Influenza virus replication is affected by glutaredoxin1-mediated protein deglutathionylation.

Several redox modifications have been described during viral infection, including influenza virus infection, but little is known about glutathionylation and this respiratory virus. Glutathionylation is a reversible, post-translational modification, in which protein cysteine forms transient disulfides with glutathione (GSH), catalyzed by cellular oxidoreductases and in particular by glutaredoxin (Grx). We show here that (i) influenza virus infection induces protein glutathionylation, including that of viral proteins such as hemagglutinin (HA); (ii) Grx1-mediated deglutathionylation is important for the viral life cycle, as its inhibition, either with an inhibitor of its enzymatic activity or by siRNA, decreases viral replication. Overall these data contribute to the characterization of the complex picture of redox regulation of the influenza virus replication cycle and could help to identify new targets to control respiratory viral infection.

Effect of Cigarette Smoking on Clinical and Molecular Endpoints in COPD Patients.

Cigarette smoking is a primary contributor to mortality risks and is associated with various diseases. Among these, COPD represents a significant contributor to global mortality and disability. The objective of this study is to investigate the effect of smoking on a selected battery of variables, with an emphasis on DNA damage. A total of 87 elderly patients diagnosed with COPD, divided into three groups based on their smoking history (current, former, never-smokers), were evaluated using a cross-sectional approach. Clinical features including mortality and inflammatory/oxidative parameters (Lymphocytes/Monocytes, Neutrophils/Lymphocytes, Platelets/Lymphocytes ratio), SII, MDA, 8-Oxo-dG, and IL6 (ELISA assay), as well as DNA damage (comet assay), were investigated. Virus infection, i.e., influenza A virus subtype H1N1, JC polyomavirus (JCPyV), BK polyomavirus (BKPyV), and Torquetenovirus (TTV), was also tested. Current smokers exhibit higher levels of comorbidity (CIRS; p < 0.001), Platelets/Lymphocytes ratio (p < 0.001), systemic immune inflammation (p < 0.05), and DNA damage (p < 0.001). Former smokers also showed higher values for parameters associated with oxidative damage and showed a much lower probability of surviving over 5 years compared to never- and current smokers (p < 0.0017). This study showed a clear interaction between events which are relevant to the oxidative pathway and cigarette smoking. A category of particular interest is represented by former smokers, especially for lower survival, possibly due to the presence of more health problems. Our findings raise also the attention to other parameters which are significantly affected by smoking and are useful to monitor COPD patients starting a program of pulmonary rehabilitation (DNA damage, inflammation parameters, and selected viral infections).

Redox-Modulating Agents in the Treatment of Viral Infections.

Viruses use cell machinery to replicate their genome and produce viral proteins. For this reason, several intracellular factors, including the redox state, might directly or indirectly affect the progression and outcome of viral infection. In physiological conditions, the redox balance between oxidant and antioxidant species is maintained by enzymatic and non-enzymatic systems, and it finely regulates several cell functions. Different viruses break this equilibrium and induce an oxidative stress that in turn facilitates specific steps of the virus lifecycle and activates an inflammatory response. In this context, many studies highlighted the importance of redox-sensitive pathways as novel cell-based targets for therapies aimed at blocking both viral replication and virus-induced inflammation. In the review, we discuss the most recent findings in this field. In particular, we describe the effects of natural or synthetic redox-modulating molecules in inhibiting DNA or RNA virus replication as well as inflammatory pathways. The importance of the antioxidant transcription factor Nrf2 is also discussed. Most of the data reported here are on influenza virus infection. We believe that this approach could be usefully applied to fight other acute respiratory viral infections characterized by a strong inflammatory response, like COVID-19.

Regioselective IBX-Mediated Synthesis of Coumarin Derivatives with Antioxidant and Anti-influenza Activities.

Different catechol and pyrogallol derivatives have been synthesized by oxidation of coumarins with 2-iodoxybenzoic acid (IBX) in DMSO at 25 °C. A high regioselectivity was observed in accordance with the stability order of the incipient carbocation or radical benzylic-like intermediate. The oxidation was also effective in water under heterogeneous conditions by using IBX supported on polystyrene. The new derivatives showed improved antioxidant effects in the DPPH test and inhibitory activity against the influenza A/PR8/H1N1 virus. These data represent a new entry for highly oxidized coumarins showing an antiviral activity possibly based on the control of the intracellular redox value.

Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal.

