter activity,
but not AP-1, CRE, NFAT and SRE (Figure 1). NF-B pathway plays an important
role in cellular response to a variety of extracellular stimuli, such as viral infection.
Up to date, NF-B has been shown to be activated by a number of families of virus
including HIV-1, HTLV-1, influenza virus, EBV, HBV and HCV. The activation of
NF-B serves various functions to promote viral replication, to prevent virus-induced
apoptosis, and to mediate immune response induced by the invading pathogen (17).
Several lines of evidence have demonstrated that NF-B plays a major role in Dengue
virus-induced IL-8 secretion (3, 6, 32, 33). Our findings indicate that DEN2 core
protein may play an important role in NF-B activation in viral infection.
In the majority of studies where P-TEFb localization has been investigated,
both cyclin T1 and CDK9 were found in the nucleus in a speckled pattern (16, 27, 35).
Their foci are coincident with those defined by the localization of the SC35 protein,
the hallmark of nuclear speckles. However, the location of cyclin T1 appears distinct
from nuclear speckles, being spatially juxtaposed in most cases. Recently, Marcello
and co-workers showed that cyclin T1 is recruited to nuclear bodies through specific
protein interactions with PML protein (28). In our studies, in the presence of DEN2
core protein, the nuclear localization of cyclin T1 is altered. The majority population
of cyclin T1 is present in nucleolus (Figure 5B-I). Notably, this distribution of cyclin
T1 was not the same with one in the absence of DEN2 core protein (data not shown).
These observations indicate that the alternation of cyclin T1 localization may be
caused by its association with DEN2 core protein. Moreover, co-immunoprecipiation
results reveal that P-TEFb is able to associate with cyclin T1 in vivo (Figure 5A).
Taken together, these results strongly suggest that P-TEFb is capable of interact
directly with DEN2 core protein.
The recruitment of P-TEFb to HIV-1 LTR promoter by the HIV-1 Tat protein
to efficiently synthesize the viral RNA has been extensively studied. P-TEFb is a
component of pre-initiation complex in HIV-1 LTR promoter and associates with Tat
protein and TAR RNA during elongation (40, 41). The kinase activity of P-TEFb
stimulates transcriptional elongation by phosphorylating three proteins: CTD of the
largest subunit of the RNA pol II, Spt5 and NELFe (14, 22, 39). It was reported
recently that CDK9 is essential for the transcriptional activation of EBNA2 of EBV
(5). However, there is no clear evidence to show that EBNA2 directly interact with PTEFb yet. Nevertheless, both viruses hijack P-TEFb, via their own proteins Tat and
EBNA2 respectively, to facilitate the production of their own mRNA. In addition, a
number of studies show that many transcription factors, including MyoD, c-Myc,
androgen receptor (AR) and NF-B, can target P-TEFb to the promoters of their
target genes, implicating that P-TEFb participates in the regulation of cellular
processes (4, 13, 24, 47). In our study, the results showed clearly that P-TEFb is
required to DEN2 core protein-mediated IL-8 promoter activation by binding with
DEN2 core protein. To our knowledge, this is the first report demonstrating that PTEFb is directly involved in virus-induced host gene expression by interacting with
the viral protein.
The dependency of DEN2 core protein-mediated transactivation on P-TEFb
raise the possibility that CDK9 inhibitor and the anticancer drug, flavopiridol could be
used as an anti-Dengue agent (10). Flavopiridol isolated from an Indian plant is a
small molecule for inhibiting the activity of cyclin-dependent kinases. It has been
reported that it is a highly selective inhibitor of CDK9 with IC50 of 3 nM (10). The
potential use of this drug as an anti-HIV agent is being studied (46). Our findings
revealing an essential role for P-TEFb in DEN2 core protein activation mechanism
pinpoint a stage of elevated level of cytokines in DHF/DSS patients that could be
targeted by flavopiridol to repress DEN infection-induced cytokine induction.
References:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Aggarwal, B. B. 2004. Nuclear factor-kappaB: the enemy within. Cancer Cell
6:203-8.
Amini, S., A. Clavo, Y. Nadraga, A. Giordano, K. Khalili, and B. E. Sawaya.
2002. Interplay between cdk9 and NF-kappaB factors determines the level of
HIV-1 gene transcription in astrocytic cells. Oncogene 21:5797-803.
Avirutnan, P., P. Malasit, B. Seliger, S. Bhakdi, and M. Husmann. 1998. Dengue
virus infection of human endothelial cells leads to chemokine production,
complement activation, and apoptosis. J Immunol 161:6338-46.
