INTRODUCTION
Dengue virus (DENV), a member of the
Evidence suggests that DENV infection notably impacts mitochondrial metabolic pathways in the host cell. For instance, fatty acid β-oxidation is induced in the infected cells to provide the energy needed for optimal viral particle production (3–6). In agreement, we have previously demonstrated that, in DENV-infected hepatocytes, glucose is mobilized for anaplerosis to favor mitochondria’s capacity for oxidizing fatty acids, while glutamine oxidation is inhibited (7). However, although we have shown that different parameters of mitochondrial bioenergetics are affected during DENV infection in distinct cellular models (2, 7–10), it was still unknown whether infection could directly impact the activity of mitochondrial respiratory complexes at a molecular level.
Here, we performed a proteomic screening in isolated mitochondria from DENV-infected hepatocytes. The analyses showed six DENV proteins associated with the mitochondria, with most of the peptides identified belonging to NS3. DENV NS3 is a multi-functional protein with approximately 70 kDa, formed by three structural domains comprising different enzymatic activities, namely the N-terminal trypsin-like serine protease domain (NS3pro), the nucleoside triphosphatase (NTPase) domain, and the C-terminal helicase and RNA 5′-triphosphatase domains (11–14). Thus, besides processing the viral polyprotein, NS3 participates in viral RNA replication (unwinding step) and capping. Additionally, it has been shown that NS3 interacts with and stimulates the activity of fatty acid synthase (FASN), redistributing FASN to sites of viral replication and increasing fatty acid synthesis locally (15, 16). Furthermore, it has recently been shown that DENV NS2B-NS3 migrates to the nucleus, where it cleaves EDRF1, a transcriptional factor involved in platelet formation (17). At the same time, the NS3pro (without the NS2B cofactor) is imported into the mitochondrial matrix, where it cleaves the protein GrpEL1, a mitochondrial Hsp70 co-chaperon (17, 18). All these findings show that DENV NS3 plays multiple key roles in viral replication, being considered one of the most attractive targets for DENV antiviral therapy (19).
In this context, we investigated the mitochondrial bioenergetics and the specific function of the electron transport system (ETS) protein complexes in isolated mouse liver mitochondria incubated with recombinant DENV NS3 protein constructs. We found that NS3 impairs complex I (NADH:ubiquinone oxidoreductase) activity likely in a protease-dependent manner, but not complex II (succinate dehydrogenase), complex III (ubiquinone/cytochrome
RESULTS
DENV NS3 localizes in the mitochondria of infected cells
We and others have shown that DENV infection affects mitochondrial function in the host cells, altering the preference for oxidizing energetic substrates in this organelle (2, 7, 8). To better understand the viral molecular players that mediate these effects, we performed a proteomic analysis of mitochondria isolated from DENV-infected Huh7 cells. In this screening, we identified peptides from six DENV proteins in the mitochondria preparation, namely E, NS1, NS2A, NS3, NS4A, and NS5 (Table 1). Most of the viral peptides identified by mass spectrometry belonged to NS3 (44.4%), which covered 46.6% of the whole protein, spanning both protease and helicase domains (Fig. 1A and B). We also noticed that about half of the proteins that appear in lower abundance in the mitochondria of infected cells (at least twofold compared to mock control) were related to metabolism, according to the function classification in the UniProt database (Fig. 1C). Of these, one-third belonged to the mitochondrial electron transport system (Fig. 1C; Table S1). Thus, we hypothesized that NS3 could directly interfere with the mitochondrial respiration of the host cells.
Fig 1
Proteomics screening of isolated mitochondria from DENV-infected Huh7 cells. (A) Pie chart showing the percentage of DENV peptides identified by mass spectrometry. (B) NS3 sequence, indicating the coverage of the peptides identified by mass spectrometry (highlighted in gray), which corresponded to 46.60% of the protein (288 out of 618 aa). Uniprot information: Entry: P29990; Entry name: POLG_DEN26; Protein name: Genome polyprotein; Organism: Dengue virus type 2 (strain Thailand/16681/1984) (DENV-2); Length: 3391 aa. (C) Pie charts with the downregulated proteins (at least twofold) in the mitochondria of DENV-infected cells relative to mock, categorized by function (according to the UniProt database). ETS, electron transport system.
