Dengue virus non-structural protein 3 inhibits mitochondrial respiration by impairing complex I function.

Dengue virus (DENV) infection is known to affect host cell metabolism, but the molecular players involved are still poorly known. Using a proteomics approach, we identified six DENV proteins associated with mitochondria isolated from infected hepatocytes, and most of the peptides identified were from NS3. We also found an at least twofold decrease of several electron transport system (ETS) host proteins. Thus, we investigated whether NS3 could modulate the ETS function by incubating recombinant DENV NS3 constructs in mitochondria isolated from mouse liver. We found that NS3pro (NS3 protease domain), but not the correspondent catalytically inactive mutant (NS3proS135A), impairs complex I (CI)-dependent NADH:ubiquinone oxidoreductase activity, but not the activities of complexes II, III, IV, or V. Accordingly, using high-resolution respirometry, we found that both NS3pro and full-length NS3 decrease the respiratory rates associated with malate/pyruvate oxidation in mitochondria. The NS3-induced impairment in mitochondrial respiration occurs without altering either leak respiration or mitochondria's capacity to maintain membrane potential, suggesting that NS3 does not deeply affect mitochondrial integrity. Remarkably, CI activity is also inhibited in DENV-infected cells, supporting that the NS3 effects observed in isolated mitochondria may be relevant in the context of the infection. Finally, in silico analyses revealed the presence of potential NS3 cleavage sites in 17 subunits of mouse CI and 16 subunits of human CI, most of them located on the CI surface, suggesting that CI is prone to undergo proteolysis by NS3. Our findings suggest that DENV NS3 can modulate mitochondrial bioenergetics by directly affecting CI function. IMPORTANCE: Dengue virus (DENV) infection is a major public health problem worldwide, affecting about 400 million people yearly. Despite its importance, many molecular aspects of dengue pathogenesis remain poorly known. For several years, our group has been investigating DENV-induced metabolic alterations in the host cells, focusing on the bioenergetics of mitochondrial respiration. The results of the present study reveal that the DENV non-structural protein 3 (NS3) is found in the mitochondria of infected cells, impairing mitochondrial respiration by directly targeting one of the components of the electron transport system, the respiratory complex I (CI). NS3 acts as the viral protease during the DENV replication cycle, and its proteolytic activity seems necessary for inhibiting CI function. Our findings uncover new nuances of DENV-induced metabolic alterations, highlighting NS3 as an important player in the modulation of mitochondria function during infection.

Dengue Virus dependence on glucokinase activity and glycolysis Confers Sensitivity to NAD(H) biosynthesis inhibitors.

Viruses have developed sophisticated strategies to control metabolic activity of infected cells in order to supply replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, underlying molecular mechanisms are still poorly characterized and dependence of DENV on this pathway has not been investigated in details yet. Here, we identified an interaction between the non-structural protein 3 (NS3) of DENV and glucokinase regulator protein (GCKR), a host protein that inhibits the liver-specific hexokinase GCK. NS3 expression was found to increase glucose consumption and lactate secretion in hepatic cell line expressing GCK. Interestingly, we observed that GCKR interaction with GCK decreases DENV replication, indicating the dependence of DENV to GCK activity and supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, in the same cells, DENV replication both induces and depends on glycolysis. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), we decreased cellular glycolytic activity and inhibited DENV replication in hepatic cells. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6-AN. Altogether, our results show that DENV has evolved strategies to control glycolysis in the liver, which could account for hepatic dysfunctions associated to infection. Besides, our findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.

Bloodmeals fuel dengue virus replication in the female mosquito Aedes aegypti.

