Deciphering the Role of Epstein-Barr Virus Latent Membrane Protein 1 in Immune Modulation: A Multifaced Signalling Perspective.

The disruption of antiviral sensors and the evasion of immune defences by various tactics are hallmarks of EBV infection. One of the EBV latent gene products, LMP1, was shown to induce the activation of signalling pathways, such as NF-κB, MAPK (JNK, ERK1/2, p38), JAK/STAT and PI3K/Akt, via three subdomains of its C-terminal domain, regulating the expression of several cytokines responsible for modulation of the immune response and therefore promoting viral persistence. The aim of this review is to summarise the current knowledge on the EBV-mediated induction of immunomodulatory molecules by the activation of signal transduction pathways with a particular focus on LMP1-mediated mechanisms. A more detailed understanding of the cytokine biology molecular landscape in EBV infections could contribute to the more complete understanding of diseases associated with this virus.

Computational and experimental identification of keystone interactions in Ebola virus matrix protein VP40 dimer formation.

The Ebola virus (EBOV) is a lipid-enveloped virus with a negative sense RNA genome that can cause severe and often fatal viral hemorrhagic fever. The assembly and budding of EBOV is regulated by the matrix protein, VP40, which is a peripheral protein that associates with anionic lipids at the inner leaflet of the plasma membrane. VP40 is sufficient to form virus-like particles (VLPs) from cells, which are nearly indistinguishable from authentic virions. Due to the restrictions of studying EBOV in BSL-4 facilities, VP40 has served as a surrogate in cellular studies to examine the EBOV assembly and budding process from the host cell plasma membrane. VP40 is a dimer where inhibition of dimer formation halts budding and formation of new VLPs as well as VP40 localization to the plasma membrane inner leaflet. To better understand VP40 dimer stability and critical amino acids to VP40 dimer formation, we integrated computational approaches with experimental validation. Site saturation/alanine scanning calculation, combined with molecular mechanics-based generalized Born with Poisson-Boltzmann surface area (MM-GB/PBSA) method and molecular dynamics simulations were used to predict the energetic contribution of amino acids to VP40 dimer stability and the hydrogen bonding network across the dimer interface. These studies revealed several previously unknown interactions and critical residues predicted to impact VP40 dimer formation. In vitro and cellular studies were then pursued for a subset of VP40 mutations demonstrating reduction in dimer formation (in vitro) or plasma membrane localization (in cells). Together, the computational and experimental approaches revealed critical residues for VP40 dimer stability in an alpha-helical interface (between residues 106-117) as well as in a loop region (between residues 52-61) below this alpha-helical region. This study sheds light on the structural origins of VP40 dimer formation and may inform the design of a small molecule that can disrupt VP40 dimer stability.

Epstein-Barr virus-driven metabolic alterations contribute to the viral lytic reactivation and tumor progression in nasopharyngeal carcinoma.

Metabolic reprogramming induced by Epstein-Barr virus (EBV) often mirrors metabolic changes observed in cancer cells. Accumulating evidence suggests that lytic reactivation is crucial in EBV-associated oncogenesis. The aim of this study was to explore the role of metabolite changes in EBV-associated malignancies and viral life cycle control. We first revealed that EBV (LMP1) accelerates the secretion of the oncometabolite D-2HG, and serum D-2HG level is a potential diagnostic biomarker for NPC. EBV (LMP1)-driven metabolite changes disrupts the homeostasis of global DNA methylation and demethylation, which have a significantly inhibitory effect on active DNA demethylation and 5hmC content. We found that loss of 5hmC indicates a poor prognosis for NPC patients, and that 5hmC modification is a restriction factor of EBV reactivation. We confirmed a novel EBV reactivation inhibitor, α-KG, which inhibits the expression of EBV lytic genes with CpG-containing ZREs and the latent-lytic switch by enhancing 5hmC modification. Our results demonstrate a novel mechanism of which metabolite abnormality driven by EBV controls the viral lytic reactivation through epigenetic modification. This study presents a potential strategy for blocking EBV reactivation, and provides potential targets for the diagnosis and therapy of NPC.

Epstein-Barr virus latency patterns in polymorphic lymphoproliferative disorders and lymphomas in immunodeficiency settings: Diagnostic implications.