NS3pro, but not NS3proS135A, inhibits CI activity
To investigate whether DENV NS3 interferes with the function of respiratory complexes, here we used three constructs of the recombinant NS3: the protease domain (NS3pro, residues 1–180); a mutant of this domain containing the serine residue of the catalytic site replaced by an alanine residue (NS3proS135A); and the full-length protein (NS3prohel, residues 1–618) (Fig. S1A through C). Since the NS3 protease domain was shown to be imported into mitochondria more efficiently than the full-length protein (18), we used NS3pro and the catalytically inactive mutant in our first set of experiments, whose results were then confirmed using the full-length protein or in the context of infection. Although it has been shown that the
To address whether NS3pro could directly impair the activity of each ETS complex, we incubated isolated mitochondria from mouse liver with recombinant NS3pro or NS3proS135A for 1 h on ice before assaying the activities of complex I (CI; NADH:ubiquinone oxidoreductase), complex II (CII; succinate dehydrogenase), complex III (CII; ubiquinone/cytochrome
To measure CI activity, we provided NADH (electron donor) and ubiquinone Q1 (electron acceptor) to the mitochondria preparation and measured the rotenone (CI inhibitor)-dependent NADH consumption photometrically (Fig. 2A). Our results showed that mitochondria incubation with NS3pro decreases NADH-ubiquinone oxidoreductase activity in a dose-dependent manner, reaching significant inhibition rates of 26.9% (S.D. ± 6.4%,
Fig 2
NS3pro inhibits CI, but not CII, CIII, CIV, or CV activities. Protein extracts from isolated mitochondria pre-incubated with 300 nM NS3pro or NS3proS135A (when indicated) were assayed for: (A) NADH:ubiquinone oxidoreductase activity (
M: site found in mouse ; H: site found in human; (-) cleavage site not possible to be represented the in the structure.
DISCUSSION
DENV infection is known to elicit a variety of metabolic alterations in the host cells, including changes in the preference for oxidizing energetic substrates in mitochondria (2, 7). Here, we show that DENV NS3 impairs CI activity directly, likely by promoting proteolytic cleavage of CI subunits. Accordingly, we also demonstrate that NS3 affects mitochondria’s capacity to utilize malate and pyruvate as respiratory substrates, which feeds the ETS by generating NADH, the CI substrate. These results agree with our previous findings demonstrating that the main respiratory substrate used in DENV-infected cells shifts from glutamine to palmitate (7), as electrons from fatty acids feed ETS from acyl-CoA dehydrogenase/electron transfer flavoprotein (ETF)/electron flavoprotein dehydrogenase (ETFDH) directly to ubiquinone, thus bypassing CI. Therefore, we suggest that DENV NS3 directly interferes with the mitochondrial ETS capacity in a substrate-specific manner and may be one of the molecular players that mediate the metabolic alterations in DENV-infected cells.