Barboric, M., R. M. Nissen, S. Kanazawa, N. Jabrane-Ferrat, and B. M. Peterlin.
2001. NF-kappaB binds P-TEFb to stimulate transcriptional elongation by RNA
polymerase II. Mol Cell 8:327-37.
Bark-Jones, S. J., H. M. Webb, and M. J. West. 2006. EBV EBNA 2 stimulates
CDK9-dependent transcription and RNA polymerase II phosphorylation on
serine 5. Oncogene 25:1775-85.
Bosch, I., K. Xhaja, L. Estevez, G. Raines, H. Melichar, R. V. Warke, M. V.
Fournier, F. A. Ennis, and A. L. Rothman. 2002. Increased production of
interleukin-8 in primary human monocytes and in human epithelial and
endothelial cell lines after dengue virus challenge. J Virol 76:5588-97.
Bulich, R., and J. G. Aaskov. 1992. Nuclear localization of dengue 2 virus core
protein detected with monoclonal antibodies. J Gen Virol 73 ( Pt 11):2999-3003.
Chambers, T. J., C. S. Hahn, R. Galler, and C. M. Rice. 1990. Flavivirus
genome organization, expression, and replication. Annu Rev Microbiol 44:64988.
Chang, C. J., H. W. Luh, S. H. Wang, H. J. Lin, S. C. Lee, and S. T. Hu. 2001.
The heterogeneous nuclear ribonucleoprotein K (hnRNP K) interacts with
dengue virus core protein. DNA Cell Biol 20:569-77.
Chao, S. H., K. Fujinaga, J. E. Marion, R. Taube, E. A. Sausville, A. M.
Senderowicz, B. M. Peterlin, and D. H. Price. 2000. Flavopiridol inhibits PTEFb and blocks HIV-1 replication. J Biol Chem 275:28345-8.
Chen, Y. C., and S. Y. Wang. 2002. Activation of terminally differentiated
human monocytes/macrophages by dengue virus: productive infection,
hierarchical production of innate cytokines and chemokines, and the synergistic
effect of lipopolysaccharide. J Virol 76:9877-87.
Chiu, Y. L., H. Cao, J. M. Jacque, M. Stevenson, and T. M. Rana. 2004.
Inhibition of human immunodeficiency virus type 1 replication by RNA
interference directed against human transcription elongation factor P-TEFb
(CDK9/CyclinT1). J Virol 78:2517-29.
Eberhardy, S. R., and P. J. Farnham. 2002. Myc recruits P-TEFb to mediate the
final step in the transcriptional activation of the cad promoter. J Biol Chem
277:40156-62.
Fujinaga, K., D. Irwin, Y. Huang, R. Taube, T. Kurosu, and B. M. Peterlin. 2004.
Dynamics of human immunodeficiency virus transcription: P-TEFb
phosphorylates RD and dissociates negative effectors from the transactivation
response element. Mol Cell Biol 24:787-95.
Gubler, D. J., and G. G. Clark. 1995. Dengue/dengue hemorrhagic fever: the
emergence of a global health problem. Emerg Infect Dis 1:55-7.
Herrmann, C. H., and M. A. Mancini. 2001. The Cdk9 and cyclin T subunits of
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
TAK/P-TEFb localize to splicing factor-rich nuclear speckle regions. J Cell Sci
114:1491-503.
Hiscott, J., H. Kwon, and P. Genin. 2001. Hostile takeovers: viral appropriation
of the NF-kappaB pathway. J Clin Invest 107:143-51.
Hoffmann, E., O. Dittrich-Breiholz, H. Holtmann, and M. Kracht. 2002.
Multiple control of interleukin-8 gene expression. J Leukoc Biol 72:847-55.
Jones, C. T., L. Ma, J. W. Burgner, T. D. Groesch, C. B. Post, and R. J. Kuhn.
2003. Flavivirus capsid is a dimeric alpha-helical protein. J Virol 77:7143-9.
Juffrie, M., G. M. van Der Meer, C. E. Hack, K. Haasnoot, Sutaryo, A. J.
Veerman, and L. G. Thijs. 2000. Inflammatory mediators in dengue virus
infection in children: interleukin-8 and its relationship to neutrophil
degranulation. Infect Immun 68:702-7.
Khabar, K. S., F. Al-Zoghaibi, M. N. Al-Ahdal, T. Murayama, M. Dhalla, N.