Epstein-Barr virus (EBV) is responsible for many B cell lymphoproliferative disorders (LPD) spanning subclinical infection to immunodeficiency-related neoplasms. EBV establishes a latent infection in the host B cell as defined histologically by the expression of EBV latent membrane proteins and nuclear antigens. Herein, we characterize the latency patterns of immunodeficiency-related neoplasms including post-transplant lymphoproliferative disorders (PTLD) and therapy-related LPD (formerly iatrogenic) with latent membrane protein-1 (LMP-1) and EBV nuclear antigen-2 (EBNA-2) immunohistochemistry. The latency pattern was correlated with immunodeficiency and dysregulation (IDD) status and time from transplant procedure. 38 cases of EBV+ PTLD in comparison to 27 cases of classic Hodgkin lymphoma (CHL) and diffuse large B cell lymphoma (DLBCL) arising in either the therapy-related immunodeficiency setting (n = 12) or without an identified immunodeficiency (n = 15) were evaluated for EBV-encoded small RNAs by in situ hybridization (EBER-ISH) and for LMP-1 and EBNA-2 by immunohistochemistry. A full spectrum of EBV latency patterns was observed across PTLD in contrast to CHL and DLBCL arising in the therapy-related immunodeficiency setting. Polymorphic-PTLD (12 of 16 cases, 75 %) and DLBCL-PTLD (9 of 11 cases, 82 %) showed the greatest proportion of cases with latency III pattern. Whereas, EBV+ CHL in an immunocompetent patient showed exclusively latency II pattern (13 of 13 cases, 100 %). The majority of EBV+ PTLD occurred by three years of transplant procedure date and were enriched for latency III pattern (21 of 22 cases, 95 %). Immunohistochemical identification of EBV latency by LMP-1 and EBNA-2 can help classify PTLD in comparison to other EBV+ B cell LPD and lymphomas arising in therapy-related immunodeficiency and non-immunodeficiency settings.

Tackling Influenza A virus by M2 ion channel blockers: Latest progress and limitations.

Influenza outbreaks cause pandemics in millions of people. The treatment of influenza remains a challenge due to significant genetic polymorphism in the influenza virus. Also, developing vaccines to protect against seasonal and pandemic influenza infections is constantly impeded. Thus, antibiotics are the only first line of defense against antigenically distinct strains or new subtypes of influenza viruses. Among several anti-influenza targets, the M2 protein of the influenza virus performs several activities. M2 protein is an ion channel that permits proton conductance through the virion envelope and the deacidification of the Golgi apparatus. Both these functions are critical for viral replication. Thus, targeting the M2 protein of the influenza virus is an essential target. Rimantadine and amantadine are two well-known drugs that act on the M2 protein. However, these drugs acquired resistance to influenza and thus are not recommended to treat influenza infections. This review discusses an overview of anti-influenza therapy, M2 ion channel functions, and its working principle. It also discusses the M2 structure and its role, and the change in the structure leads to mutant variants of influenza A virus. We also shed light on the recently identified compounds acting against wild-type and mutated M2 proteins of influenza virus A. These scaffolds could be an alternative to M2 inhibitors and be developed as antibiotics for treating influenza infections.

[Mechanism about LMP1 of EB Virus Promoting Plasma Blast Differentiation of DLBCL Cell via mTORC1].

OBJECTIVE: To investigate possible mechanism on protien LMP1 expressed by EBV inducing plasmablast differentiation of DLBCL cell via the mTORC1 pathway. METHODS: The expression levels of LMP1 protein, CD38 and the phosphorylation levels of p70S6K in EBV+ and EBV- DLBCL cell lines were detected by Western blot. Cell lines overexpressing LMP1 gene stablely were constructed and LMP1 gene was silenced by RNAi. The expression of LMP1 gene was verified by RT-qPCR. The expression levels of LMP1 and CD38 and the phosphorylation levels of p70S6K in each group were detected by Western blot. RESULTS: Compared with EBV-DLBCL cells, the expression of LMP1 was detected on EBV +DLBCL cells (P =0.0008), EBV +DLBCL cells had higher phosphorylation levels of p70S6K (P =0.0072) and expression levels of CD38(P =0.0091). Compared with vector group, the cells of LMP1OE group had higher expression levels of LMP1 and CD38 (P =0.0353; P <0.0001), meanwhile molecular p70S6K was phosphorylated much more(P =0.0065); expression of LMP1 mRNA was verified(P <0.0001). Compared with si-NC group, expression level of LMP1 protein(P =0.0129) was not detected and phosphorylated p70S6K disappeared of LMP1KO group (P =0.0228); meanwhile, expression of CD38 decreased,although there was no significant difference (P =0.2377). CONCLUSION: LMP1 promotes DLBCL cells plasmablast differentiation via activating mTORC1 signal pathway.