NS3 is a trypsin-like serine protease highly conserved among flavivirus (27). During viral polyprotein processing, flavivirus NS3 protease is believed to be fully active when interacting with its cofactor NS2B (12, 20, 26). However, NS2B was not found among the six DENV proteins we identified in the mitochondrial fraction of infected cells (NS3, NS1, NS2A, NS4A, NS5, and E). Nonetheless, it is possible that NS3 binding to specific host substrates allows conformational changes that favor its activity irrespective of NS2B. In agreement, recent studies showed that NS3pro alone can cleave the mitochondrial Hsp70 co-chaperone GrpEl1 after being imported to isolated mouse liver mitochondria (17, 18). In these studies, the authors also demonstrated that NS3pro, but not NS3proS135A (the same construct we used here for the catalytically inactive NS3), cleaved recombinant GrpEl1
After being cleaved from the viral polyprotein, NS2B remains an integral ER membrane protein. In this context, NS2B was shown to be essential for recruiting Zika virus (also a flavivirus) NS3 to the viral replication complex (29). Although most DENV NS3 associates with the ER during infection (30), it possesses a mitochondrial import signal, allowing it to be imported into the mitochondrial matrix (18). Accordingly, NS3 is enriched in the mitochondria fraction of DENV-infected cells (31, 32), and NS3-transfected cells display decreased mitochondrial respiratory rates (17). These studies support that NS3 is also addressed to mitochondria, where it can play other functions besides its role as part of the viral replication complex. Our findings here are the first to demonstrate that NS3 can modulate mitochondrial metabolism by directly interfering with the function of an ETS complex.
CI is the largest and most intricate mitochondrial respiratory complex (33). It transfers electrons from NADH to ubiquinone in a process coupled with the translocation of protons across the inner mitochondrial membrane. The mammalian CI has 44 subunits that are organized into three functional modules: the N module (NADH oxidation module), the Q module (ubiquinone reduction module), and the P module (proton pump module), the later constituting the arm embedded in the mitochondria inner membrane (33). Among the subunits within the CI structure, 14 are considered core subunits, which play active roles in CI enzymatic activity, while the remaining 30 are accessory subunits (33, 34). Mutations in all core subunits and 12 of the accessory subunits have already been described in human diseases associated with CI deficiency (34).
Here, we showed that 16 subunits of human CI (or 17 subunits in mice CI) possess potential DENV NS3 cleavage sites. Among these cleavage sites, those located in NDUFS1, NDUFS2, NDUFV1, NDUFA2, NDUFA12, NDUFA13, NDUFB1, and NDUFB4 subunits are more exposed on the CI surface, presumably being more accessible for proteolysis by NS3. NDUFS1 and NDUFS2 are core subunits, and mutations in the accessory subunits NDUFV1, NDUFA2, NDUFA12, and NDUFA13 were associated with human diseases (34). Therefore, the cleavage of one or more subunits during DENV infection may affect CI function. It does not mean, however, that subunits localized more internally in the CI structure are not accessible to NS3 since the interaction NS3/CI could expose regions normally hidden in the native CI structure. Thus, the proteolysis of the more internally localized NDUFS1, NDUFS7, and NDUFV1 subunits, which possess iron-sulfur centers and are essential to the electron transport within the CI structure, could also dramatically impair CI function.
A caveat in our data is that we cannot guarantee that the effects of NS3pro on CI activity depend on proteolysis of CI subunits since we did not demonstrate it directly. Even though NS3proS135A contains a mutation in the catalytic amino acid triad and has impaired activity, the mutation may disturb the protein conformation, thus preventing a presumably protease-independent inhibition of CI. Therefore, more evidence is required to confirm whether some of the 44 CI subunit is indeed a target of DENV NS3 proteolysis. We aim to focus on that in a future study.
Other DENV proteins were shown to localize in mitochondria. One example is the NS4B protein, which inhibits the activation of the mitochondrial fission factor DRP1, inducing mitochondrial elongation and, thus, favoring the oxidative metabolism in infected cells (31, 32). Our data suggest NS3 may also contribute to metabolic reprogramming during DENV infection. Since the overall outcomes of the infection on cellular metabolism may mask the actions of each viral protein individually, it is crucial to discriminate the effects of each protein separately to uncover new functions and potential targets for therapeutic interventions.