Vector competence defines the ability of a vector to acquire, host, and transmit a pathogen. Understanding the molecular determinants of the mosquitos' competence to host dengue virus (DENV) holds promise to prevent its transmission. To this end, we employed RNA-seq to profile mRNA transcripts of the female Aedes aegypti mosquitos feeding on naïve vs viremic mouse. While most transcripts (12,634) did not change their abundances, 360 transcripts showed decreases. Biological pathway analysis revealed representatives of the decreased transcripts involved in the wnt signaling pathway and hippo signaling pathway. One thousand three hundred fourteen transcripts showed increases in abundance and participate in 21 biological pathways including amino acid metabolism, carbon metabolism, fatty acid metabolism, and oxidative phosphorylation. Inhibition of oxidative phosphorylation with antimycin A reduced oxidative phosphorylation activity and ATP concentration associated with reduced DENV replication in the Aedes aegypti cells. Antimycin A did not affect the amounts of the non-structural proteins 3 and 5, two major components of the replication complex. Ribavirin, an agent that reduces GTP concentration, recapitulated the effects of reduced ATP concentration on DENV replication. Knocking down one of the oxidative phosphorylation components, ATP synthase subunit ß, reduced DENV replication in the mosquitos. In summary, our results suggest that DENV enhances metabolic pathways in the female Aedes aegypti mosquitos to supply nutrients and energy for virus replication. ATP synthase subunit ß knockdown might be exploited to reduce the mosquitos' competence to host and transmit DENV. IMPORTANCE: Through evolution, the mosquito-borne viruses have adapted to the blood-feeding behaviors of their opportunist hosts to fulfill a complete lifecycle in humans and mosquitos. Disruption in the mosquitos' ability to host these viruses offers strategies to prevent diseases caused by them. With the advent of genomic tools, we discovered that dengue virus (DENV) benefited from the female mosquitos' bloodmeals for metabolic and energetic supplies for replication. Chemical or genetic disruption in these supplies reduced DENV replication in the female mosquitos. Our discovery can be exploited to produce genetically modified mosquitos, in which DENV infection leads to disruption in the supplies and thereby reduces replication and transmission. Our discovery might be extrapolated to prevent mosquito-borne virus transmission and the diseases they cause.

Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein.

Severe dengue infections are characterized by endothelial dysfunction shown to be associated with the secreted nonstructural protein 1 (sNS1), making it an attractive vaccine antigen and biotherapeutic target. To uncover the biologically relevant structure of sNS1, we obtained infection-derived sNS1 (isNS1) from dengue virus (DENV)-infected Vero cells through immunoaffinity purification instead of recombinant sNS1 (rsNS1) overexpressed in insect or mammalian cell lines. We found that isNS1 appeared as an approximately 250 kDa complex of NS1 and ApoA1 and further determined the cryoEM structures of isNS1 and its complex with a monoclonal antibody/Fab. Indeed, we found that the major species of isNS1 is a complex of the NS1 dimer partially embedded in a high-density lipoprotein (HDL) particle. Crosslinking mass spectrometry studies confirmed that the isNS1 interacts with the major HDL component ApoA1 through interactions that map to the NS1 wing and hydrophobic domains. Furthermore, our studies demonstrated that the sNS1 in sera from DENV-infected mice and a human patient form a similar complex as isNS1. Our results report the molecular architecture of a biological form of sNS1, which may have implications for the molecular pathogenesis of dengue.

Role of angiotensin II in cellular entry and replication of dengue virus.

Previous studies have demonstrated the relevance of several soluble molecules in the pathogenesis of dengue. In this regard, a possible role for angiotensin II (Ang II) in the pathophysiology of dengue has been suggested by the observation of a blockade of Ang II in patients with dengue, increased expression of molecules related to Ang II production in the plasma of dengue patients, increased expression of circulating cytokines and soluble molecules related to the action of Ang II, and an apparent relationship between DENV, Ang II effects, and miRNAs. In addition, in ex vivo experiments, the blockade of Ang II AT1 receptor and ACE-1 (angiotensin converting enzyme 1), both of which are involved in Ang II production and its function, inhibits infection of macrophages by DENV, suggesting a role of Ang II in viral entry or in intracellular viral replication of the virus. Here, we discuss the possible mechanisms of Ang II in the entry and replication of DENV. Ang II has the functions of increasing the expression of DENV entry receptors, creation of clathrin-coated vesicles, and increasing phagocytosis, all of which are involved in DENV entry. This hormone also modulates the expression of the Rab5 and Rab7 proteins, which are important in the endosomal processing of DENV during viral replication. This review summarizes the data related to the possible involvement of Ang II in the entry of DENV into cells and its replication.

