Lucia Nencioni

Inhibition of Influenza A Virus Replication by Resveratrol

We have previously shown that the life cycles of several viruses are influenced by host-cell redox states. Reports of the antioxidant activities of the plant polyphenol resveratrol (RV) prompted us to investigate its effects on influenza virus replication in vitro and in vivo. We found that RV strongly inhibited the replication of influenza virus in MDCK cells but that this activity was not directly related to glutathione-mediated antioxidant activity. Rather, it involved the blockade of the nuclear-cytoplasmic translocation of viral ribonucleoproteins and reduced expression of late viral proteins seemingly related to the inhibition of protein kinase C activity and its dependent pathways. RV also significantly improved survival and decreased pulmonary viral titers in influenza virus-infected mice. No toxic effects were observed in vitro or in vivo. That RV acts by inhibiting a cellular, rather than a viral, function suggests that it could be a particularly valuable anti-influenza drug.

Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2

Growing evidence indicates that viral replication is regulated by the redox state of the host cell. We demonstrate that cells of different origins display differential permissivity for influenza A virus replication, depending on their intracellular redox power as reflected by Bcl‐2 expression and glutathione (GSH) content. Bcl‐2 expressing cells were found to have higher intracellular levels of GSH and to produce lower amounts of virus than Bcl‐2 negative cells. Two different steps in the virus life‐cycle were involved in Bcl‐2/GSH mediated viral inhibition: 1) expression of late viral proteins (in particular hemagglutinin and matrix); and 2) nuclear‐cytoplasmic translocation of viral ribonucleoproteins (vRNPs). Buthionine‐sulfoximine‐induced inhibition of GSH synthesis in Bcl‐2 expressing cells caused an increase in the expression of late viral proteins but did not restore vRNP export to the cytoplasm. Collectively, our findings show that both Bcl‐2 expression and GSH content contribute to the host cells ability to down‐regulate influenza virus replication, although their effects are exerted at different stages of the viral life‐cycle. In certain cell populations, this form of down‐regulation might conceivably favor the establishment of persistent viral infection.

GSH and analogs in antiviral therapy

Reduced glutathione (GSH) is the most prevalent non-protein thiol in animal cells. Its de novo and salvage synthesis serves to maintain a reduced cellular environment. GSH is the most powerful intracellular antioxidant and plays a role in the detoxification of a variety of electrophilic compounds and peroxides via catalysis by glutathione-S-transferases (GST) and glutathione peroxidases (GPx). As a consequence, the ratio of reduced and oxidized glutathione (GSH:GSSG) serves as a representative marker of the antioxidative capacity of the cell. A deficiency in GSH puts the cell at risk for oxidative damage. An imbalance in GSH is observed in a wide range of pathologies, such as cancer, neurodegenerative diseases, cystic fibrosis (CF), several viral infections including HIV-1, as well as in aging. Several reports have provided evidence for the use of GSH and molecules able to replenish intracellular GSH levels in antiviral therapy. This non-conventional role of GSH and its analogs as antiviral drugs is discussed in this chapter.

New insights on human polyomavirus JC and pathogenesis of progressive multifocal leukoencephalopathy.

John Cunningham virus (JCV) is a member of the Polyomaviridae family. It was first isolated from the brain of a patient with Hodgkin disease in 1971, and since then the etiological agent of the progressive multifocal leukoencephalopathy (PML) was considered. Until the human immunodeficiency virus (HIV) pandemic, PML was rare: in fact HIV-induced immunodeficiency is the most common predisposing factor accounting for 85% of all instances of PML. This data led to intense research on JCV infection and resulted in better understanding of epidemiology and clinic-pathologic spectrum. Recently, cases of PML have been observed after the introduction of monoclonal antibodies, such as natalizumab, rituximab, efalizumab, and infliximab, in the treatment of autoimmune disease, underlining the important role of host immunity in PML pathogenesis. In this review current understanding of the JCV infection and the new findings relating to the pathogenesis of PML has been comprehensively revised, focusing our attention on the interaction between the cellular and viral molecular pathways implicated in the JCV infection and the modulating role of host immune surveillance in the viral reactivation from a latent state.

