Shikonin, an inhibitor of inflammasomes, inhibits Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV) is a highly prevalent human herpesvirus that persists for life in more than 95% of the adult population. EBV usually establishes an asymptomatic life-long infection, but it is also associated with malignancies affecting B lymphocytes and epithelial cells mainly. The virus alternates between a latent phase and a lytic phase, both of which contribute to the initiation of the tumor process. So far, there is only a limited number of antiviral molecules against the lytic phase, most of them targeting viral replication. Recent studies provided evidence that EBV uses components of the NLRP3 inflammasome to enter the productive phase of its cycle following activation in response to various stimuli. In the present work, we demonstrate that shikonin, a natural molecule with low toxicity which is known to inhibit inflammasome, can efficiently repress EBV reactivation. Similar results were obtained with apigenin and OLT 1177, two other NLRP3 inflammasome inhibitors. It is shown herein that shikonin repressed the transcription of reactivation-induced NLRP3 thereby inhibiting inflammasome activation and EBV lytic phase induction.

The C-Terminal Acidic Tail Modulates the Anticancer Properties of HMGB1.

Energy metabolism reprogramming was recently listed as a hallmark of cancer. In this process, the switch from pyruvate kinase isoenzyme type M1 to pyruvate kinase isoenzyme type M2 (PKM2) is believed to play a crucial role. Interestingly, the activity of the active form of PKM2 can efficiently be inhibited by the high-mobility group box 1 (HMGB1) protein, leading to a rapid blockage of glucose-dependent aerobic respiration and cancer cell death. HMGB1 is a member of the HMG protein family. It contains two DNA-binding HMG-box domains and an acidic C-terminal tail capable of positively or negatively modulating its biological properties. In this work, we report that the deletion of the C-terminal tail of HMGB1 increases its activity towards a large panel of cancer cells without affecting the viability of normal immortalized fibroblasts. Moreover, in silico analysis suggests that the truncated form of HMGB1 retains the capacity of the full-length protein to interact with PKM2. However, based on the capacity of the cells to circumvent oxidative phosphorylation inhibition, we were able to identify either a cytotoxic or cytostatic effect of the proteins. Together, our study provides new insights in the characterization of the anticancer activity of HMGB1.

Essential role of hyperacetylated microtubules in innate immunity escape orchestrated by the EBV-encoded BHRF1 protein.

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This "mito-aggresome" is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.

Methyl-qPCR: a new method to investigate Epstein-Barr virus infection in post-transplant lymphoproliferative diseases.

Epstein-Barr virus DNA viral load is used as a surrogate marker to start Rituximab in transplant recipients at risk of developing PTLD. However, an elevated EBV DNAemia does not discriminate lymphoproliferation and replication. We designed a new molecular assay (methyl-qPCR) to distinguish methylated versus unmethylated viral genomes. In blood, viral genomes were highly methylated in EBV primary infections, PTLD and 4/5 transplant recipients with high viral load. The only patient with under-methylated EBV genomes did not respond to rituximab. Methyl-qPCR is a convenient method to discriminate between latent and lytic EBV genomes and could be useful in treatment decisions.

Expression Pattern of Purinergic Signaling Components in Colorectal Cancer Cells and Differential Cellular Outcomes Induced by Extracellular ATP and Adenosine.

The purine nucleotide adenosine triphosphate (ATP) is known for its fundamental role in cellular bioenergetics. However, in the last decades, different works have described emerging functions for ATP, such as that of a danger signaling molecule acting in the extracellular space on both tumor and stromal compartments. Beside its role in immune cell signaling, several studies have shown that high concentrations of extracellular ATP can directly or indirectly act on cancer cells. Accordingly, it has been reported that purinergic receptors are widely expressed in tumor cells. However, their expression pattern is often associated with contradictory cellular outcomes. In this work, we first investigated gene expression profiles through "RNA-Sequencing" (RNA Seq) technology in four colorectal cancer (CRC) cell lines (HT29, LS513, LS174T, HCT116). Our results demonstrate that CRC cells mostly express the A2B, P2X4, P2Y1, P2Y2 and P2Y11 purinergic receptors. Among these, the P2Y1 and P2Y2 coding genes are markedly overexpressed in all CRC cells compared to the HCEC-1CT normal-like colonic cells. We then explored the cellular outcomes induced by extracellular ATP and adenosine. Our results show that in terms of cell death induction extracellular ATP is consistently more active than adenosine against CRC, while neither compound affected normal-like colonic cell survival. Intriguingly, while for the P2Y2 receptor pharmacological inhibition completely abolished the rise in cytoplasmic Ca2+ observed after ATP exposure in all CRC cell lines, Ca2+ mobilization only impacted the cellular outcome for HT29. In contrast, non-selective phosphodiesterase inhibition completely abolished the effects of extracellular ATP on CRC cells, suggesting that cAMP and/or cGMP levels might determine cellular outcome. Altogether, our study provides novel insights into the characterization of purinergic signaling in CRC.