The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1ß in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1ß. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.

Molecular mechanisms in the pathogenesis of dengue infections.

Dengue is the most rapidly emerging climate-sensitive infection, and morbidity/mortality and disease incidence are rising markedly, leading to healthcare systems being overwhelmed. There are currently no specific treatments for dengue or prognostic markers to identify those who will progress to severe disease. Owing to an increase in the burden of illness and a change in epidemiology, many patients experience severe disease. Our limited understanding of the complex mechanisms of disease pathogenesis has significantly hampered the development of safe and effective treatments, vaccines, and biomarkers. We discuss the molecular mechanisms of dengue pathogenesis, the gaps in our knowledge, and recent advances, as well as the most crucial questions to be answered to enable the development of therapeutics, biomarkers, and vaccines.

E-Protein Protonation Titration-Induced Single-Particle Chemical Force Spectroscopy for Microscopic Understanding and pI Estimation of Infectious DENV.

The ionization state of amino acids on the outer surface of a virus regulates its physicochemical properties toward the sorbent surface. Serologically different strains of the dengue virus (DENV) show different extents of infectivity depending upon their interactions with a receptor on the host cell. To understand the structural dependence of E-protein protonation over its sequence dependence, we have followed E-protein titration kinetics both experimentally and theoretically for two differentially infected dengue serotypes, namely, DENV-2 and DENV-4. We have performed E-protein protonation titration-induced single-particle chemical force spectroscopy using an atomic force microscope (AFM) to measure the surface chemistry of DENV in physiological aqueous solutions not only to understand the charge distribution dynamics on the virus surface but also to estimate the isoelectric point (pI) accurately for infectious dengue viruses. Cryo-EM structure-based theoretical pI calculations of the DENV-2 surface protein were shown to be consistent with the evaluated pI value from force spectroscopy measurements. We also highlighted here the role of the microenvironment around the titrable residues (in the 3D-folded structure of the protein) in altering the pKa. This is a comprehensive study to understand how the cumulative charge distribution on the outer surface of a specific serotype of DENV regulates a prominent role of infectivity over minute changes at the genetic level.

A mosquito salivary protein-driven influx of myeloid cells facilitates flavivirus transmission.

Mosquitoes transmit many disease-relevant flaviviruses. Efficient viral transmission to mammalian hosts requires mosquito salivary factors. However, the specific salivary components facilitating viral transmission and their mechanisms of action remain largely unknown. Here, we show that a female mosquito salivary gland-specific protein, here named A. aegypti Neutrophil Recruitment Protein (AaNRP), facilitates the transmission of Zika and dengue viruses. AaNRP promotes a rapid influx of neutrophils, followed by virus-susceptible myeloid cells toward mosquito bite sites, which facilitates establishment of local infection and systemic dissemination. Mechanistically, AaNRP engages TLR1 and TLR4 of skin-resident macrophages and activates MyD88-dependent NF-κB signaling to induce the expression of neutrophil chemoattractants. Inhibition of MyD88-NF-κB signaling with the dietary phytochemical resveratrol reduces AaNRP-mediated enhancement of flavivirus transmission by mosquitoes. These findings exemplify how salivary components can aid viral transmission, and suggest a potential prophylactic target.

Mutation of conserved histidine residues of dengue virus envelope protein impairs viral like particle maturation and secretion.

