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The metabolic resources crucial for viral replication are provided by the host. Details of the mechanisms by which viruses interact with host metabolism, altering and recruiting high free-energy molecules for their own replication, remain unknown. Sindbis virus, the prototype of and most widespread alphavirus, causes outbreaks of arthritis in humans and serves as a model for the study of the pathogenesis of neurological diseases induced by alphaviruses in mice. In this work, respirometric analysis was used to evaluate the effects of Sindbis virus infection on mitochondrial bioenergetics of a mouse neuroblastoma cell lineage, Neuro 2a. The modulation of mitochondrial functions affected cellular ATP content and this was synchronous with Sindbis virus replication cycle and cell death. At 15 h, irrespective of effects on cell viability, viral replication induced a decrease in oxygen consumption uncoupled to ATP synthesis and a 36% decrease in maximum uncoupled respiration, which led to an increase of 30% in the fraction of oxygen consumption used for ATP synthesis. Decreased proton leak associated to complex I respiration contributed to the apparent improvement of mitochondrial function. Cellular ATP content was not affected by infection. After 24 h, mitochondria dysfunction was clearly observed as maximum uncoupled respiration reduced 65%, along with a decrease in the fraction of oxygen consumption used for ATP synthesis. Suppressed respiration driven by complexes I- and II-related substrates seemed to play a role in mitochondrial dysfunction. Despite the increase in glucose uptake and glycolytic flux, these changes were followed by a 30% decrease in ATP content and neuronal death. Taken together, mitochondrial bioenergetics is modulated during Sindbis virus infection in such a way as to favor ATP synthesis required to support active viral replication. These early changes in metabolism of Neuro 2a cells may form the molecular basis of neuronal dysfunction and Sindbis virus-induced encephalitis. [ABSTRACT FROM AUTHOR]

Dengue fever is an emerging viral disease transmitted by arthropods to humans in tropical countries. Dengue hemorrhagic fever (DHF) is escalating in frequency and mortality rates. Here we studied the involvement of macrophage migration inhibitory factor (MIF) in dengue virus (DENV) infection and its pathogenesis. Patients with DHF had elevated plasma concentrations of MIF. Both leukocytes from these patients and macrophages from healthy donors infected in vitro with DENV showed a substantial amount of MIF within lipid droplets. The secretion of MIF by macrophages and hepatocytes required a productive infection and occurred without an increase in gene transcription or cell death, thus indicating active secretion from preformed stocks. In vivo infection of wild-type and mif-deficient (Mif-/-) mice demonstrated a role of MIF in dengue pathogenesis. Clinical disease was less severe in Mif-/- mice, and they exhibited a significant delay in lethality, lower viremia, and lower viral load in the spleen than wild-type mice. This reduction in all parameters of severity on DENV infection in Mif-/- mice correlated with reduced proinflammatory cytokine concentrations. These results demonstrated the contribution of MIF to the pathogenesis of dengue and pointed to a possible beneficial role of neutralizing MIF as an adjunctive therapeutic approach to treat the severe forms of the disease. [ABSTRACT FROM AUTHOR]

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val145 and His148. [ABSTRACT FROM AUTHOR]

Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established 1H-15N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method. [ABSTRACT FROM AUTHOR]

Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid – that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia. • Galactose induces mitochondrial dysfunction in yeast model of galactosemia. • Galactose-1-phosphate decreases oxidative phosphorylation. • Galactose-1-phosphate inhibits cytochrome c oxidase from yeast, rats, and humans. • Mitochondrial retrograde signaling pathway is relevant for galactose tolerance. • Compounds that rescue mitochondrial disease models increase galactose tolerance. [ABSTRACT FROM AUTHOR]

The Covid-19 pandemic, caused by SARS-CoV-2, has resulted in over 6 million reported deaths worldwide being one of the biggest challenges the world faces today. Here we present optimizations of all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay to a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals and 103 individuals without a confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals from our cohort before and after receiving ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It is also a valuable tool for elucidating the immunological consequences of new viral strains and monitoring vaccination coverage and duration of response to different immunization regimens. [ABSTRACT FROM AUTHOR]