The COVID-19 Pandemic and the Need for an Integrated and Equitable Approach: An International Expert Consensus Paper.

BACKGROUND: One year after the declaration of the coronavirus disease 2019 (COVID-19) pandemic by the World Health Organization (WHO) and despite the implementation of mandatory physical barriers and social distancing, humanity remains challenged by a long-lasting and devastating public health crisis. MANAGEMENT: Non-pharmacological interventions (NPIs) are efficient mitigation strategies. The success of these NPIs is dependent on the approval and commitment of the population. The launch of a mass vaccination program in many countries in late December 2020 with mRNA vaccines, adenovirus-based vaccines, and inactivated virus vaccines has generated hope for the end of the pandemic. CURRENT ISSUES: The continuous appearance of new pathogenic viral strains and the ability of vaccines to prevent infection and transmission raise important concerns as we try to achieve community immunity against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and its variants. The need of a second and even third generation of vaccines has already been acknowledged by the WHO and governments. PERSPECTIVES: There is a critical and urgent need for a balanced and integrated strategy for the management of the COVID-19 outbreaks organized on three axes: (1) Prevention of the SARS-CoV-2 infection, (2) Detection and early diagnosis of patients at risk of disease worsening, and (3) Anticipation of medical care (PDA). CONCLUSION: The "PDA strategy" integrated into state policy for the support and expansion of health systems and introduction of digital organizations (i.e., telemedicine, e-Health, artificial intelligence, and machine-learning technology) is of major importance for the preservation of citizens' health and life world-wide.

Epstein-Barr Virus BALF0 and BALF1 Modulate Autophagy.

Autophagy is an essential catabolic process that degrades cytoplasmic components within the lysosome, therefore ensuring cell survival and homeostasis. A growing number of viruses, including members of the Herpesviridae family, have been shown to manipulate autophagy to facilitate their persistence or optimize their replication. Previous works showed that the Epstein-Barr virus (EBV), a human transforming gammaherpesvirus, hijacked autophagy during the lytic phase of its cycle, possibly to favor the formation of viral particles. However, the viral proteins that are responsible for an EBV-mediated subversion of the autophagy pathways remain to be characterized. Here we provide the first evidence that the BALF0/1 open reading frame encodes for two conserved proteins of the Bcl-2 family, BALF0 and BALF1, that are expressed during the early phase of the lytic cycle and can modulate autophagy. A putative LC3-interacting region (LIR) has been identified that is required both for BALF1 colocalization with autophagosomes and for its ability to stimulate autophagy.

Dipyridamole as a new drug to prevent Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV) is a widely distributed gamma-herpesvirus that has been associated with various cancers mainly from lymphocytic and epithelial origin. Although EBV-mediated oncogenesis has been associated with viral oncogenes expressed during latency, a growing set of evidence suggested that antiviral treatments directed against EBV lytic phase may contribute to prevent some forms of cancers, including EBV-positive Post-Transplant Lymphoproliferative Diseases. It is shown here that dipyridamole (DIP), a safe drug with favorable and broad pharmacological properties, inhibits EBV reactivation from B-cell lines. DIP repressed immediate early and early genes expression mostly through its ability to inhibit nucleoside uptake. Considering its wide clinical use, DIP repurposing could shortly be evaluated, alone or in combination with other antivirals, to treat EBV-related diseases where lytic replication plays a deleterious role.

Detection of IgG directed against a recombinant form of Epstein-Barr virus BALF0/1 protein in patients with nasopharyngeal carcinoma.

BALF0/1 is a putative Epstein-Barr virus (EBV) protein that has been described as a modulator of apoptosis. So far, the lack of specific immunological reagents impaired the detection of native BALF0/1 in EBV-infected cells. This study describes the expression and purification of a truncated form of BALF0/1 (tBALF0) using a heterologous bacterial expression system. tBALF0 was further used as an antigen in an indirect Enzyme-linked Immunosorbent Assay (ELISA) that unraveled the presence of low titer IgGs to BALF0/1 during primary (10.0%) and past (13.3%) EBV infection. Conversely high-titer IgGs to BALF0/1 were detected in 33.3% of nasopharyngeal carcinoma (NPC) patients suggesting that BALF0/1 and/or humoral response against it may contribute to NPC pathogenesis.