Dengue virus (DENV) envelope protein plays crucial role in virus entry and maturation of virus during infection. Maturation of DENV occurs in the trans Golgi network at slightly acidic pH which is close to pKa of histidine. When exposed to the acidic environment of the late secretory pathway, dengue virus particles go through a significant conformational change, whereby interactions of structural proteins envelope (E) and prM proteins are reorganised and enable furin protease to cleave prM resulting in mature virus. In order to study the role of histidine of E protein in DENV maturation, we mutated 7 conserved histidine residues of envelope protein and assessed the percent of budding using viral like particle (VLP) system. Histidine mutants; H144A, H244A, H261A and H282A severely disrupted VLP formation without any significant change in expression in cell and its oligomerization ability. Treatment with acidotropic amine reversed the defect for all 4 mutants suggesting that these histidines could be involved in maturation and release. Over expression of capsid protein slightly enhanced VLP release of H244A and H261A. Similarly, furin over expression increased VLP release of these mutants. Co-immunoprecipitation studies revealed that prM and E interaction is lost for H244A, H261A and H282A mutants at acidic pH but not at neutral pH indicating that they could be involved in histidine switch during maturation at acidic pH. Detailed analysis of the mutants could provide novel insights on the interplay of envelop protein during maturation and aid in target for drug development.

Host serine protease ACOT2 assists DENV proliferation by hydrolyzing viral polyproteins.

Dengue fever is a mosquito-borne tropical disease caused by the dengue virus (DENV). The replication of DENV relies on the processing of its genome-encoded polyprotein by both viral protease NS3 (NS3pro) and host proteases. However, the impact of host proteases on DENV proliferation is not well understood. In this study, we utilized fluorophosphonate-based probes (FPs) to investigate the up-regulation of host serine proteases during DENV infection in detail. Among the identified proteases, acyl-CoA thioesterase 2 (ACOT2), an enzyme that hydrolyzes acyl-CoA molecules to generate fatty acids and free CoA, exhibited cleavage activity against DENV polypeptide substrates. Enzymatic assays and virological experiments confirmed that ACOT2 contributes to DENV propagation during the replication stage by cleaving the viral polyprotein. Docking models provided insights into the binding pocket of viral polypeptides and the catalytic mechanism of ACOT2. Notably, this study is the first to demonstrate that ACOT2 functions as a serine protease to hydrolyze protein substrates. These findings offer novel insights into DENV infection, host response, as well as the potential development of innovative antiviral strategies.IMPORTANCEDENV, one of the major pathogens of Dengue fever, remains a significant public health concern in tropical and subtropical regions worldwide. How DENV efficiently hijacks the host and accesses its life cycle with delicate interaction remains to be elucidated. Here, we deconvoluted that the host protease ACOT2 assists the DENV replication and characterized the ACOT2 as a serine protease involved in the hydrolysis of the DENV polypeptide substrate. Our results not only further the understanding of the DENV life cycle but also provide a possibility for the usage of activity-based proteomics to reveal host-virus interactions.

Platelet derived exosomes disrupt endothelial cell monolayer integrity and enhance vascular inflammation in dengue patients.

Background: Thrombocytopenia is the most notable phenomenon in dengue. Activation status of platelets and interaction of platelets with endothelium contribute towards dengue disease pathogenesis. Platelets are the major cell types known to release extracellular vesicles, especially exosomes in circulation. However, the role of platelet derived exosomes (PLT-EXOs) in endothelial dysfunction during dengue infection remains unknown. Methods: In this study, we recruited 28 healthy subjects and 69 dengue patients categorized as WS- (n=31), WS+ (n=29) and SD (n=9). Platelets were isolated from platelet rich plasma of dengue patients and their activation was assessed by flow cytometry. PLT-EXOs were isolated by ultracentrifugation method. Western blot analyses were performed to characterize the exosomes. Exosome uptake experiment was carried out to see the internalization of exosomes inside endothelial cells (HUVECs). To observe the effect of exosomes on endothelial cells, exosomes were added on HUVECs and expression of adherens and tight junctional proteins were examined by immunofluorescence assay and western blot. Expression levels of vascular injury markers were measured in the culture supernatants of Exosome-HUVEC coculture and sera of dengue patients by MSD-multiplex assay. Results: As compared to healthy subjects, CD41/CD61 expression was significantly reduced (p<0.0001) and CD62p expression was significantly increased (p<0.0001) on platelets in dengue patients. PLT-EXOs isolated from the dengue patients showed higher expression of CD63 and CD9 proteins than the healthy subjects. With in-vitro immunofluorescence assays, we illustrated the internalization of PLT-EXOs by the HUVECs and observed disruption of endothelial cell monolayer integrity in the presence of PLT-EXOs from WS+ and SD patients. Furthermore, the significant reduction in the expressions of ZO-2, VE-Cadherin and CD31 in endothelial cells following exposure to PLT-EXOs from the dengue patients provide direct evidence of PLT-EXOs mediated vascular permeability. PLT-EXOs stimulated the release of inflammatory markers CRP, SAA, sVCAM-1 and sICAM-1 in the supernatants of HUVEC cells. Importantly, significantly higher levels of CRP, sVCAM-1 and sICAM-1 in the sera of severe than mild dengue patients (p<0.0001) suggest their role in disease severity. Conclusions: In summary, our data suggest that PLT-EXOs promote vascular leakage via release of proinflammatory mediators and compromise vascular barrier integrity in dengue patients.