Synthesis, insertion, and characterization of SARS-CoV-2 membrane protein within lipid bilayers.

Throughout history, coronaviruses have posed challenges to both public health and the global economy; nevertheless, methods to combat them remain rudimentary, primarily due to the absence of experiments to understand the function of various viral components. Among these, membrane (M) proteins are one of the most elusive because of their small size and challenges with expression. Here, we report the development of an expression system to produce tens to hundreds of milligrams of M protein per liter of Escherichia coli culture. These large yields render many previously inaccessible structural and biophysical experiments feasible. Using cryo-electron microscopy and atomic force microscopy, we image and characterize individual membrane-incorporated M protein dimers and discover membrane thinning in the vicinity, which we validated with molecular dynamics simulations. Our results suggest that the resulting line tension, along with predicted induction of local membrane curvature, could ultimately drive viral assembly and budding.

Second site reversion of HIV-1 envelope protein baseplate mutations maps to the matrix protein.

The HIV-1 Envelope (Env) protein cytoplasmic tail (CT) recently has been shown to assemble an unusual trimeric baseplate structure that locates beneath Env ectodomain trimers. Mutations at linchpin residues that help organize the baseplate impair virus replication in restrictive T cell lines but not in permissive cell lines. We have identified and characterized a second site suppressor of these baseplate mutations, located at residue 34 in the viral matrix (MA) protein, that rescues viral replication in restrictive cells. The suppressor mutation was dependent on the CT to exert its activity and did not appear to affect Env protein traffic or fusion functions in restrictive cells. Instead, the suppressor mutation increased Env incorporation into virions 3-fold and virus infectivity in single-round infections 10-fold. We also found that a previously described suppressor of Env-incorporation defects that stabilizes the formation of MA trimers was ineffective at rescuing Env baseplate mutations. Our results support an interpretation in which changes at MA residue 34 induce conformational changes that stabilize MA lattice trimer-trimer interactions and/or direct MA-CT associations.IMPORTANCEHow HIV-1 Env trimers assemble into virus particles remains incompletely understood. In restrictive cells, viral incorporation of Env is dependent on the Env CT and on the MA protein, which assembles lattices composed of hexamers of trimers in immature and mature viruses. Recent evidence indicates that CT assembles trimeric baseplate structures that require membrane-proximal residues to interface with trimeric transmembrane domains and C-terminal helices in the CT. We found that mutations of these membrane-proximal residues impaired replication in restrictive cells. This defect was countered by a MA mutation that does not localize to any obvious interprotein regions but was only inefficiently suppressed by a MA mutation that stabilizes MA trimers and has been shown to suppress other CT-dependent Env defects. Our results suggest that efficient suppression of baseplate mutations involves stabilization of MA inter-trimer contacts and/or direct MA-CT associations. These observations shed new light on how Env assembles into virions.

The KxGxYR and DxE motifs in the C-tail of the Middle East respiratory syndrome coronavirus membrane protein are crucial for infectious virus assembly.

The coronavirus' (CoV) membrane (M) protein is the driving force during assembly, but this process remains poorly characterized. Previously, we described two motifs in the C-tail of the Middle East respiratory syndrome CoV (MERS-CoV) M protein involved in its endoplasmic reticulum (ER) exit (211DxE213) and trans-Golgi network (TGN) retention (199KxGxYR204). Here, their function in virus assembly was investigated by two different virus-like particle (VLP) assays and by mutating both motifs in an infectious MERS-CoV cDNA clone. It was shown that the 199KxGxYR204 motif was essential for VLP and infectious virus assembly. Moreover, the mislocalization of the M protein induced by mutation of this motif prevented M-E interaction. Hampering the ER export of M by mutating its 211DxE213 motif still allowed the formation of nucleocapsid-empty VLPs, but prevented the formation of fully assembled VLPs and infectious particles. Taken together, these data show that the MERS-CoV assembly process highly depends on the correct intracellular trafficking of its M protein, and hence that not only specific protein-protein interacting motifs but also correct subcellular localization of the M protein in infected cells is essential for virus formation and should be taken into consideration when studying the assembly process.