A novel programme in science education based on the participation of high school students in the activities of an international conference on experimental biology is reported. The data were collected by means of questionnaires answered by the students during and after the meeting. The results show that students autonomously develop efficient criteria for choosing which activities to attend. Such criteria are based mainly on the students' interest in the subject and on a previous evaluation of the understanding of the title of the activity. This freedom of choice seems to help the students to overcome difficulties with the vocabulary and content of activities. Results obtained from questionnaires answered eight months after the conference showed that students did acquire updated scientific information. It is our belief that scientific meetings may represent an important, but underestimated, opportunity for science education. [ABSTRACT FROM AUTHOR]

A strong association between perinatal viral infections and neurodevelopmental disorders has been established. Both the direct contact of the virus with the developing brain and the strong maternal immune response originated by viral infections can impair proper neurodevelopment. Coronavirus disease 2019 (COVID-19), caused by the highly-infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently responsible for a large global outbreak and is a major public health issue. While initial studies focused on the viral impact on the respiratory system, increasing evidence suggest that SARS-CoV-2 infects other organs and tissues including the mature brain. While studies continue to determine the neuropathology associated to COVID-19, the consequences of SARS-CoV-2 infection to the developing brain remain largely unexplored. The present review discusses evidence suggesting that SARS-CoV-2 infection may have persistent effects on the course of pregnancy and on brain development. Studies have shown that several proinflammatory mediators which are increased in the SARS-CoV-2-associated cytokine storm, are also modified in other viral infections known to increase the risk of neurodevelopmental disorders. In this sense, further studies should assess the genuine effects of SARS-CoV-2 infection during pregnancy and delivery along with an extended follow-up of the offspring, including neurocognitive, neuroimaging, and electrophysiological examination. It also remains to be determined whether and by which mechanisms SARS-CoV-2 intrauterine and early life infection could lead to an increased risk of developing neuropsychiatric disorders, such as autism (ASD) and schizophrenia (SZ), in the offspring. • There is evidence of vertical transmission of SARS-CoV-2 during pregnancy. • COVID-19 cytokine storm can cause abnormal microglial activation. • COVID-19 may be a risk factor to development of neuropsychiatric disorders in children. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a neurotropic Orthoflavivirus that causes a myriad of neurological manifestations in newborns exposed in uterus. Despite the devastating consequences of ZIKV on the developing brain, strategies to prevent or treat the consequences of viral infection are not yet available. We previously showed that short-term treatment with the TNF-α neutralizing monoclonal antibody. Infliximab could prevent seizures at acute and chronic stages of ZIKV infection, but had no impact on long-term cognitive and motor dysfunction. Due to the central role of inflammation in ZIKV-neuropathology, we hypothesized that prolonged treatment with the anti-TNF-α monoclonal antibody Infliximab could provide complete rescue of long-term behavioral deficits associated with neonatal ZIKV infection in mice. Here, neonatal (post-natal day 3) Swiss mice were submitted to subcutaneous (s.c.) injection of 106 PFU of ZIKV or mock medium and were then treated with Infliximab (20 μg/day) or sterile saline intraperitoneally (i.p.), for 40 days starting on the day of infection, and behavioral assessment started at 60 days post-infection (dpi). Infliximab prevented ZIKV-induced cognitive and motor impairments in mice. In addition, microgliosis and cell death found in mice following ZIKV infection were partially reversed by TNF-α blockage. Altogether, these results suggest that TNF-α-mediated inflammation is central for late ZIKV-induced behavioral deficits and cell death and strategies targeting this cytokine may be promising approaches to treat subjects exposed to the virus during development. • Neonatal ZIKV infection provokes long-lasting neurological consequences. • Long-term TNF-α neutralization prevents ZIKV-induced memory impairment in mice. • Long-term TNF-α neutralization prevents ZIKV-induced motor deficits in mice. • TNF-α neutralization reduces microglial activation and cell death in the hippocampus of ZIKV-infected mice. [ABSTRACT FROM AUTHOR]

