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									                   Dengue Virus


                            Kolawole Opanubi
                            MSc.Student
                            Microbial Pathogenesis
                            Dept. of Medical Microbiology
January 19, 2012
                            University of Manitoba
   Background on Dengue Virus
            (DENV)
• Flavivirus genus as part the Flaviviridae
  family.
• Enveloped viruses containing positive-
  stranded RNA genome.
• Three structural proteins termed C, prM
  and E.
• Seven nonstructural proteins called NS1,
  NS2A, NS2B, NS3, NS4A, NS4B and
  NS5.
Figure 1- Structure of the genome of Dengue virus. (Figure extracted from Clyde
et al.2006)
           Human Health.
• DENVs infect tens of millions of human
  each year. Major global public health
  importance.
• Causes a wide variety of diseases in
  humans; Dengue fever (DF), Dengue
  hemorrhagic fever (DHF) and Dengue
  shock syndrome (DSS).
Figure 2- Life cycle of the DENV (Figure extracted from Clyde et al.
2006
       Original antigenic sin
• The immune system is primed by a primary
  DENV infection and subsequent DENV infection
  always involves a different serotype.
• The response to a secondary infection is the
  proliferation of memory cells with cross reacting
  antibodies produced against the primary
  infection.
• A low avidity of the memory T and B cells for
  the secondary challenging antigen occurs. This
  concept is know as the original antigenic sin
  (OAS)
                Pathogenesis and OAS.




Figure 3. Model of immunopathogenesis of plasma leakage in DHF. During primary DENV infection, a
normal T cell response occurs. When exposed to a second heterologous DENV serotype preexisting non-
neutralizing antibodies from the primary infection may enhance infection in APCs, leading to an altered gene
expression profile. Cross-reactive T cells (colored brown) with higher avidity for the earlier infection are
preferentially activated, while cells to the current infecting serotype (colored green) are killed by activation-
induced cell death. The low avidity T cells are unable to efficiently clear the infection and cause a cascade of
immune activation that ultimately results in plasma leakage.
        CD8+ Cells and OAS
• Normally, activated T cells have various
  responses to infection; proliferation, target cell
  lysis and production of cytokines, including IFNγ.
• In OAS, partial antigen match; T cells are not
  fully activated to respond to the invading DENV.
• Studied in Peripheral blood mononuclear cells
  (PBMCs) of DENV patients.
• HLA-A*11- found in ~30% of the population of
  Southeast Asia.
       CD8+ Cells and OAS
• Epitote for NS3 protein (FSP) found on
  HLA-A*11 and used to produce FSP
  specific Cytotoxic T Lymphocyte cell line.
• HLA restriction was confirmed using CR51
  release assays.
• FSP truncation identified minimal
  recognizable epitotes to be a 11a.a
  peptide (GTS) and a 10a.a peptide
  (10GTS).
      CD8+ Cells and OAS
• GTS variants made after searched of
  published NS3 sequences.
• PMBCs of acute infected children showed
  low or no elispot response to the GTS
  peptide during acute phase.
• Samples 2 weeks and 2 months later gave
  stronger responses.
        CD8+ Cells and OAS
• Phycoerythrin labelled Tetrameric MHC1
  complexes containing the GTS epitotes were
  used to stain the PBMCc. Gave similar results.
• In acute DENV infection GTS specific T cells
  showed an activated effector phenotype and
  were proliferative.
• The low frequency of antigen specific cells at
  early acute infection was linked to apoptosis as
  GTS specific T cells from the early phase
  showed high apoptosis (80%) compared to early
  phase tetramer negative cells (15%).
        CD8+ Cells and OAS
• In later phase cells (2 weeks and 2 months)
  small amount of both tetramer positive and
  negative CD8+ cells stained positive for
  apoptosis.
• The high apoptosis and proliferation at acute
  illness (early phase-day 2) indicates proliferation
  balanced by apoptosis in the acute setting.
• System balances out as virus is cleared.
  Apoptosis stops and memory cells produced.
        CD8+ Cells and OAS
• This indicated that the initial proliferation of
  CD8+ T cells were due to memory cells
  from the first infection.
• Apoptosis was induced on the activated
  cells by an unknown mechanism likely
  related to only partial recognition/activation
  by the secondary DENV infection epitote.
  (Mongkolsapaya et al. 2003).
        Antibody dependent
       enhancement and OAS
• Antibody-dependent enhancement (ADE)
  of infection occurs upon secondary
  infection with a different serotype of DENV
• Pre-existing non-neutralizing antibodies
  opsonize and enhance virus uptake and
  replication in FcR bearing monocytes and
  macrophages which are a major site of
  DENV replication in vivo
• (Dejnirattisai et al. 2010).
  ADE-Experimental Evidence
• FcR bearing cells were infected with
  DENV2 in presence of anti-E and anti-prM
  antibodies.
• Flow cytometry method were used to
  analyze the ADE of DENV infected cells
  using anti-NS1 or anti-E antibodies.
• Found ADE of infection occurred in dose
  dependent manner.
Flow cytometry based analysis of antibody treated FcR+ cells
infected with Dengue 2 . Cells stained with anti Dengue mAB
and strepavidin-FITC.




