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Observation and quantification of neutral lipids by Oil red O staining. DENV 2 infected and mock infected (a) HEK293T/17 cells and (b) HepG2 cells cultured on glass microscope cover slips for 24 h were stained with Oil red O before observation under bright field of a Carl Zeiss LSM800 microscope. Oil droplets show as black circles. The smaller droplets in HEK293T/17 cells are arrowed. The staining was repeated on cells grown in six well plates for 24 and 36 h and Oil red O was subsequently eluted and quantified by spectrophotometry. Experiment was undertaken independently in triplicate. Bars show mean +/−SD (*; p value <0.05)
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Background
The mosquito transmitted Dengue virus (DENV) remains a significant public health problem in many tropical and subtropical countries. Increasing evidence has suggested that during the infection process cellular lipids play important roles at several stages of the replication cycle. This study sought to characterize the changes in lipid me...
Contexts in source publication
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... infected with DENV 2, and at 24 h post infection examined by light microscopy after stain- ing with Oil Red O, a diazo dye that stains neutral tri- glycerides. While neither of the examined cell types examined showed large lipid droplets, both cell types showed results consistent with the reduction of lipid droplets as a consequence of infection (Fig. 1a, b) as established by others [22]. The reduction in neutral tri- glycerides was confirmed by elution of the dye, and spectrophotometric quantitation (Fig. 1c), although the difference between DENV infected at non-infected cells was lost in HEK293T/17 cells at 36 ...
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... While neither of the examined cell types examined showed large lipid droplets, both cell types showed results consistent with the reduction of lipid droplets as a consequence of infection (Fig. 1a, b) as established by others [22]. The reduction in neutral tri- glycerides was confirmed by elution of the dye, and spectrophotometric quantitation (Fig. 1c), although the difference between DENV infected at non-infected cells was lost in HEK293T/17 cells at 36 ...
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... panel of four different siRNAs (FASN1 -FASN4) was initially investigated for the ability to reduce FASN mRNA ex- pression as assessed by real time PCR. Results showed that two siRNAs (FASN1 and FASN4) were able to largely abolish FASN expression on day 2 post transfec- tion (Additional file 2: Figure S1), with no associated ef- fects on cell viability (Additional file 2: Figure S2). Due to the very low expression detected by real time PCR for FASN1 and FASN4, the experiment was repeated inde- pendently (Additional file 2: Figure S3) and results again showed a dramatic reduction in expression with FASN1 but a more modest reduction for FASN4. ...
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... have shown that FASN is relocalized through interaction with NS3 and colocalizes with NS3 [25] we undertook a double staining to look at expression of both DENV titer (b,c,d,g) were determined. Experiments were undertaken independently in triplicate, with duplicate plaque assay. Bars show mean +/−SD (*; p value <0.05) NS3 and FASN. Results (Fig. 10) showed no observable re- duction in DENV NS3 expression in the presence of the compounds, and, markedly, no significant colocalization between DENV NS3 and FASN was observed (Pearsons correlation coefficient 0.082 ± 0.03). We additionally tried co-immunoprecipitation experiments to confirm the interaction between NS3 and FASN, but ...
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... pro- tein expression under compound treatment, HEK293T/17 cells were again infected in the presence or absence of orli- stat or mock infected, and expression of both structural (DENV E protein) and non-structural (DENV NS3) pro- teins as well as FASN were determined by western blotting in parallel with determining expression of actin. Results (Fig. 11) showed no change in levels of NS3 expression for either drug, while levels of E protein were reduced to about 50% at the highest level of orlistat used (100 μM). The re- sults are consistent with the observations taken under con- focal microscopy in which the significant decrease in virus output was not associated with a significant ...
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... re- sults are consistent with the observations taken under con- focal microscopy in which the significant decrease in virus output was not associated with a significant decrease in DENV NS3 protein expression. Interestingly, a significant reduction in FASN expression with DENV infection and orlistat treatment was seen even with treatment at 10 μM (Fig. ...
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... analysis of FASN expres- sion after siRNA treatment. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with one of four siRNAs directed to FASN (FASN1 to FASN 4). On days 1 to 4 post transfection the level of FASN protein was determined by western blot analysis. ...
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Citations
... It was found that RGNNV induced and required lipogenesis to replicate. For this process, the relocalisation of fatty acid synthase, a critical rate-limiting enzyme in lipid biosynthesis, to the viral replication complex was considered essential (Tongluan et al., 2017). The observed decrease of the fatty acid synthase plasma levels in P5 and P8 salmon in our study could thus be another indicator that PRV-1 considerably affected the lipid metabolism. ...