Modulation of brain activity is one of the main mechanisms capable of demonstrating the synchronization dynamics of neural oscillations. In epilepsy, modulation is a key concept since seizures essentially result from neural hypersynchronization and hyperexcitability. In this study, we have introduced a time-dependent index based on the Kullback-Leibler divergence to quantify the effects of phase and frequency modulations of neural oscillations in neonatal mice exhibiting epileptiform activity induced by Zika virus (ZIKV) infection. Through this index, we demonstrate that fast oscillations (gamma and beta 2) are the more susceptible modulated rhythms in terms of phase, during seizures, whereas slow waves (delta and theta) mainly undergo changes in frequency. The index also allowed detection of specific patterns associated with the interdependent modulation of phase and frequency in neural activity. Furthermore, by comparing ZIKV modulations with the general computational model Epileptors, we verify different signatures related to the brain rhythms modulation in phase and frequency. These findings instigate new studies on the effects of ZIKV infection on neuronal networks from electrophysiological activities, and how different mechanisms can trigger epilepsy. [ABSTRACT FROM AUTHOR]

Oxaliplatin (OXA) is an antineoplastic agent used for the treatment of cisplatin-resistant tumours, presenting lower incidence of nephrotoxicity and myelotoxicity than other platinum-based drugs. However, OXA treatment is highly associated with painful peripheral neuropathy, a well-known and relevant side effect caused by mitochondrial dysfunction. The transfer of functional exogenous mitochondria (mitotherapy) is a promising therapeutic strategy for mitochondrial diseases. We investigated the effect of mitotherapy on oxaliplatin-induced painful peripheral neuropathy (OIPN) in male mice. OIPN was induced by i.p. injections of oxaliplatin (3 mg/kg) over 5 consecutive days. Mechanical (von Frey test) and cold (acetone drop test) allodynia were evaluated between 7 and 17 days after the first OXA treatment. Mitochondria was isolated from donor mouse livers and mitochondrial oxidative phosphorylation was assessed with high resolution respirometry. After confirming that the isolated mitochondria were functional, the organelles were administered at the dose of 0.5 mg/kg of mitochondrial protein on days 1, 3 and 5. Treatment with OXA caused both mechanical and cold allodynia in mice that were significant 7 days after the initial injection of OXA and persisted for up to 17 days. Mitotherapy significantly prevented the development of both sensory alterations, and attenuated body weight loss induced by OXA. Mitotherapy also prevented spinal cord ERK1/2 activation, microgliosis and the increase in TLR4 mRNA levels. Mitotherapy prevented OIPN by inhibiting neuroinflammation and the consequent cellular overactivity in the spinal cord, presenting a potential therapeutic strategy for pain management in oncologic patients undergoing OXA treatment. • Functional exogenous mitochondria are incorporated into the mouse spinal cord and DRG after systemic injection. • Animal well-being and organ function are not affected by exogenous mitochondria transfer (mitotherapy). • Mitotherapy prevented the development of mechanical and cold allodynia induced by oxaliplatin treatment in mice. • The effect of mitotherapy was attributed to its ability to inhibit neuroinflammation in the spinal cord. [ABSTRACT FROM AUTHOR]

Mitochondrial oxidation of nutrients is tightly regulated in response to the cellular environment and changes in energy demands. In vitro studies evaluating the mitochondrial capacity of oxidizing different substrates are important for understanding metabolic shifts in physiological adaptations and pathological conditions, but may be influenced by the nutrients present in the culture medium or by the utilization of endogenous stores. One such influence is exemplified by the Crabtree effect (the glucose-mediated inhibition of mitochondrial respiration) as most in vitro experiments are performed in glucose-containing media. Here, using high-resolution respirometry, we evaluated the oxidation of endogenous or exogenous substrates by cell lines harboring different metabolic profiles.Wefound that a 1-h deprivation of the main energetic nutrients is an appropriate strategy to abolish interference of endogenous or undesirable exogenous substrates with the cellular capacity of oxidizing specific substrates, namely glutamine, pyruvate, glucose, or palmitate, in mitochondria. This approach primed mitochondria to immediately increase their oxygen consumption after the addition of the exogenous nutrients. All starved cells could oxidize exogenous glutamine, whereas the capacity for oxidizing palmitate was limited to human hepatocarcinoma Huh7 cells and to C2C12 mouse myoblasts that differentiated into myotubes. In the presence of exogenous glucose, starvation decreased the Crabtree effect in Huh7 and C2C12 cells and abrogated it in mouse neuroblastoma N2A cells. Interestingly, the fact that the Crabtree effect was observed only for mitochondrial basal respiration but not for the maximum respiratory capacity suggests it is not caused by a direct effect on the electron transport system. [ABSTRACT FROM AUTHOR]