Experimental procedures
Cell culture and infection
Huh7 cells, a human hepatocarcinoma cell line, were cultured in DMEM with 5 mM glucose (Gibco, USA), supplemented with 10% fetal bovine serum (FBS) (Invitrogen Corporation, USA), 100 U/mL penicillin, 100 g/mL streptomycin, 0.22% sodium bicarbonate, and 0.2% HEPES, pH 7.4, in a CO2 humid incubation chamber, at 37°C. When the cultures reached 70% confluence, the cells were infected with DENV serotype 2 (strain 16681), using an MOI of 1, or subjected to simulated infection (mock). After 24 h of infection, the cells were collected for mitochondria isolation or determination of NADH:ubiquinone oxidoreductase activity.
Isolation of mitochondria from DENV-infected HuH7 cells
Huh7 cells, seeded in 150 cm2 flasks (2 × 107 cells), were infected with DENV (MOI = 1). After 24 h, the cells were collected and lysed with a Potter-Elvehjem homogenizer (Sigma-Aldrich, MO, USA). The cell lysate was centrifuged at 700 ×
Proteomics screening
Mitochondria samples from infected Huh7 cells were incubated in a solution containing 0.2% of RapiGest (Waters, MA, USA) for 12 h, at 37°C under agitation (200 rpm) and submitted to sonication in the bath for 30 min, followed by centrifugation (12,000 rpm, for 1 min, at room temperature). Each sample was filtered and desalted using Centriprep of 3 kDa (Merck Millipore, MA, USA) and washed (three times) with 500 µL of NH4HCO3. The retained volume was dried using a Speed-Vac system, and the dried samples were resuspended in a solution containing 0.2% of RapiGest, followed by digestion steps according to the manufacturer’s instructions. The tryptic peptides, in triplicate, were loaded on a Waters nanoAcquity system (Waters, MA, USA). In total, 9 µL of the samples was injected (3 µL each time) and desalted online, using a Waters Symmetry C18 (180 µm × 20 mm, 5 µm trap column). By using a Waters ACQUITY UPLC Peptide HSS T3 C18 column (150 µm × 75 mm, 1.7 µm), a liquid chromatography step was performed using a 0.5 µL/min mobile phase flow with a linear gradient from 3% to 40% of acetonitrile containing 0.1% formic acid across a 210 min running time. Electrospray tandem mass spectra were recorded using a Waters Synapt G1 HD/MS High-Definition Mass Spectrometer (Waters, Manchester, UK) interfaced with the nanoAcquity system capillary chromatography. The parameters used during the experiments were: 3,000 V for ESI voltage, source temperature of 80°C, and cone voltage of 35 V. MassLynx data system (Version 4.1, Waters, MA, USA) was used for data acquisition and instrument control and performed by scanning from a mass-to-charge ratio (
NS3 expression and purification
NS3 activity
The activity of NS3pro or NS3S135A was performed using the fluorogenic substrate Bz-Arg-AMC·HCl (Santa Cruz Biotechnology, Dallas, USA), following an adapted protocol previously described (28). Briefly, the reaction was initiated by adding 1.8 µM NS3pro or NS3proS135A to the reaction mix [250 µM Bz-Arg-AMC·HCl, 200 mM Tris (pH 8.5)]. Blank samples were prepared by replacing the proteins’ preparations with the equivalent volume of H2O. The proteolytic activity was monitored for 45 min at 37°C with an excitation wavelength of 385 nm and an emission wavelength of 465 nm using a SpectraMax M5 (Molecular Devices, CA, USA). Relative fluorescence units (RFUs) were normalized by subtracting the blank values and subsequently by the protein content.
