Panagiota Kaimou's research while affiliated with Pasteur Hellenic Institute and other places

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Figure 1. Schematic representation of virus constructs used in this study. From top to bottom: DENV2 full-length genome; DVR2A, derived from the DENV2 full-length genome by insertion of a Renilla luciferase (R-Luc) coding sequence downstream of the capsid cyclization sequence (CAE) and upstream of a Tosea asigna virus 2A protease cleavage site; sgDVR2A, a subgenomic reporter replicon derived from the DENV2 full-length genome by insertion of R-Luc coding sequence in-between CAE and the 2A cleavage site sequence. The last 24 amino acid residues of the envelope coding region (TM) at the Nterminus of NS1 ensure proper membrane topology of the polyprotein. sgDVR2A-GND, a replicationdeficient NS5 mutant of sgDVR2A. All DENV constructs are derived from the DVs2 16681 isolate. JcR2a, a Jc1 (J6CF-JFH1 chimera) derivative containing the R-Luc gene fused N-terminally to 16 codons of the core gene (C) and C-terminally to the FMDV 2A protease cleavage site (grey striped box); Black bars in all panels indicate UTRs. Polyprotein cleavage products are labeled as specified in the introduction.
Figure 3. Low oxygen tension selectively enhances DENV RNA replication. (A) Huh7 cells preincubated at 20% or 3% O2 for 18 h were inoculated with DVR2A (MOI = 1) and lysed at the specified time-points post inoculation. Luciferase activity is expressed as RLU/µg of total protein amount and values obtained with 20% O2 cells were set for each time point to one. (B) Hypoxia does not influence viral entry. RT-qPCR analysis of intracellular DENV positive (+) strand RNA copies from Huh7 cells that were inoculated with DENV at MOI = 1 and incubated for 1 h as specified. 20→3*% O2 refers to cells that were preincubated at 20% O2 and transferred immediately after virus inoculation from 20% to 3% O2. Negative (−) strand RNA was quantified in order to indicate the absence of viral replication at 1 h post-inoculation. Values are expressed relative to the positive-strand RNA obtained at 20→20% O2. (C,D) Hypoxia increases viral RNA replication but not translation. Huh7 cells preincubated at 20% or 3% O2 for 18 h, were electroporated (5
Figure 4. (A,B) Hypoxia induces transcriptomic and metabolic reprogramming in DENV-infected cells. (A) RT-qPCR analysis of VEGFA, GLUT1, HK2, and LDH mRNA in DENV-infected (MOI = 0.5) or noninfected Huh7 cells incubated at the specified oxygen conditions and lysed at the indicated time points p.i. YWHAZ mRNA levels were used for normalization. Values obtained with infected cells at 20% O2 and lysed 8 h.p.i. were set to one. Mean values from three independent experiments are presented. * p < 0.001 vs. 20% O2 cells, # p < 0.01 vs. NI cells (Student's t-test). (B) Intracellular ATP levels of Huh7 DVR2Ainfected (MOI 0.1) and non-infected cells, incubated at the specified oxygen conditions. Cells were lysed at the indicated time points p.i. and intracellular ATP levels were expressed as RLU/µg of total protein amount. Values from cells cultured at 20% O2 at each time-point were set to 100. * p < 0.01, ** p < 0.001 vs. 20% O2 cells. (C,D) Association between glucose metabolism and low oxygen-mediated DENV
Figure 5. HIF upregulation enhances DENV replication. (A) Western blot analysis of DENV NS3 (top), HIF-1α (middle), and β-actin (bottom) of DENV-infected (MOI 0.5) or non-infected cells, incubated as specified on top of each lane and lysed at 24 h p.i. β-actin served as a loading control. A representative experiment of 3 independent repetitions is shown. (B) Activation of HRE (hypoxia response element) by low oxygen. Huh7 cells were transfected with the 9×HRE-Luc construct (0.4 µg/4 × 10 4 cells), 4 h posttransfection inoculated with DENV (MOI 0.5) for 4 h and further incubated at 20% or transferred to 3% O2 for 24 or 48 h p.i. HRE-dependent F-Luc activity was measured and expressed as RLU/µg of total protein amount. Values obtained from cells incubated at 20% O2 were set to one each time. * p < 0.001 vs. 20% O2 cells (Student's t-test). (C,D) Chemically-induced hypoxia stabilizes HIF-1α and enhances DENV replication. (C) (Top) Western blot analysis of HIF-1α (top) DENV NS3 (middle) and β-actin (bottom) of Huh7 cells inoculated with DENV (MOI 0.5) for 4 h, and subsequently treated, for 24 h, with CoCl2 (75 µM), DFO (37.5 µM), or DMOG (62.5 µM), as specified on top of each lane. β-actin served as loading control. A representative experiment is shown. C: control non-treated cells. (Bottom) Quantification of NS3 signals from 3 independent experiments, normalized to the β-actin loading control was performed and mean values are expressed relative to that obtained from control cells. (D) Luciferase activity obtained with Huh7 cells transfected with the 9×HRE-Luc construct (left) or infected with DVR2A (MOI = 0.01,
Figure 6. HIF downregulation impairs the replication of DENV but not HCV. (A-B) Huh7 cells were cotransfected with a mixture of siRNAs targeting HIF-1α and HIF-2α (20 nM each) or a control siRNA (40

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The Role of Tissue Oxygen Tension in Dengue Virus Replication
  • Article
  • Full-text available

December 2018

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201 Reads

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25 Citations

Cells

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Panagiota Kaimou

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Maria Moraiti

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Low oxygen tension exerts a profound effect on the replication of several DNA and RNA viruses. In vitro propagation of Dengue virus (DENV) has been conventionally studied under atmospheric oxygen levels despite that in vivo, the tissue microenvironment is hypoxic. Here, we compared the efficiency of DENV replication in liver cells, monocytes, and epithelial cells under hypoxic and normoxic conditions, investigated the ability of DENV to induce a hypoxia response and metabolic reprogramming and determined the underlying molecular mechanism. In DENV-infected cells, hypoxia had no effect on virus entry and RNA translation, but enhanced RNA replication. Overexpression and silencing approaches as well as chemical inhibition and energy substrate exchanging experiments showed that hypoxia-mediated enhancement of DENV replication depends on the activation of the key metabolic regulators hypoxia-inducible factors 1α/2α (HIF-1α/2α) and the serine/threonine kinase AKT. Enhanced RNA replication correlates directly with an increase in anaerobic glycolysis producing elevated ATP levels. Additionally, DENV activates HIF and anaerobic glycolysis markers. Finally, reactive oxygen species were shown to contribute, at least in part through HIF, both to the hypoxia-mediated increase of DENV replication and to virus-induced hypoxic reprogramming. These suggest that DENV manipulates hypoxia response and oxygen-dependent metabolic reprogramming for efficient viral replication.

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... Because enhanced microbial activity may reduce O 2 concentrations at the animal-water interface, it is possible that suboxic conditions may lead to viral proliferation. Indeed, several viruses replicate faster under hypoxic host cell conditions in culture by hijacking hypoxia inducible factors (Vassilaki and Frakolaki, 2017;Frakolaki et al., 2018). Whether enhanced viral load enhances the effects of hypoxic stress on tissues or through buildup of toxic compounds under suboxic conditions (Alessandra et al., 2003) remains unclear. ...

Reference:

Dynamics of the Apostichopus californicus-associated flavivirus under suboxic conditions and organic matter amendment
The Role of Tissue Oxygen Tension in Dengue Virus Replication

Cells

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Panagiota Kaimou

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Maria Moraiti

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[...]

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