The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein.

Coronaviridae is recognized as one of the most rapidly evolving virus family as a consequence of the high genomic nucleotide substitution rates and recombination. The family comprises a large number of enveloped, positive-sense single-stranded RNA viruses, causing an array of diseases of varying severity in animals and humans. To date, seven human coronaviruses (HCoV) have been identified, namely HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, which are globally circulating in the human population (seasonal HCoV, sHCoV), and the highly pathogenic SARS-CoV, MERS-CoV and SARS-CoV-2. Seasonal HCoV are estimated to contribute to 15-30% of common cold cases in humans; although diseases are generally self-limiting, sHCoV can sometimes cause severe lower respiratory infections and life-threatening diseases in a subset of patients. No specific treatment is presently available for sHCoV infections. Herein we show that the anti-infective drug nitazoxanide has a potent antiviral activity against three human endemic coronaviruses, the Alpha-coronaviruses HCoV-229E and HCoV-NL63, and the Beta-coronavirus HCoV-OC43 in cell culture with IC50 ranging between 0.05 and 0.15 µg/mL and high selectivity indexes. We found that nitazoxanide does not affect HCoV adsorption, entry or uncoating, but acts at postentry level and interferes with the spike glycoprotein maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Altogether the results indicate that nitazoxanide, due to its broad-spectrum anti-coronavirus activity, may represent a readily available useful tool in the treatment of seasonal coronavirus infections.

The roles of different forms of IL-15 in human melanoma progression.

Background: Melanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development. Methods: The expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both ex vivo and in vitro by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS). Results: Analysis of a melanoma tissue microarray shows a significant increase in the number of IL-15+ tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5+ and NKp46+ (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV. Conclusions: Membrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism.

MicroRNA analysis of Natural Killer cell-derived exosomes: the microRNA let-7b-5p is enriched in exosomes and participates in their anti-tumor effects against pancreatic cancer cells.

Natural Killer (NK) cells are important components of the immune system in the defense against tumor growth and metastasis. They release exosomes containing proteins and nucleic acids, including microRNAs (miRNAs). NK-derived exosomes play a role in the anti-tumor NK cell function since they are able to recognize and kill cancer cells. However, the involvement of exosomal miRNAs in the function of NK exosomes is poorly understood. In this study, we explored the miRNA content of NK exosomes by microarray as compared to their cellular counterparts. The expression of selected miRNAs and lytic potential of NK exosomes against childhood B acute lymphoblastic leukemia cells after co-cultures with pancreatic cancer cells were also evaluated. We identified a small subset of miRNAs, including miR-16-5p, miR-342-3p, miR-24-3p, miR-92a-3p and let-7b-5p that is highly expressed in NK exosomes. Moreover, we provide evidence that NK exosomes efficiently increase let-7b-5p expression in pancreatic cancer cells and induce inhibition of cell proliferation by targeting the cell cycle regulator CDK6. Let-7b-5p transfer by NK exosomes could represent a novel mechanism by which NK cells counteract tumor growth. However, both cytolytic activity and miRNA content of NK exosomes were reduced upon co-culture with pancreatic cancer cells. Alteration in the miRNA cargo of NK exosomes, together with their reduced cytotoxic activity, could represent another strategy exerted by cancer to evade the immune response. Our study provides new information on the molecular mechanisms used by NK exosomes to exert anti-tumor-activity and offers new clues to integrate cancer treatments with NK exosomes.

Impairment of SARS-CoV-2 spike glycoprotein maturation and fusion activity by nitazoxanide: an effect independent of spike variants emergence.

SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here, we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell-cell fusion assays, we show that nitazoxanide-induced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID-19 infections.

The proteostasis guardian HSF1 directs the transcription of its paralog and interactor HSF2 during proteasome dysfunction.

Protein homeostasis is essential for life in eukaryotes. Organisms respond to proteotoxic stress by activating heat shock transcription factors (HSFs), which play important roles in cytoprotection, longevity and development. Of six human HSFs, HSF1 acts as a proteostasis guardian regulating stress-induced transcriptional responses, whereas HSF2 has a critical role in development, in particular of brain and reproductive organs. Unlike HSF1, that is a stable protein constitutively expressed, HSF2 is a labile protein and its expression varies in different tissues; however, the mechanisms regulating HSF2 expression remain poorly understood. Herein we demonstrate that the proteasome inhibitor anticancer drug bortezomib (Velcade), at clinically relevant concentrations, triggers de novo HSF2 mRNA transcription in different types of cancers via HSF1 activation. Similar results were obtained with next-generation proteasome inhibitors ixazomib and carfilzomib, indicating that induction of HSF2 expression is a general response to proteasome dysfunction. HSF2-promoter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies unexpectedly revealed that HSF1 is recruited to a heat shock element located at 1.397 bp upstream from the transcription start site in the HSF2-promoter. More importantly, we found that HSF1 is critical for HSF2 gene transcription during proteasome dysfunction, representing an interesting example of transcription factor involved in controlling the expression of members of the same family. Moreover, bortezomib-induced HSF2 was found to localize in the nucleus, interact with HSF1, and participate in bortezomib-mediated control of cancer cell migration. The results shed light on HSF2-expression regulation, revealing a novel level of HSF1/HSF2 interplay that may lead to advances in pharmacological modulation of these fundamental transcription factors.