The emergence of Zika virus (ZIKV) infection has stimulated several research groups to study and collaborate to understand virus biology and pathogenesis. These efforts may assist with the development of antiviral drugs, vaccines and diagnostic tests, as well as to promote advancements in public health policies. Here, we aim to develop standard protocols for propagation, titration, and purification of ZIKV strains, by systematically testing different cell types, kinetics, multiplicity of infection and centrifugation protocols. ZIKV produces a productive infection in human, non-human primate, and rodents-derived cell lines, with different efficacies. The highest yield of ZIKV-AFR and ZIKV-BR infectious progeny was obtained at 7 days post infection in C6/36 cells (7 × 10 7 and 2 × 10 8 PFU/ml, respectively). However, high titers of ZIKV-AFR could be obtained at earlier time points in Vero cells (2.5 × 10 7 PFU/ml at 72 hpi), whereas ZIKV-BR titers reached 10 8 PFU/ml at 4dpi in C6/36 cells. High yield of purified virus was obtained by purification through a discontinuous sucrose gradient. This optimized procedure will certainly contribute to future studies of virus structure and vaccine development. Beyond the achievement of efficient virus propagation, the normalization of these protocols will also allow different laboratories around the world to better compare and discuss data regarding different features of ZIKV biology and disease, contributing to more efficient collaborations and progression in ZIKV research. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) infection causes severe neurological consequences in both gestationally-exposed infants and adults. Sensorial gating deficits strongly correlate to the motor, sensorial and cognitive impairments observed in ZIKV-infected patients. However, no startle response or prepulse inhibition (PPI) assessment has been made in patients or animal models. In this study, we identified different outcomes according to the age of infection and sex in mice: neonatally infected animals presented an increase in PPI and delayed startle latency. However, adult-infected male mice presented lower startle amplitude, while a PPI impairment was observed 14 days after infection in both sexes. Our data further the understanding of the functional impacts of ZIKV on the developing and mature nervous system, which could help explain other behavioral and cognitive alterations caused by the virus. With this study, we support the startle reflex testing in ZIKV-exposed patients, especially infants, allowing for early detection of functional neuromotor damage and early intervention. • ZIKV-infection changes startle response differently according to age and sex. • ZIKV increases startle latency in neonatally infected mice of both sexes. • ZIKV acutely reduces startle amplitude in adult-infected male mice. • ZIKV transiently impair PPI in adult-infected male mice. [ABSTRACT FROM AUTHOR]

Secretome analysis can be described as a subset of proteomics studies consisting in the analysis of the molecules secreted by cells or tissues. Dengue virus (DENV) infection can lead to a broad spectrum of clinical manifestations, with the severe forms of the disease characterized by hemostasis abnormalities and liver injury. The hepatocytes are a relevant site of viral replication and a major source of plasma proteins. Until now, we had limited information on the small molecules secreted by hepatic cells after infection by DENV. In the present study, we analysed a fraction of the secretome of mock- and DENV-infected hepatic cells (HepG2 cells) containing molecules with < 10 kDa, using different proteomic approaches. We identified 175 proteins, with 57 detected only in the samples from mock-infected cells, 59 only in samples from DENV-infected cells, and 59 in both conditions. Most of the peptides identified were derived from proteins larger than 10 kDa, suggesting a proteolytic processing of the secreted molecules. Using in silico analysis, we predicted consistent differences between the proteolytic processing occurring in mock and DENV-infected samples, raising, for the first time, the hypothesis that differential proteolysis of secreted molecules would be involved in the pathogenesis of dengue. Biological significance Since the liver, one of the targets of DENV infection, is responsible for producing molecules involved in distinct biological processes, the identification of proteins and peptides secreted by hepatocytes after infection would help to a better understanding of the physiopathology of dengue. Proteomic analyses of molecules with < 10 kDa secreted by HepG2 cells after infection with DENV revealed differential proteolytic processing as an effect of DENV infection. [ABSTRACT FROM AUTHOR]