Bcl-2 Expression and p38MAPK Activity in Cells Infected with Influenza A Virus IMPACT ON VIRALLY INDUCED APOPTOSIS AND VIRAL REPLICATION

Previous reports have shown that various steps in the influenza A virus life cycle are impaired in cells expressing the antiapoptotic protein Bcl-2 (Bcl-2+ cells). We demonstrated a direct link between Bcl-2 and the reduced nuclear export of viral ribonucleoprotein (vRNP) complexes in these cells. However, despite its negative impact on viral replication, Bcl-2 did not prevent host cells from undergoing virally triggered apoptosis. The proteins reduced antiapoptotic capacity was related to phosphorylation of its threonine 56 and serine 87 residues by virally activated p38MAPK. In infected Bcl-2+ cells, activated p38MAPK was found predominantly in the cytoplasm, colocalized with Bcl-2, and both Bcl-2 phosphorylation and virally induced apoptosis were diminished by specific inhibition of p38MAPK activity. In contrast, in Bcl-2-negative (Bcl-2−) cells, which are fully permissive to viral infection, p38MAPK activity was predominantly nuclear, and its inhibition decreased vRNP traffic, phosphorylation of viral nucleoprotein, and virus titers in cell supernatants, suggesting that this kinase also contributes to the regulation of vRNP export and viral replication. This could explain why in Bcl-2+ cells, where p38MAPK is active in the cytoplasm, phosphorylating Bcl-2, influenza viral replication is substantially reduced, whereas apoptosis proceeds at rates similar to those observed in Bcl-2− cells. Our findings suggest that the impact of p38MAPK on the influenza virus life cycle and the apoptotic response of host cells to infection depends on whether or not the cells express Bcl-2, highlighting the possibility that the pathological effects of the virus are partly determined by the cell type it targets.

Higher Prevalence and Abundance of Bdellovibrio bacteriovorus in the Human Gut of Healthy Subjects

Introduction Members of the human intestinal microbiota are key players in maintaining human health. Alterations in the composition of gut microbial community (dysbiosis) have been linked with important human diseases. Understanding the underlying processes that control community structure, including the bacterial interactions within the microbiota itself, is essential. Bdellovibrio bacteriovorus is a gram-negative bacterium that preys other gram-negative species for survival, acting as a population-balancer. It was found in terrestrial/aquatic ecosystems, and in animal intestines, postulating its presence also in the human gut. Methods The present study was aimed to evaluate, by end-point PCR and qPCR, the presence of B. bacteriovorus in intestinal and faecal biopsy specimens from 92 paediatric healthy subjects and patients, suffering from Inflammatory Bowel Diseases (IBD), Celiac disease and Cystic fibrosis (CF). Results i) B. bacteriovorus was present and abundant only in healthy individuals, while it was heavily reduced in patients, as in the case of IBD and Celiac, while in CF patients and relative controls we observed comparable results; ii) B. bacteriovorus seemed to be mucosa-associated, because all IBD and Celiac biopsies (and related controls) were treated with mucus-removing agents, leaving only the mucosa-attached microflora; iii) B. bacteriovorus abundance was district-dependent, with a major preponderance in duodenum, and gradually decreasing up to rectum; iv) B. bacteriovorus levels significantly dropped in disease status, in duodenum and ileum. Conclusions Results obtained in this study could represent the first step for new therapeutic strategies aimed to restore a balance in the intestinal ecosystem, utilizing Bdellovibrio as a probiotic.

Influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by NOX4-derived ROS.

An overproduction of reactive oxygen species (ROS) mediated by NADPH oxidase 2 (NOX2) has been related to airway inflammation typical of influenza infection. Virus‐induced oxidative stress may also control viral replication, but the mechanisms underlying ROS production, as well as their role in activating intracellular pathways and specific steps of viral life cycle under redox control have to be fully elucidated. In this study, we demonstrate that influenza A virus infection of lung epithelial cells causes a significant ROS increase that depends mainly on NOX4, which is upregulated at both mRNA and protein levels, while the expression of NOX2, the primary source of ROS in inflammatory cells, is downregulated. Inhibition of NOX4 activity through chemical inhibitors or RNA silencing blocks the ROS increase, prevents MAPK phosphorylation, and inhibits viral ribonucleoprotein (vRNP) nuclear export and viral release. Overall these data, obtained in cell lines and primary culture, describe a so far unrecognized role for NOX4‐derived ROS in activating redox‐regulated intracellular pathways during influenza virus infection and highlight their relevance in controlling specific steps of viral replication in epithelial cells. Pharmacological modulation of NOX4‐mediated ROS production may open the way for new therapeutic approaches to fighting influenza by targeting cell and not the virus.