Biomarkers that give quantifiable information about disease development are valuable diagnostic indicators providing opportunities to thwart adverse outcomes through timely detection and initiation of countermeasures. Whereas human medicine already relies extensively on biomarker analysis, their application in the diagnostics of fish diseases is still at a tentative beginning. However, the increased utilisation of broad-range analytical approaches such as untargeted proteomics enhances the chances to discover changes in the levels of proteins, which are connected to fish health and could serve as novel biomarkers. In the present study, proteomic analysis accompanied by biostatistical data processing was employed with the aim of identifying plasma biomarker candidates for the onset of heart and skeletal muscle inflammation (HSMI) caused by Piscine orthoreovirus (PRV)-1 infection in Atlantic salmon. HSMI is a common viral disease and threat to fish welfare and productivity in Norwegian aquaculture, so that the early detection of HSMI development would allow a timely intervention to limit the damage. In the search for biomarker candidates, salmon plasma was sampled five and eight weeks after a PRV-1 cohabitation challenge experiment and from adequate control fish, and analysed by mass spectrometry-based proteomics. The proteome dataset contained 695 proteins with >90% probability of correct identification according to the set cut-off criteria for peptide quality. Time- and condition-related differences in protein abundance levels were revealed by multivariate analysis. Comparison of the proteins identified as significant using three different biostatistical models resulted in a short list of four potential biomarkers: galectin-3-binding protein (Gal-3BP), fucolectin-6, urokinase plasminogen activator surface receptor and ryanodine receptor 3. The physiological functions described for these proteins in mammals designate them as interesting indicators of inflammatory diseases also in other species, such as PRV-1 pathogenesis in salmon. In situ-hybridisation targeting Gal-3BP and PRV-1 mRNA in heart tissue sections from the same challenge trial showed a significant upregulation of Gal-3BP mRNA in infected salmon hearts as compared to controls, supporting the findings of the proteomic analysis and suggesting a role in the onset of heart- and skeletal muscle inflammation.
... In silico approaches suggested that a fatty acid synthase (FASN) was probably a target of AM-and GM-inhibiting SARS-CoV-2 translation and replication ( Fig. 4A and B). FASN chemical inhibitors or siRNAs were correlated with the reduction of viral replications in various strains of SARS-CoV-2, as well as other viruses; VSV, HSV-1, MHV68, dengue, chikungunya, HIV and HCV [53][54][55][56]. Furthermore, AM inhibited FASN expression and activity [57], implying that FASN might be a potential target of AM and GM, reducing SARS-CoV-2 inhibition. ...
... As anticipated, we observed that both virus infection increased the expression levels of DEFA and CECG. As virus infection could modify mosquitoes' lipid metabolism to facilitate their development [81][82][83][84], we then quantified expression level of Fatty Acid Synthase 1 (FAS1), a key gene in regulating lipid metabolism, after virus infection. Interestingly we found ZIKV infection in A. aegypti could significantly down-regulate FAS1 expression while DENV did not influence it. ...
... While in previously DENV-infected A. aegypti, ZIKV copies in legs and heads have an average of 4.5-fold and 6.2-fold decrease, respectively. The stronger DENV inhibition by previous ZIKV infection in mosquitoes may relate to the significantly downregulated FAS1 expression, as it had been well proved that fatty acid synthesis in mosquitoes is crucial for DENV replication [81,84]. The exogenous small interfering RNA (siRNA) pathway is widely acknowledged as a pivotal component of the arthropod antiviral immune response [65,85,86], we therefore quantified the mRNA expression level of the Dicer2 (Dcr2) gene, which encodes one of the key enzymes involved in RNAi pathway. ...
Background
Mosquito-borne arboviruses are expanding their territory and elevating their infection prevalence due to the rapid climate change, urbanization, and increased international travel and global trade. Various significant arboviruses, including the dengue virus, Zika virus, Chikungunya virus, and yellow fever virus, are all reliant on the same primary vector, Aedes aegypti . Consequently, the occurrence of arbovirus coinfection in mosquitoes is anticipated. Arbovirus coinfection in mosquitoes has two patterns: simultaneous and sequential. Numerous studies have demonstrated that simultaneous coinfection of arboviruses in mosquitoes is unlikely to exert mutual developmental influence on these viruses. However, the viruses’ interplay within a mosquito after the sequential coinfection seems intricated and not well understood.