Dengue, due to its global burden, is the most important arthropod-borne flavivirus disease, and early detection lowers fatality rates to below 1%. Since the metabolic resources crucial for viral replication are provided by host cells, detection of changes in the metabolic profile associated with disease pathogenesis could help with the identification of markers of prognostic and diagnostic importance. We applied ¹H nuclear magnetic resonance exploratory metabolomics to study longitudinal changes in plasma metabolites in a cohort in Recife, Brazil. To gain statistical power, we used innovative paired multivariate analyses to discriminate individuals with primary and secondary infection presenting as dengue fever (DF; mild) and dengue hemorrhagic fever (DHF; severe) and subjects with a nonspecific nondengue (ND) illness (ND subjects). Our results showed that a decrease in plasma low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) discriminated dengue virus (DENV)-infected subjects from ND subjects, and also, subjects with severe infection even presented a decrease in lipoprotein concentrations compared to the concentrations in subjects with mild infection. These results add to the ongoing discussion that the manipulation of lipid metabolism is crucial for DENV replication and infection. In addition, a decrease in plasma glutamine content was characteristic of DENV infection and disease severity, and an increase in plasma acetate levels discriminated subjects with DF and DHF from ND subjects. Several other metabolites shown to be altered in DENV infection and the implications of these alterations are discussed. We hypothesize that these changes in the plasma metabolome are suggestive of liver dysfunction, could provide insights into the underlying molecular mechanisms of dengue virus pathogenesis, and could help to discriminate individuals at risk of the development of severe infection and predict disease outcome. [ABSTRACT FROM AUTHOR]

Mayaro virus (MAYV) is an arthritogenic arbovirus that causes a debilitating illness that can progress to a chronic rheumatic disease characterized by persistent viral replication in macrophages within joint tissues. Here, we report that MAYV-infected macrophages release decondensed DNA traps (DNA extracellular traps, DETs) through a mechanism driven by the production of reactive oxygen species and peptidyl arginine deiminase activation, resembling the classical mechanism of pathogen clearance by activated neutrophils. Unlike traditional pathogen clearance observed for NETs released by neutrophils, MAYV-induced DETs did not inactivate the virus. Instead, DET-ensnared viruses are internalized by neighboring uninfected macrophages, increasing the number of infected cells. Collectively, these findings suggest that MAYV-containing DETs act as a "Trojan horse" that facilitates viral dissemination within inflamed tissues, connecting macrophage-mediated inflammatory response to viral persistence in the articular tissue in chronic MAYV disease., (© 2025 Wiley Periodicals LLC.)

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., Competing Interests: The authors declare no conflict of interest.

Zika virus (ZIKV) is a neurotropic flavivirus that can persist in several tissues. The late consequences of ZIKV persistence and whether new rounds of active replication can occur, remain unaddressed. Here, we investigated whether neonatally ZIKV-infected mice are susceptible to viral reactivation in adulthood. We found that when ZIKV-infected mice are treated with immunosuppressant drugs, they present increased susceptibility to chemically induced seizures. Levels of subgenomic flavivirus RNAs (sfRNAs) were increased, relative to the amounts of genomic RNAs, in the brains of mice following immunosuppression and were associated with changes in cytokine expression. We investigated the impact of immunosuppression on the testicles and found that ZIKV genomic RNA levels are increased in mice following immunosuppression, which also caused significant testicular damage. These findings suggest that ZIKV can establish new rounds of active replication long after acute stages of disease, so exposed patients should be monitored to ensure complete viral eradication., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s).)