Isolation of mitochondria from mouse liver
Male C57BL/6 mice aged 12–20 weeks were euthanized by cervical dislocation, and the liver was quickly collected to initiate the mitochondria isolation. The experimental approaches were approved by the Committee on Ethics in Animal Use (CEUA) in Scientific Experimentation of the Health Sciences Center of the Federal University of Rio de Janeiro (protocol no. A39/23-046/22). Mitochondria isolation was performed using a method previously described (36), with some adaptations. Briefly, the tissue was lysed in isolation buffer [0.1 M Tris/MOPs (pH 7.4), 0.1 M EGTA/Tris (pH 7.4), 1 M sucrose] using Potter-Elvehjem homogenizer (Sigma-Aldrich, MO, USA) on ice. The homogenate was centrifuged at 700 ×
NADH:ubiquinone oxidoreductase activity
NADH:ubiquinone oxidoreductase (CI) activity was performed using mitochondria isolated from mouse liver, incubated or not with NS3pro or NS3proS135A at different concentrations for 1 h, and subjected to freeze-thawing cycles to produce extracts following an adapted protocol previously described (37). The activity was evaluated in reaction buffer [50 mM potassium phosphate buffer (pH 7.5), 3 mg/mL fatty acid-free BSA, 300 µM KCN, and 100 µM NADH]. The reaction was initiated with 60 µM ubiquinone, and the absorbance of the samples was monitored for 2 min at 340 nm using a Shimadzu UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan), followed by the addition of 10 µM rotenone (used to discount CI-independent NADH oxidoreductase activity). The calculation was carried out using the NADH extinction coefficient (6.2 mM⁻¹ cm⁻¹). We adapted the same protocol for DENV-infected Huh7 cells, using 80 µg of protein of cell extracts. Huh7 cells were collected 24 hpi, and the pellets were resuspended in 20 mM potassium phosphate buffer (pH 7.5). To lyse the cells, the suspension was taken up and expelled using a Hamilton syringe (Hamilton Company, NV, USA) until it became a homogeneous solution and subjected to freeze-thawing cycles.
Succinate dehydrogenase activity
The succinate dehydrogenase (CII) activity was performed with mouse liver mitochondria extracts incubated or not with NS3pro for 1 h, using an adapted protocol previously described (38). Briefly, the activity was determined using 2,6-dichlorophenolindophenol, DCPIP (Sigma-Aldrich, MO, USA), an artificial electron acceptor. The assay was conducted in 96-well plates using a SpectraMax M5 (Molecular Devices, CA, USA). Approximately 20 µg of isolated mitochondria were added to the wells and the reaction was started by adding 200 µL of reaction buffer [20 mM phosphate buffer (pH 7), 0.1% Triton X-100, 4 mM sodium azide, 5 mM succinate, and 300 mM DCPIP]. The plate was shaken for 10 s, and the DCPIP reduction was monitored for 3 min at 600 nm, 35°C. The activity calculation was performed using the reduced DCPIP absorption coefficient (21.0 mM−1 cm−1) and data represented normalized by the protein concentration of each sample.
Succinate:cytochrome
CII + CIII activity was performed using mitochondria isolated from mouse liver, incubated or not with NS3pro for 1 h, following an adapted protocol previously described (37). The samples were incubated for 10 min in reaction buffer [50 mM potassium phosphate buffer (pH 7.5), 300 µM KCN, and 10 mM succinate], at 37°C. The reaction was started with 50 µM oxidized cytochrome
Cytochrome c oxidase activity
Cytochrome
ATP synthase reverse activity
The ATP synthase (CV) reverse activity was based on ATP hydrolysis and subsequent determination of inorganic phosphate content. The activity was performed using mouse liver mitochondria extracts at 150 µg/mL protein concentration, incubated or not with NS3pro for 1 h, at 37°C. The reaction was started by adding 1 mL of reaction buffer (20 mM HEPES, 5 mM MgCl2, and 1 mM ATP) to the samples. For 30 min, aliquots were collected every 5 min, and the reaction was stopped with the addition of 20% TCA. Inorganic phosphate content was measured by the Fiske-Subbarow method (39) using a SpectraMax M5 (Molecular Devices, CA, USA). The same assay was conducted in the presence of 5 mM azide to inhibit ATP synthase and discount background signal.