 FIGURE 4. The enhancement of dengue virus infection by anti-E or anti-prM Abs. A, P388D1
 cells were infected by DENV2 in the presence of various anti-dengue mAbs. B, The dose effect of anti-
 prM and anti-E Ab-mediated ADE infection. DC2.4 cells were infected by dengue-2 virus with various
 concentrations of anti-dengue mAbs. (Figure extracted from Huang et al.2006)
                       Both FcR+ and FcR- cells are susceptible to
                                     ADE




FIGURE 5. Anti-prM Ab enhanced the dengue virus infection on cells that bear FcR (A) or cells that do not have FcR
(B). The FO (myeloma),hybridoma 185-10 (anti-E Ab), H21F8-1 (anti-HBsAb), 206-35 (anti-NS1),B cell line BJAB, A20,
and dendritic cell or macrophage cell line DC2.4,P388D1, U937 were infected by dengue-2 virus for 24 h in the presence of
anti-prM Ab (10 ug/ml). Cells that do not bear FcR (BHK-21, A549, NIH3T3, C6/36, MRC5, Jurkat T) were infected by
dengue-2 virus for 24 h with different percentages of anti-prM Ab hybridoma culture supernatants. Dengue virus-infected
cells were quantitated by flow cytometric analysis with anti-NS1 Ab cytoplasmic staining..(Figure extracted from Huang et al
2006)
 M3 epitote of prM. Dual specific
 binding of DENV and target cells.
• M3 peptide was discovered to be an
  epitote to prM antibody binding region.
• M3 reduced anti-prM ADE of infection in a
  dose dependent manner.
• HSP60, a membrane protein was found to
  be the cause of ADE in FcR- A549 and
  BHK cells.
• Dual specific binding of DENV and target
  FcR- cells in ADE of infection.
FIGURE 6. Dual specificity of anti-prM Ab. A and B, Specific binding of anti-prM Ab to BHK-21 or A549 cells. BHK-21
or A549 cells were incubated with antiprM, anti-NS, or anti-E Ab (2ug) followed by staining with FITC-labeled goat anti-
mouse IgG secondary Ab. The binding was determined by flow cytometry or immunofluorescent staining. M3 peptide dose-
dependently inhibited the anti-prM Ab binding to BHK-21 cells. C, Anti-prM Ab recognized one of the self-proteins, HSP60.
Total cell lysates of BHK-21 or A549 membrane protein extracts were detected by anti-HSP60 Ab or anti-prM Ab on Western
blot (upper panel). The immunoprecipitated membrane proteins from BHK-21 orA549 cells by anti-HSP60 were Western
blotted with anti-prM Ab, after washing away, the membrane was re-probed with anti-HSP60 Ab (lower right panel).
DENV inhibition of IFN signaling.
• A Strategy for escaping host defenses.
• IFN produced by infected cells leads to
  activation of a classical INF signaling
  pathway called the Janus Kinase /signal
  transducer and activator of transcription
  (JAK/STAT) pathway.
Figure7- Interferon receptors and activation of
  classical JAK–STAT pathways by type I and
  type II interferons. All type I interferons
 (IFNs) bind the type I IFN receptor at the
 surface of human cells. The receptor Janus
 activated kinases (JAKs) tyrosine kinase 2
 (TYK2) and JAK1, respectively.
 IFN-γ binds the type II IFN receptor, which
 is associated with JAK1 and JAK2,
 respectively.
 Activation of the JAKs associated with the
 type I IFN receptor results in
 phosphorylationof STAT2 and STAT1; this
 leads to the formation of STAT1–STAT2–
 IRF9 (IFN-regulatory factor 9) complexes,
 which are known as ISGF3 (IFN-stimulated
 gene (ISG) factor 3)
 ISGF3 translocates to the nucleus and binds
 IFN-stimulated response elements (ISREs) in
 DNA to initiate gene transcription.
 Both type I and type II IFNs also induce the
 formation of STAT1–STAT1 homodimers
 that translocate to the nucleus and bind GAS
 (IFN-γ-activated site) elements that are
 present in the promoter of certain ISGs.
 (Figure extracted from Platanias.2005).
Figure8-Sensing Dengue Virus Infection by the Host Cell. (a) Recognition of DENV RNA by TLR7occurs in endosomal
compartments during early infection of dendritic cells, leading to IFNa/b\ production. Induction of IFNa/b through TLR7/8/9 is
mediated by the adaptor molecule myeloid differentiation primary response protein 88 (MyD88), resulting in downstream activation
of IRF7,IKKa/b/g and MAPK cascades, leading to activation of NFkB and AP-1. IRF7 activates IFNa and IFNb expression.
(b)TLR3, a broad sensor of dsRNA intermediates during viral infections, is upregulated in human umbilical endothelial cells
(HUVEC) by DENV infection. IRF3 leads to activation of IFNb. (c) Cytoplasmic sensing of DENV occurs through RIG-I and
MDA5, resulting in activation of a macromolecular signaling complex that stimulates IRF3; in turn this activation induces the IFNb
promoter. (d) Secreted IFNa/b activate the IFNa/b receptor and the JAK/STAT pathway, leading to phosphorylation and dimerization
of STAT1/2 and formation of the macromolecular factor ISGF3, which translocates to the nucleus and activates ISREs. The
nonstructural proteins NS4B, NS5 and to a lesser extent NS2A and NS4A, impair parts of the JAK/STATpathway and reduce
activation of ISREs. (Figure extracted from Munoz-Jordan. 2010)
 DENV NS4B protein blocks INF
         signaling
• NS4B interferes with STAT1 function.
• NS4A and NS2A do so to a lesser extent.
• Studied using HA tagged DENV proteins
  produced in vitro to test the ability of each of
  the10 protein to block IFN signaling.
• Used IFN sensitive Newcastle disease virus .
• NDV replication is inhibited in transfected CEF
  cell. Co-transfection with plasmids expressing
  IFN antagonists, such as the Influenza A virus
  NS1 protein, enhances NDV replication. Tried
  with DENV non-structural proteins.
NS4a, NS4B and NS2a DENV proteins inhibit IFN signaling in
CEFs and A549 cells.