Arthritogenic alphaviruses are mosquito-borne viruses that cause a debilitating rheumatic disease characterized by fever, headache, rash, myalgia, and polyarthralgia with the potential to evolve into a severe and very prolonged illness. Although these viruses have been geographically restricted by vector hosts and reservoirs, recent epidemics have revealed the risks of their spread worldwide. In this review, we aim to discuss the protective and pathological roles of macrophages during the development of arthritis caused by alphaviruses. The progression to the chronic phase of the disease is related to the extension of viral replication and the maintenance of articular inflammation, in which the cellular infiltrate is predominantly composed of macrophages. We explore the possible implications of macrophage polarization to M1/M2 activation phenotypes, drawing a parallel between alphavirus arthritis and rheumatoid arthritis (RA), a chronic inflammatory disease that also affects articular tissues. In RA, it is well established that M1 macrophages contribute to tissue damage and inflammation, while M2 macrophages have a role in cartilage repair, so modulating the M1/M2 macrophage ratio is being considered as a strategy in the treatment of this disease. In the case of alphavirus-induced arthritis, the picture is more complex, as proinflammatory factors derived from M1 macrophages contribute to the antiviral response but cause tissue damage, while M2 macrophages may contribute to tissue repair but impair viral clearance., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Dengue virus belongs to the Flaviviridae family, being responsible for an endemic arboviral disease in humans. It is an enveloped virus, whose genome is a positive-stranded RNA packaged by the capsid protein. Dengue virus capsid protein (DENVC) forms homodimers in solution organized in 4 α-helices and an intrinsically disordered N-terminal region. The N-terminal region is involved in the binding of membranous structures in host cells and in the recognition of nucleotides. Here we report the 1 H, 15 N and 13 C resonance assignments of the DENVC with the deletion of the first 19 intrinsically disordered residues. The backbone chemical shift perturbations suggest changes in the α1 and α2 helices between full length and the truncated proteins., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Cognitive dysfunction is often reported in patients with post-coronavirus disease 2019 (COVID-19) syndrome, but its underlying mechanisms are not completely understood. Evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein or its fragments are released from cells during infection, reaching different tissues, including the CNS, irrespective of the presence of the viral RNA. Here, we demonstrate that brain infusion of Spike protein in mice has a late impact on cognitive function, recapitulating post-COVID-19 syndrome. We also show that neuroinflammation and hippocampal microgliosis mediate Spike-induced memory dysfunction via complement-dependent engulfment of synapses. Genetic or pharmacological blockage of Toll-like receptor 4 (TLR4) signaling protects animals against synapse elimination and memory dysfunction induced by Spike brain infusion. Accordingly, in a cohort of 86 patients who recovered from mild COVID-19, the genotype GG TLR4-2604G>A (rs10759931) is associated with poor cognitive outcome. These results identify TLR4 as a key target to investigate the long-term cognitive dysfunction after COVID-19 infection in humans and rodents., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Nucleocapsid (NC) assembly is an essential step of the virus replication cycle. It ensures genome protection and transmission among hosts. Flaviviruses are human viruses for which envelope structure is well known, whereas no information on NC organization is available. Here we designed a dengue virus capsid protein (DENVC) mutant in which a highly positive spot conferred by arginine 85 in α4-helix was replaced by a cysteine residue, simultaneously removing the positive charge and restricting the intermolecular motion through the formation of a disulfide cross-link. We showed that the mutant self-assembles into capsid-like particles (CLP) in solution without nucleic acids. Using biophysical techniques, we investigated capsid assembly thermodynamics, showing that an efficient assembly is related to an increased DENVC stability due to α4/α4' motion restriction. To our knowledge, this is the first time that flaviviruses' empty capsid assembly is obtained in solution, revealing the R85C mutant as a powerful tool to understand the NC assembly mechanism., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to β-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com , we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form., Competing Interests: CH was employed by Signals GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Altincekic, Korn, Qureshi, Dujardin, Ninot-Pedrosa, Abele, Abi Saad, Alfano, Almeida, Alshamleh, de Amorim, Anderson, Anobom, Anorma, Bains, Bax, Blackledge, Blechar, Böckmann, Brigandat, Bula, Bütikofer, Camacho-Zarco, Carlomagno, Caruso, Ceylan, Chaikuad, Chu, Cole, Crosby, de Jesus, Dhamotharan, Felli, Ferner, Fleischmann, Fogeron, Fourkiotis, Fuks, Fürtig, Gallo, Gande, Gerez, Ghosh, Gomes-Neto, Gorbatyuk, Guseva, Hacker, Häfner, Hao, Hargittay, Henzler-Wildman, Hoch, Hohmann, Hutchison, Jaudzems, Jović, Kaderli, Kalniņš, Kaņepe, Kirchdoerfer, Kirkpatrick, Knapp, Krishnathas, Kutz, zur Lage, Lambertz, Lang, Laurents, Lecoq, Linhard, Löhr, Malki, Bessa, Martin, Matzel, Maurin, McNutt, Mebus-Antunes, Meier, Meiser, Mompeán, Monaca, Montserret, Mariño Perez, Moser, Muhle-Goll, Neves-Martins, Ni, Norton-Baker, Pierattelli, Pontoriero, Pustovalova, Ohlenschläger, Orts, Da Poian, Pyper, Richter, Riek, Rienstra, Robertson, Pinheiro, Sabbatella, Salvi, Saxena, Schulte, Schiavina, Schwalbe, Silber, Almeida, Sprague-Piercy, Spyroulias, Sreeramulu, Tants, Tārs, Torres, Töws, Treviño, Trucks, Tsika, Varga, Wang, Weber, Weigand, Wiedemann, Wirmer-Bartoschek, Wirtz Martin, Zehnder, Hengesbach and Schlundt.)