Selective targeting and clustering of phosphatidylserine lipids by RSV M protein is critical for virus particle production.

Human respiratory syncytial virus (RSV) is the leading cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. The elderly and the immunosuppressed are also affected. It is a major unmet target for vaccines and antiviral drugs. RSV assembles and buds from the host cell plasma membrane by forming infectious viral particles which are mostly filamentous. A key interaction during RSV assembly is the interaction of the matrix (M) protein with cell plasma membrane lipids forming a layer at assembly sites. Although the structure of RSV M protein dimer is known, it is unclear how the viral M proteins interact with cell membrane lipids, and with which one, to promote viral assembly. Here, we demonstrate that M proteins are able to cluster at the plasma membrane by selectively binding with phosphatidylserine (PS). Our in vitro studies suggest that M binds PS lipid as a dimer and upon M oligomerization, PS clustering is observed. In contrast, the presence of other negatively charged lipids like PI(4, 5)P2 does not enhance M binding beyond control zwitterionic lipids, while cholesterol negatively affects M interaction with membrane lipids. Moreover, we show that the initial binding of the RSV M protein with PS lipids is independent of the cytoplasmic tail of the fusion (F) glycoprotein (FCT). Here, we highlight that M binding on membranes occurs directly through PS lipids, this interaction is electrostatic in nature, and M oligomerization generates PS clusters.

Anoikis resistance and immune escape mediated by Epstein-Barr virus-encoded latent membrane protein 1-induced stabilization of PGC-1α promotes invasion and metastasis of nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr virus (EBV) is the first discovered human tumor virus that is associated with a variety of malignancies of both lymphoid and epithelial origin including nasopharyngeal carcinoma (NPC). The EBV-encoded latent membrane protein 1 (LMP1) has been well-defined as a potent oncogenic protein, which is intimately correlated with NPC pathogenesis. Anoikis is considered to be a physiological barrier to metastasis, and avoiding anoikis is a major hallmark of metastasis. However, the role of LMP1 in anoikis-resistance and metastasis of NPC has not been fully identified. METHODS: Trypan blue staining, colony formation assay, flow cytometry, and TUNEL staining, as well as the detection of apoptosis and anoikis resistance-related markers was applied to evaluate the anoikis-resistant capability of NPC cells cultured in ultra-low adhesion condition. Co-immunoprecipitation (Co-IP) experiment was performed to determine the interaction among LMP1, PRMT1 and PGC-1α. Ex vivo ubiquitination assay was used to detect the ubiquitination level of PGC-1α. Anoikis- resistant LMP1-positive NPC cell lines were established and applied for the xenograft and metastatic animal experiments. RESULTS: Our current findings reveal the role of LMP1-stabilized peroxisome proliferator activated receptor coactivator-1a (PGC-1α) in anoikis resistance and immune escape to support the invasion and metastasis of NPC. Mechanistically, LMP1 enhances PGC-1α protein stability by promoting the interaction between arginine methyltransferase 1 (PRMT1) and PGC-1α to elevate the methylation modification of PGC-1α, thus endowing NPC cells with anoikis-resistance. Meanwhile, PGC-1α mediates the immune escape induced by LMP1 by coactivating with STAT3 to transcriptionally up-regulate PD-L1 expression. CONCLUSION: Our work provides insights into how virus-encoded proteins recruit and interact with host regulatory elements to facilitate the malignant progression of NPC. Therefore, targeting PGC-1α or PRMT1-PGC-1α interaction might be exploited for therapeutic gain for EBV-associated malignancies.

The matrix protein of respiratory syncytial virus suppresses interferon signaling via RACK1 association.

IMPORTANCE: Respiratory syncytial virus (RSV) matrix (M) protein is indispensable for virion assembly and release. It is localized to the nucleus during early infection to perturb host transcription. However, the function of RSV M protein in other cellular activities remains poorly understood. In this study, several interferon response-associated host factors, including RACK1, were identified by proteomic analysis as RSV M interactors. Knockdown of RACK1 attenuates RSV-restricted IFN signaling leading to enhanced host defense against RSV infection, unraveling a role of M protein in antagonizing IFN response via association with RACK1. Our study uncovers a previously unrecognized mechanism of immune evasion by RSV M protein and identifies RACK1 as a novel host factor recruited by RSV, highlighting RACK1 as a potential new target for RSV therapeutics development.