Mukaida, M. Taha, S. T. Al-Sedairy, Y. Siddiqui, G. Kessie, and K. Matsushima.
1997. The alpha chemokine, interleukin 8, inhibits the antiviral action of
interferon alpha. J Exp Med 186:1077-85.
Kim, J. B., and P. A. Sharp. 2001. Positive transcription elongation factor B
phosphorylates hSPT5 and RNA polymerase II carboxyl-terminal domain
independently of cyclin-dependent kinase-activating kinase. J Biol Chem
276:12317-23.
Kuhn, R. J., W. Zhang, M. G. Rossmann, S. V. Pletnev, J. Corver, E. Lenches, C.
T. Jones, S. Mukhopadhyay, P. R. Chipman, E. G. Strauss, T. S. Baker, and J. H.
Strauss. 2002. Structure of dengue virus: implications for flavivirus organization,
maturation, and fusion. Cell 108:717-25.
Lee, D. K., H. O. Duan, and C. Chang. 2001. Androgen receptor interacts with
the positive elongation factor P-TEFb and enhances the efficiency of
transcriptional elongation. J Biol Chem 276:9978-84.
Ma, L., C. T. Jones, T. D. Groesch, R. J. Kuhn, and C. B. Post. 2004. Solution
structure of dengue virus capsid protein reveals another fold. Proc Natl Acad Sci
U S A 101:3414-9.
Mahalingam, S., J. S. Friedland, M. T. Heise, N. E. Rulli, J. Meanger, and B. A.
Lidbury. 2003. Chemokines and viruses: friends or foes? Trends Microbiol
11:383-91.
Marcello, A., R. A. Cinelli, A. Ferrari, A. Signorelli, M. Tyagi, V. Pellegrini, F.
Beltram, and M. Giacca. 2001. Visualization of in vivo direct interaction
between HIV-1 TAT and human cyclin T1 in specific subcellular compartments
by fluorescence resonance energy transfer. J Biol Chem 276:39220-5.
Marcello, A., A. Ferrari, V. Pellegrini, G. Pegoraro, M. Lusic, F. Beltram, and M.
Giacca. 2003. Recruitment of human cyclin T1 to nuclear bodies through direct
interaction with the PML protein. Embo J 22:2156-66.
Marianneau, P., A. Cardona, L. Edelman, V. Deubel, and P. Despres. 1997.
Dengue virus replication in human hepatoma cells activates NF-kappaB which
in turn induces apoptotic cell death. J Virol 71:3244-9.
Marshall, N. F., J. Peng, Z. Xie, and D. H. Price. 1996. Control of RNA
polymerase II elongation potential by a novel carboxyl-terminal domain kinase.
J Biol Chem 271:27176-27183.
Marshall, N. F., and D. H. Price. 1992. Control of formation of two distinct
classes of RNA polymerase II elongation complexes. Mol Cell Biol 12:20782090.
Medin, C. L., K. A. Fitzgerald, and A. L. Rothman. 2005. Dengue virus
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
nonstructural protein NS5 induces interleukin-8 transcription and secretion. J
Virol 79:11053-61.
Medin, C. L., and A. L. Rothman. 2006. Cell type-specific mechanisms of
interleukin-8 induction by dengue virus and differential response to drug
treatment. J Infect Dis 193:1070-7.
Moreno-Altamirano, M. M., M. Romano, M. Legorreta-Herrera, F. J. SanchezGarcia, and M. J. Colston. 2004. Gene expression in human macrophages
infected with dengue virus serotype-2. Scand J Immunol 60:631-8.
Napolitano, G., P. Licciardo, R. Carbone, B. Majello, and L. Lania. 2002. CDK9
has the intrinsic property to shuttle between nucleus and cytoplasm, and
enhanced expression of cyclin T1 promotes its nuclear localization. J Cell
Physiol 192:209-15.
Nguyen, T. H., H. Y. Lei, T. L. Nguyen, Y. S. Lin, K. J. Huang, B. L. Le, C. F.
Lin, T. M. Yeh, Q. H. Do, T. Q. Vu, L. C. Chen, J. H. Huang, T. M. Lam, C. C.
Liu, and S. B. Halstead. 2004. Dengue hemorrhagic fever in infants: a study of
clinical and cytokine profiles. J Infect Dis 189:221-32.
Peng, J., Y. Zhu, J. T. Milton, and D. H. Price. 1998. Identification of multiple
cyclin subunits of human P-TEFb. Genes Dev 12:755-62.