The Flaviviridae family comprises important human pathogens, including Dengue, Zika, West Nile, Yellow Fever and Japanese Encephalitis viruses. The viral genome, a positive-sense single-stranded RNA, is packaged by a single protein, the capsid protein, which is a small and highly basic protein that form intertwined homodimers in solution. Atomic-resolution structures of four flaviviruses capsid proteins were solved either in solution by nuclear magnetic resonance spectroscopy, or after protein crystallization by X-ray diffraction. Analyses of these structures revealed very particular properties, namely (i) the predominance of quaternary contacts maintaining the structure; (ii) a highly electropositive surface throughout the protein; and (iii) a flexible helix (α1). The goal of this review is to discuss the role of these features in protein structure-function relationship., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Neurological complications affecting the central nervous system have been reported in adult patients infected by Zika virus (ZIKV) but the underlying mechanisms remain unknown. Here, we report that ZIKV replicates in human and mouse adult brain tissue, targeting mature neurons. ZIKV preferentially targets memory-related brain regions, inhibits hippocampal long-term potentiation and induces memory impairment in adult mice. TNF-α upregulation, microgliosis and upregulation of complement system proteins, C1q and C3, are induced by ZIKV infection. Microglia are found to engulf hippocampal presynaptic terminals during acute infection. Neutralization of TNF-α signaling, blockage of microglial activation or of C1q/C3 prevent synapse and memory impairment in ZIKV-infected mice. Results suggest that ZIKV induces synapse and memory dysfunction via aberrant activation of TNF-α, microglia and complement. Our findings establish a mechanism by which ZIKV affects the adult brain, and point to the need of evaluating cognitive deficits as a potential comorbidity in ZIKV-infected adults.

The global situation of diseases transmitted by arthropod-borne viruses such as Dengue (DENV), Yellow Fever (YFV), Chikungunya (CHIKV) and Zika (ZIKV) viruses is alarming and treatment of human infection by these arboviruses faces several challenges. The discovery of broad-spectrum antiviral molecules, able to inactivate different groups of viruses, is an interesting approach. The viral envelope is a common structure among arboviruses, being a potential target for antivirals. Porphyrins are amphipathic molecules able to interact with membranes and absorb light, being widely used in photodynamic therapy. Previously, we showed that heme, Co-protoporphyrin IX (CoPPIX) and Sn-protoporphyrin IX (SnPPIX) directly inactivate DENV and YFV infectious particles. Here we demonstrate that the antiviral activity of these porphyrins can be broadened to CHIKV, ZIKV, Mayaro virus, Sindbis virus and Vesicular Stomatitis virus. Porphyrin treatment causes viral envelope protein loss, affecting viral morphology, adsorption and entry into target cells. Also, light-stimulation enhanced the SnPPIX activity against all tested arboviruses. In summary, CoPPIX and SnPPIX were shown to be efficient broad-spectrum compounds to inactivate medically and veterinary important viruses.