Sentencia sobre responsabilidad empresarial en un caso de trabajador con enfermedad de Dengue / Legal judgment about company responsability and a case of worker with Dengue disease

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Oxido nitrico en la patologia del dengue / Nitric oxide in dengue pathology

El óxido nítrico (ON) es una molécula pleyotropica, que interviene tanto en procesos citoprotectivos como citotóxicos, su síntesis debe estar altamente regulada por la célula, ya que la alteración en su producción esta asociada a una gran variedad de patologías, entre las que se encuentran el shock séptico, anafiláctico y hemorrágico. La infección con el virus dengue produce una enfermedad que clínicamente presenta un espectro que va desde un polo benigno conocido como fiebre dengue, hasta un polo severo denominado fiebre hemorrágica que puede progresar y producir el síndrome de shock por dengue, que en la mayoría de los casos determina la muerte del paciente. En el presente trabajo se discuten evidencias que involucran al óxido nítrico en la patología del polo severo de la enfermedad producida por este virus.

Dengue fever with thrombocytopenia: studies towards defining vulnerability of bleeding

PURPOSE: To define the period of greater vulnerability of bleeding in patients with Dengue fever in reference to the onset of their constitutional symptoms and the laboratory abnormalities. PATIENTS AND METHODS: In a retrospective study we reviewed the records of all patients admitted to San Pablo Medical Center in 1991 with a diagnosis of Dengue Fever or Hemorrhagic Dengue. All patients with a platelet count of less than 125,000 were included for analysis. The exclusion criteria included the presence of systemic disorders which may influence the platelet count, and patients without documentation regarding the presence of constitutional symptoms suggestive of viral illness. RESULTS: A total of 101 patients were analyzed of which only 74 were included in the study. All patients had fever and chills; skin rash, asthenia and general malaise was seen in over 50 of patients. Over 70 of patients had recovery of their platelet count, and most had their maximal thrombocytopenia within the 5th day and 8th day from the onset of constitutional symptoms. Leukopenia was seen in over 70 of patients with its lowest level within the 5th and 8th day from the onset of the constitutional symptoms. Significantly prolonged partial thromboplastin time was seen in 11 of the patients. Proteinuria was seen in 22 of the patients, 38 of which had it within the first 4 days of the onset of constitutional symptoms and also noted on the 5th and 6th day. Alteration in liver enzymes were noted in 47 of patients, with a maximal severity distributed in all time frames. Hypoalbuminemia was present in 28 of the patients, of these 67 presented within the first 4 days from the onset of constitutional symptoms. The pulse rate was usually normal in spite of the patient's dehydration and fever. CONCLUSIONS: We identified three phases that define the sequence of events seen in the majority of patients with Dengue Fever and Thrombocytopenia. These are: 1. proteinuria and hypoalbuminemia; 2. maximal cytopenia; 3. bradycardia and liver enzyme elevation. We believe this information is useful in the management of patients with Dengue Fever and thrombocytopenia