Figure 9- Identification of DEN-2 proteins that facilitate NDV-GFP replication in the
presence of an IFN response. CEF (A) and A549 (B) cells were transfected with pCAGGS
plasmids expressing HA-tagged DEN-2 proteins (C, prM, E, NS1, NS2A, NS2B, NS3, NS4A,
NS4B, or NS5), influenza A virus NS1 (fNS1), empty pCAGGS (P), or untransfected (UT).
Twenty-four hours later, cells were infected with NDV-GFP, and green fluorescence was
visualized 24 h after infection under the fluorescence microscope.( Figure extracted from Munoz-
Jordan et al. 2003)
INF stimulated Response element -
             54 study.
• Probed ability of DENV proteins to block
  IFN induced by viral infection or IFN
  stimulated signal transduction.
• CAT reporter gene controlled by ISRE-54
  promoter used to monitor ISG expression.
• ISRE-54 was chosen because its viral
  induced activation is known to be
  mediated by IRF-3.
   RT-PCR analysis of IFN inhibition by DENV NS proteins.




Figure10- Inhibition of virus- and IFN--mediated induction of ISRE by DEN-2proteins. (A)
Fold induction of ISRE-54-CAT reporter gene on infection with SeV. 293T cells were transfected
with pCAGGS plasmids expressing the indicated DEN-2 proteins, pCAGGS-fNS1, or empty
pCAGGS (P), as well as with ISRE-54-CAT and pCAGGS-FL reporter plasmids. Twenty-four
hours posttransfection,cells were infected with SeV (moi of 2) (SeV) or mock-infected (SeV).
CAT and luciferase activities were measured 24 h later. The CAT activities were normalized to
the corresponding FL activities to determine the fold induction. (B) Fold induction of ISRE-54-
CAT reporter gene on treatment with IFN-. Vero cells were transfected with each of the indicated
plasmids as in A. As positive control pCAGGS-NipV was used. Twenty-four hours
posttransfection, cells were treated with 1,000 units of human IFN (IFN) or mock-treated (IFN).
CAT and luciferase activities were measured 24 h posttreatment (Figure extracted from Munoz-
Jordan et al.2003)
    ISRE 9-27 promoter study
• ISRE 9-27 promoter which is stimulated by
  IFN but not by virus mediated activation of
  IRFs.
• ISRE 9-27 signaling was reduced similar
  to the experiment with ISRE 54.
• Ability of NS4B to inhibit TNF alpha was
  tested. No effect found.
Figure 11-DEN-2 NS4B blocks IFN- signaling (A) Percentage of inhibition of CAT activity due to
expression of the indicated DEN-2 proteins in Vero cells transfected with reporter constructs and incubated
with human IFN-beta. (B) Vero cells were transfected with plasmids expressing DEN-2 NS4B or IBDN and a
reporter plasmid expressing FL from an NF-kB-responsive promoter. A plasmid expressing RL under the
control of the HSV TK promoter was cotransfected to normalize FL values. 24 hours posttransfection, cells
were incubated with TNF for 30 min, and FL and RL activities were measured. (Figure extracted from
Munoz-Jordan et al 2003).
 DENV inhibits INF signaling by
  reducing STAT2 expression
• Used human cell lines stably expressing
  self replicating sub-genomic dengue virus
  RNA (replicons) containing DENV
  nonstructural proteins.
• Mimics DENV infection.
• Replicon containing cells inhibited
  classical ISG genes MxA and PKR along
  with other ISGs.
  Figure12 Schematic showing construction of plasmid pDENCprME-PAC2A. (A) Dengue virus type 2
  infectious clone cDNA (in plasmid pDVWS601 (B) pDENCprME. A large in-frame deletion was introduced
  within the region carrying the structural genes. (C) pDENCprME-PAC2A. An antibiotic selection cassette
  encoding PAC and the foot-and-mouth disease virus protein 2A was cloned in place of the deleted structural
  genes. (Figure extracted from Jones et al. 2005)