Methodology/principal findings
We conducted experiments aimed at examining the phenomenon of arbovirus sequential coinfection in both mosquito cell line (C6/36) and A . aegypti , specifically focusing on dengue virus (DENV, serotype 2) and Zika virus (ZIKV). We firstly observed that DENV and ZIKV can sequentially infect mosquito C6/36 cell line, but the replication level of the subsequently infected ZIKV was significantly suppressed. Similarly, A . aegypti mosquitoes can be sequentially coinfected by these two arboviruses, regardless of the order of virus exposure. However, the replication, dissemination, and the transmission potential of the secondary virus were significantly inhibited. We preliminarily explored the underlying mechanisms, revealing that arbovirus-infected mosquitoes exhibited activated innate immunity, disrupted lipid metabolism, and enhanced RNAi pathway, leading to reduced susceptibility to the secondary arbovirus infections.
Conclusions/significance
Our findings suggest that, in contrast to simultaneous arbovirus coinfection in mosquitoes that can promote the transmission and co-circulation of these viruses, sequential coinfection appears to have limited influence on arbovirus transmission dynamics. However, it is important to note that more experimental investigations are needed to refine and expand upon this conclusion.
... FASN promotes palmitate synthesis, which can be used as a raw material for other lipid syntheses, thereby promoting the construction of viral envelopes and replication organelles [83]. Some enveloped viruses, including hepatitis B virus [84], dengue virus [85,86], hepatitis C virus [87], and West Nile virus [88,89], upregulate the expression and enhance FASN activity, whereas the knockout of FASN significantly impairs SARS-CoV-2 replication [83,90], while exogenous FA supplementation can rescue this damage [91]. The antiviral effects of cholesterol and its metabolizing enzymes or corresponding natural products are closely associated with various steps in CoV replication [11]. ...
Coronaviruses (CoVs) are emerging pathogens with a significant potential to cause life-threatening harm to human health. Since the beginning of the 21st century, three highly pathogenic and transmissible human CoVs have emerged, triggering epidemics and posing major threats to global public health. CoVs are enveloped viruses encased in a lipid bilayer. As fundamental components of cells, lipids can play an integral role in many physiological processes, which have been reported to play important roles in the life cycle of CoVs, including viral entry, uncoating, replication, assembly, and release. Therefore, research on the role of lipids in the CoV life cycle can provide a basis for a better understanding of the infection mechanism of CoVs and provide lipid targets for the development of new antiviral strategies. In this review, research advances on the role of lipids in different stages of viral infection and the possible targets of lipids that interfere with the viral life cycle are discussed.
... [15][16][17][18] Thus, inhibition of the fatty acid metabolism pathway can block virus entry, replication and assembly. 19,20 Several studies have indicated that inhibition of fatty acid synthase (FAS), which are rate-limiting enzymes in palmitic acid biosynthesis pathway, is capable of blocking multiple viral infections, including dengue virus, 21 28 rhinoviruses, 29 respiratory syncytial viruses 30 and SARS-CoV-2. 31,32 These researches mean that inhibition of FAS is expected to become a broad-spectrum antiviral approach. ...
Infectious diseases caused by viruses are one of the major diseases that seriously endanger global health. The development of broad-spectrum antiviral drugs for effective control of emerging and recurrent viral infectious diseases is urgently needed. Fatty acid synthase (FAS) has been reported as a valuable target for developing broad-spectrum antiviral drugs. In this study, we aimed to screen potent FAS inhibitors as broad-spectrum antiviral agents through computational approach. A pharmacophore model was generated with crystal structure of FAS in complex with Orlistat using Pharmit server, and 2,215 compounds were screened from ZINC15 database against the pharmacophore model. Next, 12 compounds exhibited more affinity to FAS than the control compound (Orlistat) were screened using molecular docking. The top three compounds (ZINC6146291, ZINC8925941 and ZINC3257427), which showed docking affinity to FAS with −8.19 kcal/mol, −8.14 kcal/mol and −8.07 kcal/mol, respectively, were subjected to 200 ns molecular dynamics simulation to evaluate the stability of docked complexes and their interaction mechanisms. The simulation trajectory analysis showed the stable conformation observed in FAS bound of the compounds and the screened compounds were firmly bound of the active sites of FAS throughout molecular dynamics simulation. The screened FAS candidate inhibitors can be used as broad-spectrum antiviral agents for in vitro-in vivo evaluations.
... Heaton and Randall (2010) showed that DENV infection not only leads to the processing of cellular lipid droplets and triglycerides, but also that the depletion of lipid droplets correlates to an increase of autophagosomes and stimulation of b-oxidation in infected cells, providing energy for DENV virus replication. While some authors have shown this reduction of lipid droplets in response to DENV infection, acting as an "energy sink" that is tapped during viral replication (Heaton and Randall, 2010;Tongluan et al., 2017), others have reported an increase in lipid droplets (Samsa et al., 2009;Soto-Acosta et al., 2014), that act as binding sites for the DENV C protein during DENV assembly Martins et al., 2012). ...
