Counteraction of HCV-induced oxidative stress concurs to establish chronic infection in liver cell cultures

Hepatitis C virus (HCV) is a blood-borne pathogen causing acute and chronic hepatitis. A significant number of people chronically infected with HCV develop cirrhosis and/or liver cancer. The pathophysiologic mechanisms of hepatocyte damage associated with chronic HCV infection are not fully understood yet, mainly due to the lack of an in vitro system able to recapitulate the stages of infection in vivo. Several studies underline that HCV virus replication depends on redox-sensitive cellular pathways; in addition, it is known that virus itself induces alterations of the cellular redox state. However, the exact interplay between HCV replication and oxidative stress has not been elucidated. In particular, the role of reduced glutathione (GSH) in HCV replication and infection is still not clear. We set up an in vitro system, based on low m.o.i. of Huh7.5 cell line with a HCV infectious clone (J6/JFH1), that reproduced the acute and persistent phases of HCV infection up to 76 days of culture. We demonstrated that the acute phase of HCV infection is characterized by the elevated levels of reactive oxygen species (ROS) associated in part with an increase of NADPH-oxidase transcripts and activity and a depletion of GSH accompanied by high rates of viral replication and apoptotic cell death. Conversely, the chronic phase is characterized by a reestablishment of reduced environment due to a decreased ROS production and increased GSH content in infected cells that might concur to the establishment of viral persistence. Treatment with the prooxidant auranofin of the persistently infected cultures induced the increase of viral RNA titer, suggesting that a prooxidant state could favor the reactivation of HCV viral replication that in turn caused cell damage and death. Our results suggest that targeting the redox-sensitive host-cells pathways essential for viral replication and/or persistence may represent a promising option for contrasting HCV infection

A polyphenol rich extract from Solanum melongena L. DR2 peel exhibits antioxidant properties and anti-herpes simplex virus type 1 activity in vitro

DR2B and DR2C extracts, obtained by ethanolic maceration of peel from commercially and physiologically ripe aubergine berries, were studied for the antioxidative cytoprotective properties and anti-HSV-1 activity, in line with the evidence that several antioxidants can impair viral replication by maintaining reducing conditions in host cells. The antioxidative cytoprotective effects against tBOOH-induced damage were assessed in Caco2 cells, while antiviral activity was studied in Vero cells; polyphenolic fingerprints were characterized by integrated phytochemical methods. Results highlighted different compositions of the extracts, with chlorogenic acid and delphinidin-3-rutinoside as the major constituents; other peculiar phytochemicals were also identified. Both samples reduced reactive oxygen species (ROS) production and exhibited scavenging and chelating properties. DR2C partly counteracted the tBOOH-induced cytotoxicity, with a remarkable lowering of lactate metabolism under both normoxia and hypoxia; interestingly, it increased intracellular GSH levels. Furthermore, DR2C inhibited the HSV-1 replication when added for 24 h after viral adsorption, as also confirmed by the reduction of many viral proteins’ expression. Since DR2C was able to reduce NOX4 expression during HSV-1 infection, its antiviral activity may be correlated to its antioxidant properties. Although further studies are needed to better characterize DR2C activity, the results suggest this extract as a promising new anti-HSV-1 agent

Influenza Virus Down-Modulates G6PD Expression and Activity to Induce Oxidative Stress and Promote Its Replication