Figure12- Induction of classical ISGs by IFN- is inhibited in dengue virus replicon-containing cells.
K562 cells (black bars) and K562.CprME-PAC2A cells (white bars) were stimulated with 100 IU of IFN for 6
and 24 h. MxA (A) and PKR (B) gene transcription was measured by real-time PCR and normalized to
GAPDH. .(Figure extracted from Jones et al. 2005)
  DENV reduces STAT2 levels
• STAT1-P was seen by immunobloting to
  be reduced in replicon containing cells
  following 30’ treatment with IFNα.
• STAT2 steady state levels were markedly
  reduced in response to IFNα.
• NO reduction STAT1P after treatment with
  INF gamma, which acts independently of
  STAT2.
Figure 13. Dengue virus RNA replication inhibits STAT1 and STAT2phosphorylation in response to
IFN- and reduces steady-state levels of STAT2. (A) K562, K562.CprME-PAC2A, and cured K562 cells;
(B)THP-1 and THP-1.CprME-PAC2A cells. Cells were left untreated or treated with 100 IU of IFNa or
IFNg per ml for 30 min and then lysed in SDS loading buffer. Proteins were separated by SDS-PAGE and
thenanalyzed by immunoblotting with specific antibodies for STAT1, phosphorylatedSTAT1, STAT2, and
phosphorylated STAT2, as indicated.(C) Dengue virus infection reduces STAT2 levels. K562 cells were
infected with dengue virus for 48 h. Cell lysates were separated by SDS-PAGE and then analyzed by
immunoblotting with specific antibodies for dengue virus NS1, STAT1, and STAT2, as indicated. Mock-
infected cells were included for comparison
(Figure extracted from Jones et al.2005)
  NS5 mediates STAT2 binding
       and degradation.
.
• Studied Interactions of NS5 and STAT2
  using tagged recombinant proteins.
• 293 cells were co-transfected with
  plasmids expressing FLAG tagged STAT-
  1 + 2 and HA tagged NS5 or empty vector.
• Found NS5 specifically interacts with
  STAT2.
Figure14- DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-
tagged STAT1(STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid empty) or plasmid expressing HA-
tagged DENV NS5 (NS5-HA),DENV core (CORE-HA), or NiV-V (HA–NiV-V) proteins. Lysates were then
immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-
FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were
cotransfected with plasmids expressingNS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then
immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and
anti-FLAG antibodies.TCE, total cell extracts were subjected to Western blotting
using anti-HA, anti-FLAG, and anti-GAPDH antibodies. (Figure extracted from Ashour et al 2009).
      STAT2 degradation requires
         proteasomal activity
• Proteasomal activity is used by cells for many protein
  degrading functions such as recycling of older proteins.
• Inhibitors of the proteasomal degradation pathway were
  used to probe STAT2 degradation by NS5.
• Vero cells expressing NS5 replicons were treated with
  proteasomal inhibitors.
• Some cells contransfected with uncleaved NS5-HA and
  NS2B-3 to show requirement of a cleaved mature NS5
  for STAT2 effect.
• STAT2 not degraded and ubiquitin accumulated.
              STAT2 degradation requires proteasomal activities.




Figure 15- Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells
stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed
and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were
transfected with HA-ubiquitin, STAT2- FLAG, STAT1-GFP, NS2b-3, and either E-clvNS5-HA or an empty vector plasmid. Cells were
then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA,
and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3–HA and the plasmids indicated at the top. Ten hours
posttransfection, cells were treated with the indicated amounts of lactacystin.Twenty-four hours posttransfection, cells were sorted for
GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific
antibodies.(Figure extracted from Ashour et al. 2009)
               Summary
• DENV is increasing in incidence and is of
  concern to public health.
• DENV pathogenicity may be enhanced by
  OAS of CTL and ADE.
• DENV uses many methods to evade
  antiviral IFN responses.
• NS4A inhibition of STAT1P and NS5B
  degradation of STAT2.
???Questions???
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