The clinical outcome of DENV and other Flaviviruses infections represents a spectrum of severity that ranges from mild manifestations to severe disease, which can ultimately lead to death. Nonetheless, most of these infections result in an asymptomatic outcome that may play an important role in the persistent circulation of these viruses. Also, although little is known about the mechanisms that lead to these asymptomatic infections, they are likely the result of a complex interplay between viral and host factors. Specific characteristics of the infecting viral strain, such as its replicating efficiency, coupled with host factors, like gene expression of key molecules involved in the immune response or in the protection against disease, are among crucial factors to study. This review revisits recent data on factors that may contribute to the asymptomatic outcome of the world’s widespread DENV, highlighting the importance of silent infections in the transmission of this pathogen and the immune status of the host.
... Others have found that acylcarnitines, a class of lipids, are altered in mosquitos by the presence of Wolbachia (47). Acylcarnitines have been found to help provide energy for the cell by transporting long-chain fatty acids into mitochondria (48), which leads to B-oxidation, found to be important for flavivirus replication (49). In Aedes aegypti mosquito cells, acylcarnitines have been found to be decreased in Wolbachia-containing cells, and depletion of acylcarnitines demonstrated increased Wolbachia density, while diminishing ZikV replication (47). ...
Zika virus is a member of the arbovirus Flaviviridae family transmitted by Aedes mosquitos and it is associated with microcephaly in infants born to infected mothers. Wolbachia is an intracellular gram-negative alpha-proteobacteria that infects many species of arthropods, including mosquitos. The presence of Wolbachia in mosquitos has been shown to control the vector population and suppress arbovirus transmission. One mechanism of Wolbachia -mediated interference with virus replication is competition over host resources between Wolbachia and the virus. We hypothesize that cholesterol metabolism is involved in Wolbachia -mediated virus suppression due to its important role in Zika virus replication. In this study, we determined that Wolbachia impacted virus replication by altering cholesterol biosynthesis in Aedes albopictus C6/36 cells, diverting resources from the host cell mevalonate (MVA) pathway to fulfill the needs of the bacteria. This resulted in a decrease of total cholesterol, increased Wolbachia loads, and decreased viral titers. Inhibition of the MVA pathway using fluvastatin decreased total cholesterol and viral titers, mimicking the effects of Wolbachia on the virus in Wolbachia- free cells. We also found that Wolbachia- infected cells had depleted lipid droplets, the main component of which is cholesterol esters. We confirmed that cholesterol esterases were upregulated in response to virus infection in C6/36 cells. Functional analysis showed that alteration of cholesterol metabolism simulated Wolbachia -mediated inhibition of virus infection in C6/36 cells. Our study provides a mechanism behind Wolbachia -induced interference of arbovirus replication and could help advance strategies to control arbovirus pathogens in insect vectors and human infections.
IMPORTANCE
Arthropod-borne viruses are emerging pathogens that are spread widely by mosquitos. Zika virus is an arbovirus that can infect humans and be transmitted from an infected mother to the fetus, potentially leading to microcephaly in infants. One promising strategy to prevent disease caused by arboviruses is to target the insect vector population. Recent field studies have shown that mosquito populations infected with Wolbachia bacteria suppress arbovirus replication and transmission. Here, we describe how intracellular bacteria redirect resources within their host cells and suppress Zika virus replication at the cellular level. Understanding the mechanism behind Wolbachia -induced interference of arbovirus replication could help advance strategies to control arbovirus pathogens in insect vectors and human populations.
... FAS, a major enzyme in lipogenesis, is activated in tumor cells for sustaining proliferation and in virus-infected cells for virion morphogenesis [45][46][47] . We previously found that FAS inhibition impairs virion morphogenesis but not viral protein expression 23 . ...
In addition to the Warburg effect, which increases the availability of energy and biosynthetic building blocks in WSSV-infected shrimp, WSSV also induces both lipolysis at the viral genome replication stage (12 hpi) to provide material and energy for the virus replication, and lipogenesis at the viral late stage (24 hpi) to complete virus morphogenesis by supplying particular species of long-chain fatty acids (LCFAs). Here, we further show that WSSV causes a reduction in lipid droplets (LDs) in hemocytes at the viral genome replication stage, and an increase in LDs in the nuclei of WSSV-infected hemocytes at the viral late stage. In the hepatopancreas, lipolysis is triggered by WSSV infection, and this leads to fatty acids being released into the hemolymph. β-oxidation inhibition experiment reveals that the fatty acids generated by WSSV-induced lipolysis can be diverted into β-oxidation for energy production. At the viral late stage, WSSV infection leads to lipogenesis in both the stomach and hepatopancreas, suggesting that fatty acids are in high demand at this stage for virion morphogenesis. Our results demonstrate that WSSV modulates lipid metabolism specifically at different stages to facilitate its replication.