TLR9 activation is triggered by the excess of stimulatory versus inhibitory motifs present in Trypanosomatidae DNA.

DNA sequences purified from distinct organisms, e.g. non vertebrate versus vertebrate ones, were shown to differ in their TLR9 signalling properties especially when either mouse bone marrow-derived- or human dendritic cells (DCs) are probed as target cells. Here we found that the DC-targeting immunostimulatory property of Leishmania major DNA is shared by other Trypanosomatidae DNA, suggesting that this is a general trait of these eukaryotic single-celled parasites. We first documented, in vitro, that the low level of immunostimulatory activity by vertebrate DNA is not due to its limited access to DCs' TLR9. In addition, vertebrate DNA inhibits the activation induced by the parasite DNA. This inhibition could result from the presence of competing elements for TLR9 activation and suggests that DNA from different species can be discriminated by mouse and human DCs. Second, using computational analysis of genomic DNA sequences, it was possible to detect the presence of over-represented inhibitory and under-represented stimulatory sequences in the vertebrate genomes, whereas L. major genome displays the opposite trend. Interestingly, this contrasting features between L. major and vertebrate genomes in the frequency of these motifs are shared by other Trypanosomatidae genomes (Trypanosoma cruzi, brucei and vivax). We also addressed the possibility that proteins expressed in DCs could interact with DNA and promote TLR9 activation. We found that TLR9 is specifically activated with L. major HMGB1-bound DNA and that HMGB1 preferentially binds to L. major compared to mouse DNA. Our results highlight that both DNA sequence and vertebrate DNA-binding proteins, such as the mouse HMGB1, allow the TLR9-signaling to be initiated and achieved by Trypanosomatidae DNA.

Stepwise release of biologically active HMGB1 during HSV-2 infection.

BACKGROUND: High mobility group box 1 protein (HMGB1) is a major endogenous danger signal that triggers inflammation and immunity during septic and aseptic stresses. HMGB1 recently emerged as a key soluble factor in the pathogenesis of various infectious diseases, but nothing is known of its behaviour during herpesvirus infection. We therefore investigated the dynamics and biological effects of HMGB1 during HSV-2 infection of epithelial HEC-1 cells. METHODOLOGY/PRINCIPAL FINDINGS: Despite a transcriptional shutdown of HMGB1 gene expression during infection, the intracellular pool of HMGB1 protein remained unaffected, indicating its remarkable stability. However, the dynamics of HMGB1 was deeply modified in infected cells. Whereas viral multiplication was concomitant with apoptosis and HMGB1 retention on chromatin, a subsequent release of HMGB1 was observed in response to HSV-2 mediated necrosis. Importantly, extracellular HMGB1 was biologically active. Indeed, HMGB1-containing supernatants from HSV-2 infected cells induced the migration of fibroblasts from murine or human origin, and reactivated HIV-1 from latently infected T lymphocytes. These effects were specifically linked to HMGB1 since they were blocked by glycyrrhizin or by a neutralizing anti-HMGB1 antibody, and were mediated through TLR2 and the receptor for Advanced Glycation End-products (RAGE). Finally, we show that genital HSV-2 active infections also promote HMGB1 release in vivo, strengthening the clinical relevance of our experimental data. CONCLUSIONS: These observations target HMGB1 as an important actor during HSV-2 genital infection, notably in the setting of HSV-HIV co-infection.

High-mobility group protein HMGB2 regulates human erythroid differentiation through trans-activation of GFI1B transcription.

Gfi-1B is a transcriptional repressor that is crucial for erythroid differentiation: inactivation of the GFI1B gene in mice leads to embryonic death due to failure to produce differentiated red cells. Accordingly, GFI1B expression is tightly regulated during erythropoiesis, but the mechanisms involved in such regulation remain partially understood. We here identify HMGB2, a high-mobility group HMG protein, as a key regulator of GFI1B transcription. HMGB2 binds to the GFI1B promoter in vivo and up-regulates its trans-activation most likely by enhancing the binding of Oct-1 and, to a lesser extent, of GATA-1 and NF-Y to the GFI1B promoter. HMGB2 expression increases during erythroid differentiation concomitantly to the increase of GfI1B transcription. Importantly, knockdown of HMGB2 in immature hematopoietic progenitor cells leads to decreased Gfi-1B expression and impairs their erythroid differentiation. We propose that HMGB2 potentiates GATA-1-dependent transcription of GFI1B by Oct-1 and thereby controls erythroid differentiation.