The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the -SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.

Synthesis of 2'-deoxy-1'-homo-N-nucleosides with anti-influenza activity by catalytic methyltrioxorhenium (MTO)/H2O2 oxyfunctionalization.

This paper describes a new route for the synthesis of 1'-homo-N-nucleoside derivatives by means of either methyltrioxorhenium (MTO) or supported MTO catalysts, with H(2)O(2) as the primary oxidant. Under these selective conditions, the oxyfunctionalization of the heterocyclic ring and the N heteroatom oxidation were operative processes, regardless of the type of substrate used, that is, purine or pyrimidine derivatives. In addition, the oxidation of 1'-homo-N-thionucleosides, showed the occurrence of site-specific oxidative nucleophilic substitutions of the heterocyclic ring. The MTO/H(2)O(2) system showed, in general, high reactivity under both homogeneous and heterogeneous conditions, affording the final products with high conversion values of substrates and from medium to high yields. Many of the novel 1'-homo-N-nucleoside analogues were active against the influenza A virus, without any cytotoxic effects, retaining their activity in both protected and unprotected forms.

The Environmental Pollutant Cadmium Promotes Influenza Virus Replication in MDCK Cells by Altering Their Redox State.

Cadmium (Cd) is a toxic heavy metal that is considered an environmental contaminant. Several sources of human exposure to Cd, including employment in primary metal industries, production of certain batteries, foods, soil and cigarette smoke, are known. Its inhalation has been related to different respiratory diseases and toxic effects, among which alterations of the physiological redox state in individuals exposed to the metal have been described. Host-cell redox changes characteristic of oxidative stress facilitate the progression of viral infection through different mechanisms. In this paper, we have demonstrated that pre-treatment with CdCl(2) of MDCK cells increased influenza virus replication in a dose-dependent manner. This phenomenon was related to increased viral protein expression (about 40% compared with untreated cells). The concentration of CdCl(2), able to raise the virus titer, also induced oxidative stress. The addition of two antioxidants, a glutathione (GSH) derivative or the GSH precursor, N-acetyl-L-cysteine, to Cd pre-treated and infected cells restored the intracellular redox state and significantly inhibited viral replication. In conclusion, our data demonstrate that Cd-induced oxidative stress directly increases the ability of influenza virus to replicate in the host-cell, thus suggesting that exposure to heavy metals, such as this, could be a risk factor for individuals exposed to a greater extent to the contaminant, resulting in increased severity of virus-induced respiratory diseases.

Exploring the Role of CD74 and D-Dopachrome Tautomerase in COVID-19: Insights from Transcriptomic and Serum Analyses.

The COVID-19 pandemic has posed a significant threat to public health worldwide. While some patients experience only mild symptoms or no symptoms at all, others develop severe illness, which can lead to death. The host immune response is believed to play a crucial role in determining disease severity. In this study, we investigated the involvement of CD74 and D-DT in COVID-19 patients with different disease severities, by employing an in silico analysis of a publicly available transcriptomic dataset and by measuring their serum levels by ELISA. Our results showed a significant increase in MIF levels in PBMCs from COVID-19 patients, as well as a significant increase in the D-DT levels in PBMCs. However, we observed no modulation in the serum levels of D-DT. We also observed a concordant reduction in the serum levels and PBMCs expression levels of CD74. Furthermore, we found a negative correlation between CD74 serum levels and IL-13. In conclusion, our study sheds light on the involvement of CD74 and D-DT in COVID-19, with potential implications for disease severity and treatment. Further studies are needed to fully elucidate the mechanisms underlying these observations and to explore the potential therapeutic value of targeting CD74 and IL-13 in COVID-19.

Effects of polyphenol compounds on influenza A virus replication and definition of their mechanism of action.

A set of polyphenol compounds was synthesized and assayed for their ability in inhibiting influenza A virus replication. A sub-set of them showed low toxicity. The best compounds within this sub-set were 4 and 6g, which inhibited the viral replication in a dose-dependent manner. The antiviral activity of these molecules was demonstrated to be caused by their interference with intracellular pathways exploited for viral replication: (1) MAP kinases controlling nuclear-cytoplasmic traffic of viral ribonucleoprotein complex; (2) redox-sensitive pathways, involved in maturation of viral hemagglutinin protein.

Pepstatin A alters host cell autophagic machinery and leads to a decrease in influenza A virus production.