Dengue virus (DENV) infection affects millions of people and is becoming a major global disease for which there is no specific available treatment. pep14-23 is a recently designed peptide, based on a conserved segment of DENV capsid (C) protein. It inhibits the interaction of DENV C with host intracellular lipid droplets (LDs), which is crucial for viral replication. Combining bioinformatics and biophysics, here, we analyzed pep14-23 structure and ability to bind different phospholipids, relating that information with the full-length DENV C. We show that pep14-23 acquires α-helical conformation upon binding to negatively charged phospholipid membranes, displaying an asymmetric charge distribution structural arrangement. Structure prediction for the N-terminal segment reveals four viable homodimer orientations that alternatively shield or expose the DENV C hydrophobic pocket. Taken together, these findings suggest a new biological role for the disordered N-terminal region, which may function as an autoinhibitory domain mediating DENV C interaction with its biological targets. The results fit with our current understanding of DENV C and pep14-23 structure and function, paving the way for similar approaches to understanding disordered proteins and improved peptidomimetics drug development strategies against DENV and similar Flavivirus infections.

Dengue affects millions of people worldwide. No specific treatment is currently available, in part due to an incomplete understanding of the viral components' interactions with host cellular structures. We tested dengue virus (DENV) capsid protein (C) interaction with low- and very low-density lipoproteins (LDL and VLDL, respectively) using atomic force microscopy-based force spectroscopy, dynamic light scattering, NMR and computational analysis. Data reveal a specific DENV C interaction with VLDL, but not LDL. This binding is potassium-dependent and involves the DENV C N-terminal region, as previously observed for the DENV C-lipid droplets (LDs) interaction. A successful inhibition of DENV C-VLDL binding was achieved with a peptide drug lead. The similarities between LDs and VLDL, and between perilipin 3 (DENV C target on LDs) and ApoE, indicate ApoE as the molecular target on VLDL. We hypothesize that DENV may form lipoviroparticles, which would constitute a novel step on DENV life cycle., From the Clinical Editor: Using atomic force microscopy-based force spectroscopy, dynamic light scattering, NMR, and computational analysis, these authors demonstrate that dengue viral capsid proteins (DENV C) bind to very low density lipoprotein surfaces, but not to LDLs, in a potassium-dependent manner. This observation suggests the formation of lipo-viroparticles, which may be a novel step in its life cycle, and may offer potential therapeutic interventions directed to this step., (© 2013.)

A misconception regarding the human metabolism has been shown to be widespread among high school students. The students consider glucose as the sole metabolic fuel, disregarding that lipids and amino acids can be oxidized for ATP production by human cells. This misconception seems to be a consequence of formal teaching in grade and high schools. The present study reports the evaluation of a teaching strategy based on the use of a dialogic teaching methodology within a conceptual change approach to remediate that misconception. Students were stimulated to formulate hypotheses, outline experiments, and to discuss their outcomes. The results showed that students were able to reformulate their original concepts immediately after teaching. The majority of the students showed adequate learning of the topic eight months after the application of the teaching strategy, although some level of misconception recurrence was observed. The educational consequences of the teaching unit are discussed in the context of the possible reasons for its success as well as the need for similar initiatives at grade school to avoid the establishment of the misconception., (Copyright © 2013 Wiley Periodicals, Inc.)

Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in the VSV G protein, comprising the residues 145-164, directly involved in membrane interaction and fusion. In the present work we studied the interaction of pep[145-164] with membranes using NMR to solve the structure of the peptide in two membrane-mimetic systems: SDS micelles and liposomes composed of phosphatidylcholine and phosphatidylserine (PC:PS vesicles). The presence of medium-range NOEs showed that the peptide has a tendency to form N- and C-terminal helical segments in the presence of SDS micelles. Analysis of the chemical shift index indicated helix-coil equilibrium for the C-terminal helix under all conditions studied. At pH 7.0, the N-terminal helix also displayed a helix-coil equilibrium when pep[145-164] was free in solution or in the presence of PC:PS. Remarkably, at the fusogenic pH, the region of the N-terminal helix in the presence of SDS or PC:PS presented a third conformational species that was in equilibrium with the helix and random coil. The N-terminal helix content decreases pH and the minor beta-structured conformation becomes more prevalent at the fusogenic pH. These data point to a beta-conformation as the fusogenic active structure-which is in agreement with the X-ray structure, which shows a beta-hairpin for the region corresponding to pep[145-164].