Current Advances in Anti-Influenza Therapy

Every year, influenza epidemics cause numerous deaths and millions of hospitalizations, but the most frightening effects are seen when new strains of the virus emerge from different species (e.g. the swine-origin influenza A/H1N1 virus), causing world-wide outbreaks of infection. Several antiviral compounds have been developed against influenza virus to interfere with specific events in the replication cycle. Among them, the inhibitors of viral uncoating (amantadine), nucleoside inhibitors (ribavirin), viral transcription and neuraminidase inhibitors (zanamivir and oseltamivir) are reported as examples of traditional virus-based antiviral strategies. However, for most of them the efficacy is often limited by toxicity and the almost inevitable selection of drug-resistant viral mutants. Thus, the discovery of novel anti-influenza drugs that target general cell signaling pathways essential for viral replication, irrespective to the specific origin of the virus, would decrease the emergence of drug resistance and increase the effectiveness towards different strains of influenza virus. In this context, virus-activated intracellular cascades, finely regulated by small changes in the intracellular redox state, can contribute to inhibit influenza virus replication and pathogenesis of virus-induced disease. This novel therapeutic approach involves advanced cell-based antiviral strategies. In this review current advances in the anti-influenza therapy for both traditional virus-based antiviral strategies as well as for alternative cell-based antiviral strategies are described focusing on the last 10 years. Anti-influenza compounds are classified on the basis of their chemical structure with a special attention to describe their synthetic pathways and the corresponding structure activity relationships.

Novel Quinolinonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design, Synthesis, and Biological Activities

Novel quinolinonyl diketo acids were designed to obtain integrase (IN) inhibitors selectively active against the strand transfer (ST) step of the HIV integration process. Those new compounds are characterized by a single aryl diketo acid (DKA) chain in comparison to 4, a bifunctional diketo acid reported by our group as an anti-IN agent highly potent against both the 3′-processing and ST steps. Compound 6d was the most potent derivative in IN enzyme assays, while 6i showed the highest potency against HIV-1 in acutely infected cells. The selective inhibition of ST suggested the newly designed monofunctional DKAs bind the IN−DNA acceptor site without affecting the DNA donor site.

Sex differences in the response to viral infections: TLR8 and TLR9 ligand stimulation induce higher IL10 production in males.

Background Susceptibility to viral infections as well as their severity are higher in men than in women. Heightened antiviral responses typical of women are effective for rapid virus clearance, but if excessively high or prolonged, can result in chronic/inflammatory pathologies. We investigated whether this variability could be in part attributable to differences in the response to the Toll-Like Receptors (TLR) more involved in the virus recognition. Methods Cytokine production by peripheral blood mononuclear cells (PBMCs) from male and female healthy donors after stimulation with Toll-like receptors (TLR) 3, 7, 8, 9 ligands or with viruses (influenza and Herpes-simplex-1) was evaluated. Results Compared to females, PBMCs from males produced not only lower amounts of IFN-α in response to TLR7 ligands but also higher amounts of the immunosuppressive cytokine IL10 after stimulation with TLR8 and TLR9 ligands or viruses. IL10 production after TLR9 ligands or HSV-1 stimulation was significantly related with plasma levels of sex hormones in both groups, whereas no correlation was found in cytokines produced following TLR7 and TLR8 stimulation. Conclusions Given the role of an early production of IL10 by cells of innate immunity in modulating innate and adaptive immune response to viruses, we suggest that sex-related difference in its production following viral nucleic acid stimulation of TLRs may be involved in the sex-related variability in response to viral infections.