none10no: Influenza virus infection induces oxidative stress in host cells by decreasing the intracellular content of glutathione (GSH) and increasing reactive oxygen species (ROS) level. Glucose-6-phosphate dehydrogenase (G6PD) is responsible for the production of reducing equivalents of nicotinamide adenine dinucleotide phosphate (NADPH) that is used to regenerate the reduced form of GSH, thus restoring redox homeostasis. Cells deficient in G6PD display elevated levels of ROS and an increased susceptibility to viral infection, although the consequences of G6PD modulation during viral infection remain to be elucidated. In this study, we demonstrated that influenza virus infection decreases G6PD expression and activity, resulting in an increase in oxidative stress and virus replication. Moreover, the down regulation of G6PD correlated with a decrease in the expression of nuclear factor erythroid 2-related factor 2 (NRF2), a key transcription factor that regulates the expression of the antioxidant response gene network. Also down-regulated in influenza virus infected cells was sirtuin 2 (SIRT2), a NADPH-dependent deacetylase involved in the regulation of G6PD activity. Acetylation of G6PD increased during influenza virus infection in a manner that was strictly dependent on SIRT2 expression. Furthermore, the use of a pharmacological activator of SIRT2 rescued GSH production and NRF2 expression, leading to decreased influenza virus replication. Overall, these data identify a novel strategy used by influenza virus to induce oxidative stress and to favor its replication in host cells. These observations furthermore suggest that manipulation of metabolic and oxidative stress pathways could define new therapeutic strategies to interfere with influenza virus infection.openDe Angelis, Marta; Amatore, Donatella; Checconi, Paola; Zevini, Alessandra; Fraternale, Alessandra; Magnani, Mauro; Hiscott, John; De Chiara, Giovanna; Palamara, Anna Teresa; Nencioni, LuciaDe Angelis, Marta; Amatore, Donatella; Checconi, Paola; Zevini, Alessandra; Fraternale, Alessandra; Magnani, Mauro; Hiscott, John; De Chiara, Giovanna; Palamara, Anna Teresa; Nencioni, Luci

Differential redox state contributes to sex disparities in the response to influenza virus infection in male and female mice

Influenza virus replicates intracellularly exploiting several pathways involved in the regulation of host responses. The outcome and the severity of the infection are thus strongly conditioned by multiple host factors, including age, sex, metabolic, and redox conditions of the target cells. Hormones are also important determinants of host immune responses to influenza and are recently proposed in the prophylaxis and treatment. This study shows that female mice are less susceptible than males to mouse-adapted influenza virus (A/PR8/H1N1). Compared with males, PR8-infected females display higher survival rate (+36%), milder clinical disease, and less weight loss. They also have milder histopathological signs, especially free alveolar area is higher than that in males, even if pro-inflammatory cytokine production shows slight differences between sexes; hormone levels, moreover, do not vary significantly with infection in our model. Importantly, viral loads (both in terms of viral M1 RNA copies and tissue culture infectious dose 50%) are lower in PR8-infected females. An analysis of the mechanisms contributing to sex disparities observed during infection reveals that the female animals have higher total antioxidant power in serum and their lungs are characterized by increase in (i) the content and biosynthesis of glutathione, (ii) the expression and activity of antioxidant enzymes (peroxiredoxin 1, catalase, and glutathione peroxidase), and (iii) the expression of the anti-apoptotic protein Bcl-2. By contrast, infected males are characterized by high expression of NADPH oxidase 4 oxidase and phosphorylation of p38 MAPK, both enzymes promoting viral replication. All these factors are critical for cell homeostasis and susceptibility to infection. Reappraisal of the importance of the host cell redox state and sex-related effects may be useful in the attempt to develop more tailored therapeutic interventions in the fight against influenza

Diketo acid inhibitors of nsp13 of SARS-CoV-2 block viral replication

For RNA viruses, RNA helicases have long been recognized to play critical roles during virus replication cycles, facilitating proper folding and replication of viral RNAs, therefore representing an ideal target for drug discovery. SARS-CoV-2 helicase, the non-structural protein 13 (nsp13) is a highly conserved protein among all known coronaviruses, and, at the moment, is one of the most explored viral targets to identify new possible antiviral agents. In the present study, we present six diketo acids (DKAs) as nsp13 inhibitors able to block both SARS-CoV-2 nsp13 enzymatic functions. Among them four compounds were able to inhibit viral replication in the low micromolar range, being active also on other human coronaviruses such as HCoV229E and MERS CoV. The experimental investigation of the binding mode revealed ATP-non-competitive kinetics of inhibition, not affected by substrate-displacement effect, suggesting an allosteric binding mode that was further supported by molecular modelling calculations predicting the binding into an allosteric conserved site located in the RecA2 domain