Epstein-Barr virus LMP1 enhances levels of large extracellular vesicle-associated PD-L1.

IMPORTANCE: A growing body of evidence has supported the notion that viruses utilize EVs and associated pathways to incorporate viral products. This allows for the evasion of an immune response while enabling viral spread within the host. Given that viral proteins often elicit strong antigenic peptides that are recognized by T cells, the regulation of the PD-L1 pathway through the overexpression of lEV-associated PD-L1 may serve as a strategy for immune evasion by viruses. The discovery that EBV LMP1 increases the secretion of PD-L1 in larger EVs identifies a new potential target for immune blockade therapy in EBV-associated cancers. Our findings may help to clarify the mechanism of LMP1-mediated enhancement of PD-L1 packaging into lEVs and may lead to the identification of more specific targets for treatment. Additionally, the identification of lEV biomarkers that predict a viral origin of disease could allow for more targeted therapies to be developed.

Human herpesvirus 6A nuclear matrix protein U37 interacts with heat shock transcription factor 1 and activates the heat shock response.

Nascent nucleocapsids of herpesviruses acquire a primary envelope during their nuclear export by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This process is mediated by a conserved viral heterodimeric complex designated the nuclear egress complex, which consists of the nuclear matrix protein and the nuclear membrane protein. In addition to its essential roles during nuclear egress, the nuclear matrix protein has been shown to interact with intracellular signaling pathway molecules including NF-κB and IFN-ß to affect viral or cellular gene expression. The human herpesvirus 6A (HHV-6A) U37 gene encodes a nuclear matrix protein, the role of which has not been analyzed. Here, we show that HHV-6A U37 activates the heat shock element promoter and induces the accumulation of the molecular chaperone Hsp90. Mechanistically, HHV-6A U37 interacts with heat shock transcription factor 1 (HSF1) and induces its phosphorylation at Ser-326. We report that pharmacological inhibition of HSF1, Hsp70, or Hsp90 decreases viral protein accumulation and viral replication. Taken together, our results lead us to propose a model in which HHV-6A U37 activates the heat shock response to support viral gene expression and replication. IMPORTANCE Human herpesvirus 6A (HHV-6A) is a dsDNA virus belonging to the Roseolovirus genus within the Betaherpesvirinae subfamily. It is frequently found in patients with neuroinflammatory disease, although its pathogenetic role, if any, awaits elucidation. The heat shock response is important for cell survival under stressful conditions that disrupt homeostasis. Our results indicate that HHV-6A U37 activates the heat shock element promoter and leads to the accumulation of heat shock proteins. Next, we show that the heat shock response is important for viral replication. Overall, our findings provide new insights into the function of HHV-6A U37 in host cell signaling and identify potential cellular targets involved in HHV-6A pathogenesis and replication.

The C-terminus of Sudan ebolavirus VP40 contains a functionally important CXnC motif, a target for redox modifications.

The Ebola virus matrix protein VP40 mediates viral budding and negatively regulates viral RNA synthesis. The mechanisms by which these two functions are exerted and regulated are unknown. Using a high-resolution crystal structure of Sudan ebolavirus (SUDV) VP40, we show here that two cysteines in the flexible C-terminal arm of VP40 form a stabilizing disulfide bridge. Notably, the two cysteines are targets of posttranslational redox modifications and interact directly with the host`s thioredoxin system. Mutation of the cysteines impaired the budding function of VP40 and relaxed its inhibitory role for viral RNA synthesis. In line with these results, the growth of recombinant Ebola viruses carrying cysteine mutations was impaired and the released viral particles were elongated. Our results revealed the exact positions of the cysteines in the C-terminal arm of SUDV VP40. The cysteines and/or their redox status are critically involved in the differential regulation of viral budding and viral RNA synthesis.

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-ß Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

Novel Bifunctional Affibody Molecules with Specific Binding to Both EBV LMP1 and LMP2 for Targeted Therapy of Nasopharyngeal Carcinoma.