... While there is no approved antiviral drug for ZIKV, we have previously shown anti-ZIKV activity of the FDA approved anti-obesity drug orlistat (tetrahydrolipstatin) [52], as well as established the EC 50 of this drug against the closely related flavivirus, dengue virus (DENV) [53]. ...
... Results showed that viral production decreased in a dosedependent manner (Fig. 2B), with an EC 50 of 109.68 µM, giving a selectivity index (SI) of 13.82. These results were consistent with our previous observations [52,53], and in particular the EC 50 value for ZIKV is very close to that previously determined for DENV (84.79 µM at 24 h posttreatment; [53]). ...
... Results showed that viral production decreased in a dosedependent manner (Fig. 2B), with an EC 50 of 109.68 µM, giving a selectivity index (SI) of 13.82. These results were consistent with our previous observations [52,53], and in particular the EC 50 value for ZIKV is very close to that previously determined for DENV (84.79 µM at 24 h posttreatment; [53]). ...
Background
Zika virus (ZIKV) is a mosquito transmitted virus spread primarily by Aedes species mosquitoes that can cause disease in humans, particularly when infection occurs in pregnancy where the virus can have a significant impact on the developing fetus. Despite this, there remains no prophylactic agent or therapeutic treatment for infection. Baicalein is a trihydroxyflavone, that is found in some traditional medicines commonly used in Asia, and has been shown to have several activities including antiviral properties. Importantly, studies have shown baicalein to be safe and well tolerated in humans, increasing its potential utilization.
Methods
This study sought to determine the anti-ZIKV activity of baicalein using a human cell line (A549). Cytotoxicity of baicalein was determined by the MTT assay, and the effect on ZIKV infection determined by treating A549 cells with baicalien at different time points in the infection process. Parameters including level of infection, virus production, viral protein expression and genome copy number were assessed by flow cytometry, plaque assay, western blot and quantitative RT-PCR, respectively.
Results
The results showed that baicalein had a half-maximal cytotoxic concentration (CC50) of > 800 µM, and a half-maximal effective concentration (EC50) of 124.88 µM. Time-of-addition analysis showed that baicalein had an inhibitory effect on ZIKV infection at the adsorption and post-adsorption stages. Moreover, baicalein also exerted a significant viral inactivation activity on ZIKV (as well as on dengue virus and Japanese encephalitis virus) virions.
Conclusion
Baicalein has now been shown to possess anti-ZIKV activity in a human cell line.
... Autophagy inhibitor 3-methyladenine (3MA) or siRNAs targeting autophagy gene expression compromised viral infection [11]. In addition, a small molecule inhibitor of FAS (C75) was reported to inhibit DENV replication in cell culture [14]. However, another FAS inhibitor (Orlistat) has weak antiviral activity and weak interaction between FAS and NS3 [15]. ...
... Lipids are widely used during dengue virus proliferation and viral assembly [14]. Therefore, we attempted to clarify which process was affected by CP640186. ...
Dengue fever is the most common mosquito-borne viral disease and is caused by the dengue virus (DENV). There is still a lack of efficient drugs against DENV infection, so it is urgent to develop new inhibitors for future clinical use. Our previous research indicated the role of VEGFR2/AMPK in regulating cellular metabolism during DENV infection, while acetyl-CoA carboxylase (ACC) is located downstream of AMPK and plays a crucial role in mediating cellular lipid synthesis; therefore, we speculated that an ACC inhibitor could serve as an antiviral agent against DENV. Luckily, we found that CP640186, a reported noncompetitive ACC inhibitor, significantly inhibited DENV proliferation, and CP640186 clearly reduced DENV2 proliferation at an early stage with an EC50 of 0.50 μM. A mechanism study indicated that CP640186 inhibited ACC activation and destroyed the cellular lipid environment for viral proliferation. In the DENV2 infection mice model, oral CP640186 administration (10 mg/kg/day) significantly improved the mice survival rate after DENV2 infection. In summary, our research suggests that lipid synthesis plays an important role during DENV2 proliferation and indicates that CP640186 is a promising drug candidate against DNEV2 in the future.