The biogenesis of SARS-CoV-2 spike glycoprotein: multiple targets for host-directed antiviral therapy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 (coronavirus disease-19), represents a far more serious threat to public health than SARS and MERS coronaviruses, due to its ability to spread more efficiently than its predecessors. Currently, there is no worldwide-approved effective treatment for COVID-19, urging the scientific community to intense efforts to accelerate the discovery and development of prophylactic and therapeutic solutions against SARS-CoV-2 infection. In particular, effective antiviral drugs are urgently needed. With few exceptions, therapeutic approaches to combat viral infections have traditionally focused on targeting unique viral components or enzymes; however, it has now become evident that this strategy often fails due to the rapid emergence of drug-resistant viruses. Targeting host factors that are essential for the virus life cycle, but are dispensable for the host, has recently received increasing attention. The spike glycoprotein, a component of the viral envelope that decorates the virion surface as a distinctive crown ("corona") and is essential for SARS-CoV-2 entry into host cells, represents a key target for developing therapeutics capable of blocking virus invasion. This review highlights aspects of the SARS-CoV-2 spike biogenesis that may be amenable to host-directed antiviral targeting.

The Zinc-Finger AN1-Type Domain 2a Gene Acts as a Regulator of Cell Survival in Human Melanoma: Role of E3-Ligase cIAP2.

The zinc-finger AN1-type domain-2a gene, also known as AIRAP (arsenite-inducible RNA-associated protein), was initially described as an arsenite-inducible gene in Caenorhabditis elegans and mammalian cells. Differently from the AIRAP worm homologue, aip-1, a gene known to play an important role in preserving animal lifespan and buffering arsenic-induced proteotoxicity, mammals have a second, constitutively expressed, AIRAP-like gene (AIRAPL), recently implicated in myeloid transformation. We have identified human AIRAP as a canonical heat-shock gene, whose expression, differently from AIRAPL, is strictly dependent on the proteotoxic-stress regulator heat-shock factor 1 (HSF1). AIRAP function is still not well defined and there is no information on AIRAP in cancer. Herein we show that bortezomib and next-generation proteasome inhibitors ixazomib and carfilzomib markedly induce AIRAP expression in human melanoma at concentrations comparable to plasma-levels in treated patients. AIRAP-downregulation leads to bortezomib sensitization, whereas AIRAP-overexpression protects melanoma cells from the drug, identifying AIRAP as a novel HSF1-regulated marker of chemotherapy resistance. More importantly, this study unexpectedly revealed that, also in the absence of drugs, AIRAP-silencing hinders melanoma clonogenic potential and spheroid growth, promoting caspase activation and apoptotic cell death, an effect independent of AIRAPL and linked to downregulation of the antiapoptotic protein cIAP2. Interestingly, AIRAP was found to interact with cIAP2, regulating its stability in melanoma. Taken together, the results identify AIRAP as a novel HSF1-dependent regulator of prosurvival networks in melanoma cells, opening new therapeutic perspectives in chemoresistant melanoma treatment. IMPLICATIONS: The findings identify ZFAND2A/AIRAP as a novel stress-regulated survival factor implicated in the stabilization of the antiapoptotic protein cIAP2 and as a new potential therapeutic target in melanoma.

DOF AFFECTING GERMINATION 2 is a positive regulator of light-mediated seed germination and is repressed by DOF AFFECTING GERMINATION 1.

BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of the light-mediated seed germination process. DAG1 acts downstream PHYTOCHROME INTERACTING FACTOR3-LIKE 5 (PIL5), the master repressor, and negatively regulates gibberellin biosynthesis by directly repressing the biosynthetic gene AtGA3ox1. The Dof protein DOF AFFECTING GERMINATION (DAG2) shares a high degree of aminoacidic identity with DAG1. While DAG1 inactivation considerably increases the germination capability of seeds, the dag2 mutant has seeds with a germination potential substantially lower than the wild-type, indicating that these factors may play opposite roles in seed germination. RESULTS: We show here that DAG2 expression is positively regulated by environmental factors triggering germination, whereas its expression is repressed by PIL5 and DAG1; by Chromatin Immuno Precipitation (ChIP) analysis we prove that DAG1 directly regulates DAG2. In addition, we show that Red light significantly reduces germination of dag2 mutant seeds. CONCLUSIONS: In agreement with the seed germination phenotype of the dag2 mutant previously published, the present data prove that DAG2 is a positive regulator of the light-mediated seed germination process, and particularly reveal that this protein plays its main role downstream of PIL5 and DAG1 in the phytochrome B (phyB)-mediated pathway.

Independent and interactive effects of DOF affecting germination 1 (DAG1) and the Della proteins GA insensitive (GAI) and Repressor of ga1-3 (RGA) in embryo development and seed germination.

BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of seed germination acting downstream of the master repressor PHYTOCROME INTERACTING FACTOR3-LIKE 5 (PIL5). Among others, PIL5 induces the expression of the genes encoding the two DELLA proteins GA INSENSITIVE 1 (GAI) and REPRESSOR OF ga1-3 (RGA). RESULTS: Based on the properties of gai-t6 and rga28 mutant seeds, we show here that the absence of RGA severely increases dormancy, while lack of GAI only partially compensates RGA inactivation. In addition, the germination properties of the dag1rga28 double mutant are different from those of the dag1 and rga28 single mutants, suggesting that RGA and DAG1 act in independent branches of the PIL5-controlled germination pathway. Surprisingly, the dag1gai-t6 double mutant proved embryo-lethal, suggesting an unexpected involvement of (a possible complex between) DAG1 and GAI in embryo development. CONCLUSIONS: Rather than overlapping functions as previously suggested, we show that RGA and GAI play distinct roles in seed germination, and that GAI interacts with DAG1 in embryo development.