Peterlin, B. M., and D. H. Price. 2006. Controlling the elongation phase of
transcription with P-TEFb. Mol Cell 23:297-305.
Ping, Y. H., and T. M. Rana. 2001. DSIF and NELF interact with RNA
polymerase II elongation complex and HIV-1 Tat stimulates P-TEFb-mediated
phosphorylation of RNA polymerase II and DSIF during transcription
elongation. J Biol Chem 276:12951-8.
Ping, Y. H., and T. M. Rana. 1999. Tat-associated kinase (P-TEFb): a
component of transcription preinitiation and elongation complexes. J Biol Chem
274:7399-404.
Price, D. H. 2000. P-TEFb, a cyclin-dependent kinase controlling elongation by
RNA polymerase II. Mol Cell Biol 20:2629-34.
Raghupathy, R., U. C. Chaturvedi, H. Al-Sayer, E. A. Elbishbishi, R. Agarwal,
R. Nagar, S. Kapoor, A. Misra, A. Mathur, H. Nusrat, F. Azizieh, M. A. Khan,
and A. S. Mustafa. 1998. Elevated levels of IL-8 in dengue hemorrhagic fever. J
Med Virol 56:280-5.
Richter, S., Y. H. Ping, and T. M. Rana. 2002. TAR RNA loop: a scaffold for the
assembly of a regulatory switch in HIV replication. Proc Natl Acad Sci U S A
99:7928-33.
Rigau-Perez, J. G., G. G. Clark, D. J. Gubler, P. Reiter, E. J. Sanders, and A. V.
Vorndam. 1998. Dengue and dengue haemorrhagic fever. Lancet 352:971-7.
Rothman, A. L., and F. A. Ennis. 1999. Immunopathogenesis of Dengue
hemorrhagic fever. Virology 257:1-6.
Sadaie, M. R., R. Mayner, and J. Doniger. 2004. A novel approach to develop
anti-HIV drugs: adapting non-nucleoside anticancer chemotherapeutics.
Antiviral Res 61:1-18.
Simone, C., and A. Giordano. 2001. New insight in cdk9 function: from Tat to
MyoD. Front Biosci 6:D1073-82.
Sims, R. J., 3rd, R. Belotserkovskaya, and D. Reinberg. 2004. Elongation by
RNA polymerase II: the short and long of it. Genes Dev 18:2437-68.
Spain-Santana, T. A., S. Marglin, F. A. Ennis, and A. L. Rothman. 2001. MIP-1
alpha and MIP-1 beta induction by dengue virus. J Med Virol 65:324-30.
Wang, S. H., W. J. Syu, and S. T. Hu. 2004. Identification of the homotypic
interaction domain of the core protein of dengue virus type 2. J Gen Virol
85:2307-14.
51. Wang, S. H., W. J. Syu, K. J. Huang, H. Y. Lei, C. W. Yao, C. C. King, and S. T.
Hu. 2002. Intracellular localization and determination of a nuclear localization
signal of the core protein of dengue virus. J Gen Virol 83:3093-102.
52. Warke, R. V., K. Xhaja, K. J. Martin, M. F. Fournier, S. K. Shaw, N. Brizuela, N.
de Bosch, D. Lapointe, F. A. Ennis, A. L. Rothman, and I. Bosch. 2003. Dengue
virus induces novel changes in gene expression of human umbilical vein
endothelial cells. J Virol 77:11822-32.
53. Wei, P., M. E. Garber, S. M. Fang, W. H. Fischer, and K. A. Jones. 1998. A
novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and
mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92:451-62.
54. Yamamoto, Y., and R. B. Gaynor. 2004. IkappaB kinases: key regulators of the
NF-kappaB pathway. Trends Biochem Sci 29:72-9.
55. Yang, X., M. O. Gold, D. N. Tang, D. E. Lewis, E. Aguilar-Cordova, A. P. Rice,
and C. H. Herrmann. 1997. TAK, an HIV Tat-associated kinase, is a member of
the cyclin-dependent family of protein kinases and is induced by activation of
peripheral blood lymphocytes and differentiation of promonocytic cell lines.
Proc Natl Acad Sci U S A 94:12331-6.
56. Zhou, Q., and J. H. Yik. 2006. The Yin and Yang of P-TEFb regulation:
implications for human immunodeficiency virus gene expression and global
control of cell growth and differentiation. Microbiol Mol Biol Rev 70:646-59.