Autophagy is a survival mechanism that can take place in cells under metabolic stress and through which cells can recycle waste material. Disturbances in autophagic processes appear to be associated with a number of human pathologies, including viral infections. It has been hypothesized that viruses can subvert autophagy in order to penetrate the host cell and replicate. Because it has been suggested that autophagy is involved in influenza A virus replication, we analyzed the effects of two inhibitors of lysosomal proteases on the cellular control of influenza A virus replication. In particular, we used biochemical and morphological analyses to evaluate the modulation of influenza A/Puerto Rico/8/34 H1N1 virus production in the presence of CA074 and Pepstatin A, inhibitors of cathepsin proteases B and D, respectively. We found that Pepstatin A, but not CA074, significantly hindered influenza virus replication, probably by modulating host cell autophagic/apoptotic responses. These results are of potential interest to provide useful insights into the molecular pathways exploited by the influenza in order to replicate and to identify further cellular factors as targets for the development of innovative antiviral strategies.

BK Polyomavirus Activates HSF1 Stimulating Human Kidney Hek293 Cell Proliferation.

OBJECTIVES: Some DNA viruses, such as BKPyV, are capable of inducing neoplastic transformation in human tissues through still unclear mechanisms. The goal of this study is to investigate the carcinogenic potential of BK polyomavirus (BKPyV) in human embryonic kidney 293 (Hek293) cells, dissecting the molecular mechanism that determines the neoplastic transformation. MATERIALS AND METHODS: BKPyV, isolated from urine samples of infected patients, was used to infect monolayers of Hek293 cells. Subsequently, intracellular redox changes, GSH/GSSH concentration by HPLC, and reactive oxygen/nitrogen species (ROS/RNS) production were monitored. Moreover, to understand the signaling pathway underlying the neoplastic transformation, the redox-sensitive HFS1-Hsp27 molecular axis was examined using the flavonoid quercetin and polishort hairpin RNA technologies. RESULTS: The data obtained show that while BKPyV replication is closely linked to the transcription factor p53, the increase in Hek293 cell proliferation is due to the activation of the signaling pathway mediated by HSF1-Hsp27. In fact, its inhibition blocks viral replication and cell growth, respectively. CONCLUSIONS: The HSF1-Hsp27 signaling pathway is involved in BKPyV infection and cellular replication and its activation, which could be involved in cell transformation.

Protective Role of Combined Polyphenols and Micronutrients against Influenza A Virus and SARS-CoV-2 Infection In Vitro.

Polyphenols have been widely studied for their antiviral effect against respiratory virus infections. Among these, resveratrol (RV) has been demonstrated to inhibit influenza virus replication and more recently, it has been tested together with pterostilbene against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In the present work, we evaluated the antiviral activity of polydatin, an RV precursor, and a mixture of polyphenols and other micronutrients, named A5+, against influenza virus and SARS-CoV-2 infections. To this end, we infected Vero E6 cells and analyzed the replication of both respiratory viruses in terms of viral proteins synthesis and viral titration. We demonstrated that A5+ showed a higher efficacy in inhibiting both influenza virus and SARS-CoV-2 infections compared to polydatin treatment alone. Indeed, post infection treatment significantly decreased viral proteins expression and viral release, probably by interfering with any step of virus replicative cycle. Intriguingly, A5+ treatment strongly reduced IL-6 cytokine production in influenza virus-infected cells, suggesting its potential anti-inflammatory properties during the infection. Overall, these results demonstrate the synergic and innovative antiviral efficacy of A5+ mixture, although further studies are needed to clarify the mechanisms underlying its inhibitory effect.

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

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.

SARS-CoV-2: Comparative analysis of different RNA extraction methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.

Merkel Cell Polyomavirus (MCPyV) in the Context of Immunosuppression: Genetic Analysis of Noncoding Control Region (NCCR) Variability among a HIV-1-Positive Population.