Expression of Epstein-Barr virus EBNA1 protein in Escherichia coli: purification under nondenaturing conditions and use in DNA-binding studies.

Epstein-Barr virus nuclear antigen 1 (EBNA1) is a viral protein required for stable replication and segregation of DNA episomes containing the Epstein-Barr virus (EBV) origin of replication, OriP. Overproduction of EBNA1 protein in Escherichia coli has previously been shown to be difficult due to the large number of codons in EBNA1 gene that are infrequently used in E. coli. Here we changed the 26 rare codons that are found among the first 78 codons of EBNA1 gene, and replaced them with codons that encode the same amino-acids but are abundant in E. coli. This led to a significant improvement of EBNA1 expression in a standard E. coli strain. Partial EBNA1 polypeptides of 11.5-16 kDa extending from the N-terminus to the second arginine and glycine-rich region were extremely abundant in the extract, however, resulting in a second limitation of the level of EBNA1 expression. EBNA1 was expressed as a fusion with a C-terminal six-histidine tag in order to get rid of the short polypeptides by Ni-NTA affinity purification, and salt conditions were used that allowed us to extract and purify EBNA1 without resorting to protein denaturing reagents. The purified protein was used in DNA-binding experiments with DNA fragments containing specific EBNA1 sites. The E. coli-expressed protein formed specific DNA-protein complexes that could be analyzed in polyacrylamide gels without showing the aggregation, or linking, phenomenon that is usually observed with EBNA1 expressed in eukaryotic cells. EBNA1 protein expressed in E. coli should therefore prove useful to further study the biochemical properties of this crucial Epstein-Barr virus protein.

A high-sensitivity method for detection and measurement of HMGB1 protein concentration by high-affinity binding to DNA hemicatenanes.

BACKGROUND: Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration. METHODOLOGY/PRINCIPAL FINDINGS: In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum. CONCLUSION: The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies.

High-mobility group box 1 protein induces HIV-1 expression from persistently infected cells.

BACKGROUND: Necrosis is a frequent condition during AIDS, notably in organs targetted by opportunistic infections. Soluble factors released by necrotic cells are important for signalling cell damage, but little is known concerning their effect on HIV-1 replication. We focused on HMGB1, an abundant component of the chromatin that is released from necrotic cells and can act as a pro-inflammatory mediator. MATERIALS AND METHODS: A native form of HMGB1 was obtained from necrotic Hela cells, whereas a purified recombinant HMGB1 was generated in Escherichia coli. ACH-2 and U1 cells were used as models of persistent HIV-1 infection in lymphocytes and monocytes. Reactivation from latency was also investigated ex vivo using peripheral blood mononuclear cells (PBMC) collected from HIV-1-infected patients controlled by HAART. HIV-1 expression was quantified by enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction and branched DNA techniques. Flow cytometry and blocking experiments were used to identify the receptor used by HMGB1. Chromatin immunoprecipitation was used to investigate long-terminal repeat activation upon stimulation by HMGB1. RESULTS: HMGB1 increased HIV-1 transcription in chronically infected cells, a process that did not require de-novo protein synthesis. HIV-1 induction relied on HMGB1 interaction with the receptor for advanced glycation end-products. The activation pathway involved p38 and extracellular signal-related kinase as well as nuclear factor kappa B binding to the HIV-1 promoter. Finally, HMGB1 reactivated HIV-1 from latently infected PBMC collected in aviraemic HIV-infected patients. CONCLUSION: This work establishes for the first time a link between necrosis and HIV-1 replication, which involves HMGB1, a soluble mediator released by damaged cells.

Cell cycle arrest in G2 induces human immunodeficiency virus type 1 transcriptional activation through histone acetylation and recruitment of CBP, NF-kappaB, and c-Jun to the long terminal repeat promoter.

In human immunodeficiency virus type 1 (HIV-1)-infected cells, a cell cycle arrest in G(2) increases viral expression and may represent a strategy for the virus to optimize its expression. In latently infected cells, balance between viral silencing and reactivation relies on the nucleosomal organization of the integrated long terminal repeat (LTR). It is shown here that nucleosome nuc-1, which is located downstream of the TATA box, is specifically modified when latently infected cells are arrested in G(2) by chemical inducers. Notably, histones H3 and H4 are hyperacetylated, and this modification is associated with an increased LTR-driven transcription. nuc-1 hyperacetylation is also associated with the recruitment of histone acetyltransferase CBP and transcription factors NF-kappaB and c-Jun. NF-kappaB and/or c-Jun binding to the LTR in G(2)-arrested cells appears to be required for CBP recruitment as well as for nuc-1 remodeling and viral reactivation.