High-resolution respirometry
Mitochondrial OCRs were assessed by high-resolution respirometry using the Oxygraph-2k system (Oroboros Instruments, Innsbruck, Austria) and DatLab 7.4.0.4 software (Oroboros Instruments, Innsbruck, Austria). The experiments were performed with isolated mitochondria from mouse liver (50 µg protein), incubated or not with NS3pro, NS3proS135A, or NS3prohel for 1 h, in a final volume of 2 mL of MIR05, at 37°C, stirring at 750 rpm. Different substrates and inhibitors were added in the following sequence and concentrations. For complexes I + III + IV-dependent respiration, 5 mM malate and 10 mM pyruvate were added, followed by 2.5 mM ADP, and then 0.25 µM rotenone. Respiratory state 2 represents the OCR after the addition of substrates (in this case malate and pyruvate) but in the absence of ADP. Respiratory state 3 represents the OCR value reached after ADP addition. Rotenone was added to determine the residual rate of non-mitochondrial oxygen consumption, which is subtracted from OCR values for the analyses. For the multisubstrate protocol, 5 mM malate, 10 mM pyruvate, and 10 mM succinate were added, followed by 2.5 mM ADP, and then 0.25 µM rotenone and 0.5 mM antimycin. Leak respiration, a non-phosphorylating resting state when substrates are oxidized but ADP is not supplied, was calculated by subtracting residual oxygen consumption (in the presence of rotenone and antimycin) from the respiratory state 2.
Mitochondrial membrane potential
The mitochondrial membrane potential (Δψm) was analyzed using safranin O (Sigma-Aldrich, MO, USA). This lipophilic cationic dye self-quenches its fluorescence through its potential-dependent distribution between the external environment and the intramitochondrial compartment. The assay was carried out in a Varian Cary Eclipse fluorometer (Agilent, CA, USA), with an excitation wavelength of 495 nm, an emission wavelength of 587 nm and high-speed stirring at 37°C. The cuvettes were prepared with 1 mL of MIR05, isolated mitochondria (50 µg protein) incubated or not with NS3pro for 1 h, and 0.5 µM safranin O. The assay started with the addition of 5 mM malate, 10 mM pyruvate, and 10 mM succinate, followed by 200 µM ADP, 0.1 µg/mL oligomycin and 0.05 µM FCCP. The maximum membrane potential was calculated by subtracting the value of safranin O fluorescence intensity obtained after oligomycin addition (hyperpolarization) from the minimum fluorescence intensity obtained after the depolarization with FCCP. The percentage of the maximum mitochondrial membrane potential maintained in the phosphorylating state was obtained by calculating the difference between the fluorescence value obtained after ADP addition and after FCCP addition, divided by the maximum membrane potential, and then multiplied by 100.
Prediction of viral protease cleavage sites in CI subunits
The presence of viral protease cleavage sites in the mitochondrial CI subunits was evaluated using the SitePrediction website (https://www.dmbr.ugent.be/prx/bioit2-public/SitePrediction/) (24). The searches were performed by entering the 12 combinations of amino acids that create potential cleavage sites at positions P2-P1xP1′ (KRA, KRG, KRS, RRA, RRG, RRS, RKA, RKG, RKS, QRA, QRG, and QRS), considering that the NS2B-NS3 cleavage motifs present in flavivirus polyproteins consist of a pair of basic amino acid residues (KR, RR, RK, or occasionally QR) at the canonical positions P2 and P1 followed by a small amino acid (G, S, or A) in position P1′ (20, 25). We entered the protein sequences of the 44 subunits from mouse or human CI as substrate inputs. Only sites with 100% similarity to the researched sites were considered for each substrate sequence. The potential cleavage sites in CI were represented in the complex structure (PDB: 5XTD) using PyMOL.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.0.1 (GraphPad Software, CA, USA) and data were expressed as mean ± standard deviation. Differences between means were analyzed using one-way ANOVA with Sidak’s post hoc test or Student’s
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Abstract
ABSTRACT
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,
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.
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