BACKGROUND: Since limited data are available about the prevalence of Merkel cell polyomavirus (MCPyV) and the genetic variability of its noncoding control region (NCCR) in the context of immunosuppression, this study aimed to investigate the distribution of MCPyV in anatomical sites other than the skin and the behavior of NCCR among an HIV-1-positive population. METHODS: Urine, plasma, and rectal swabs specimens from a cohort of 66 HIV-1-positive patients were collected and subjected to quantitative real-time polymerase chain reaction (qPCR) for MCPyV DNA detection. MCPyV-positive samples were amplified by nested PCR targeting the NCCR, and NCCRs alignment was carried out to evaluate the occurrence of mutations and to identify putative binding sites for cellular factors. RESULTS: MCPyV DNA was detected in 10/66 urine, in 7/66 plasma, and in 23/66 rectal samples, with a median value of 5 × 102 copies/mL, 1.5 × 102 copies/mL, and 2.3 × 103 copies/mL, respectively. NCCR sequence analysis revealed a high degree of homology with the MCC350 reference strain in urine, whereas transitions, transversions, and single or double deletions were observed in plasma and rectal swabs. In these latter samples, representative GTT and GTTGA insertions were also observed. Search for putative binding sites of cellular transcription factors showed that in several strains, deletions, insertions, or single base substitutions altered the NCCR canonical configuration. CONCLUSIONS: Sequencing analysis revealed the presence of numerous mutations in the NCCR, including insertions and deletions. Whether these mutations may have an impact on the pathogenic features of the virus remains to be determined. qPCR measured on average a low viral load in the specimens analyzed, with the exception of those with the GTTGA insertion.

Glutathione and Nitric Oxide: Key Team Players in Use and Disuse of Skeletal Muscle.

Glutathione (GSH) is the main non-enzymatic antioxidant playing an important role in detoxification, signal transduction by modulation of protein thiols redox status and direct scavenging of radicals. The latter function is not only performed against reactive oxygen species (ROS) but GSH also has a fundamental role in buffering nitric oxide (NO), a physiologically-produced molecule having-multifaceted functions. The efficient rate of GSH synthesis and high levels of GSH-dependent enzymes are characteristic features of healthy skeletal muscle where, besides the canonical functions, it is also involved in muscle contraction regulation. Moreover, NO production in skeletal muscle is a direct consequence of contractile activity and influences several metabolic myocyte pathways under both physiological and pathological conditions. In this review, we will consider the homeostasis and intersection of GSH with NO and then we will restrict the discussion on their role in processes related to skeletal muscle function and degeneration.

Role of Glutathionylation in Infection and Inflammation.

Glutathionylation, that is, the formation of mixed disulfides between protein cysteines and glutathione (GSH) cysteines, is a reversible post-translational modification catalyzed by different cellular oxidoreductases, by which the redox state of the cell modulates protein function. So far, most studies on the identification of glutathionylated proteins have focused on cellular proteins, including proteins involved in host response to infection, but there is a growing number of reports showing that microbial proteins also undergo glutathionylation, with modification of their characteristics and functions. In the present review, we highlight the signaling role of GSH through glutathionylation, particularly focusing on microbial (viral and bacterial) glutathionylated proteins (GSSPs) and host GSSPs involved in the immune/inflammatory response to infection; moreover, we discuss the biological role of the process in microbial infections and related host responses.

Corrigendum: GSH-C4 Acts as Anti-inflammatory Drug in Different Models of Canonical and Cell Autonomous Inflammation Through NFκB Inhibition.

[This corrects the article DOI: 10.3389/fimmu.2019.00155.].

Glutathione increase by the n-butanoyl glutathione derivative (GSH-C4) inhibits viral replication and induces a predominant Th1 immune profile in old mice infected with influenza virus.

During aging, glutathione (GSH) content declines and the immune system undergoes a deficiency in the induction of Th1 response. Reduced secretion of Th1 cytokines, which is associated with GSH depletion, could weaken the host defenses against viral infections. We first evaluated the concentration of GSH and cysteine in organs of old mice; then, the effect of the administration of the N-butanoyl GSH derivative (GSH-C4) on the response of aged mice infected with influenza A PR8/H1N1 virus was studied through the determination of GSH concentration in organs, lung viral titer, IgA and IgG1/IgG2a production, and Th1/Th2 cytokine profile. Old mice had lower GSH than young mice in organs. Also the gene expression of endoplasmic reticulum (ER) stress markers involved in GSH metabolism and folding of proteins, that is, Nrf2 and PDI, was reduced. Following infection, GSH content remained low and neither infection nor GSH-C4 treatment affected Nrf2 expression. In contrast, PDI expression was upregulated during infection and appeared counterbalanced by GSH-C4. Moreover, the treatment with GSH-C4 increased GSH content in organs, reduced viral replication and induced a predominant Th1 response. In conclusion, GSH-C4 treatment could be used in the elderly to contrast influenza virus infection by inducing immune response, in particular the Th1 profile.