Antibodies are considered highly specific therapeutic agents in cancer medicines, and numerous formats have been developed. Among them, bispecific antibodies (BsAbs) have gained a lot of attention as a next-generation strategy for cancer therapy. However, poor tumor penetration is a major challenge because of their large size and thus contributes to suboptimal responses within cancer cells. On the other hand, affibody molecules are a new class of engineered affinity proteins and have achieved several promising results with their applications in molecular imaging diagnostics and targeted tumor therapy. In this study, an alternative format for bispecific molecules was constructed and investigated, named ZLMP110-277 and ZLMP277-110, that targets Epstein-Barr virus latent membrane protein 1 (LMP1) and latent membrane protein 2 (LMP2). Surface plasmon resonance (SPR), indirect immunofluorescence assay, co-immunoprecipitation, and near-infrared (NIR) imaging clearly demonstrated that ZLMP110-277 and ZLMP277-110 have good binding affinity and specificity for both LMP1 and LMP2 in vitro and in vivo. Moreover, ZLMP110-277 and ZLMP277-110, especially ZLMP277-110, significantly reduced the cell viability of C666-1 and CNE-2Z as compared to their monospecific counterparts. ZLMP110-277 and ZLMP277-110 could inhibit phosphorylation of proteins modulated by the MEK/ERK/p90RSK signaling pathway, ultimately leading to suppression of oncogene nuclear translocations. Furthermore, ZLMP110-277 and ZLMP277-110 showed significant antitumor efficacy in nasopharyngeal carcinoma-bearing nude mice. Overall, our results demonstrated that ZLMP110-277 and ZLMP277-110, especially ZLMP277-110, are promising novel prognostic indicators for molecular imaging and targeted tumor therapy of EBV-associated nasopharyngeal carcinoma.

Epstein-Barr virus LMP1 protein promotes proliferation and inhibits differentiation of epithelial cells via activation of YAP and TAZ.

Latent Epstein-Barr virus (EBV) infection promotes undifferentiated nasopharyngeal carcinomas (NPCs) in humans, but the mechanism(s) for this effect has been difficult to study because EBV cannot transform normal epithelial cells in vitro and the EBV genome is often lost when NPC cells are grown in culture. Here we show that the latent EBV protein, LMP1 (Latent membrane protein 1), induces cellular proliferation and inhibits spontaneous differentiation of telomerase-immortalized normal oral keratinocytes (NOKs) in growth factor-deficient conditions by increasing the activity of the Hippo pathway effectors, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif). We demonstrate that LMP1 enhances YAP and TAZ activity in NOKs both by decreasing Hippo pathway-mediated serine phosphorylation of YAP and TAZ and increasing Src kinase-mediated Y357 phosphorylation of YAP. Furthermore, knockdown of YAP and TAZ is sufficient to reduce proliferation and promote differentiation in EBV-infected NOKs. We find that YAP and TAZ are also required for LMP1-induced epithelial-to-mesenchymal transition. Importantly, we demonstrate that ibrutinib (an FDA-approved BTK inhibitor that blocks YAP and TAZ activity through an off-target effect) restores spontaneous differentiation and inhibits proliferation of EBV-infected NOKs at clinically relevant doses. These results suggest that LMP1-induced YAP and TAZ activity contributes to the development of NPC.

Cruciferous vegetable-derived indole-3-carbinol prevents coronavirus cell egression mechanisms in tracheal and intestinal 3D in vitro models.

The potential antiviral effects of indole-3-carbinol (I3C), a phytochemical found in Cruciferous vegetables, were investigated. Fibroblasts and epithelial cells were co-cultured on Alvetex® scaffolds, to obtain ad hoc 3D in vitro platforms able to mimic the trachea and intestinal mucosae, which represent the primary structures involved in the coronavirus pathogenesis. The two barriers generated in vitro were treated with various concentrations of I3C for different incubation periods. A protective effect of I3C on both intestinal and trachea models was demonstrated. A significant reduction in the transcription of the two main genes belonging to the Homologous to E6AP C-terminus (HECT)-E3 ligase family members, namely NEDD4 E3 Ubiquitin Protein Ligase (NEDD4) and WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1), which promote virus matrix protein ubiquitination and inhibit viral egression, were detected. These findings indicate I3C potential effect in preventing coronavirus cell egression processes that inhibit viral production. Although further studies are needed to clarify the molecular mechanisms whereby HECT family members control virus life cycle, this work paves the way to the possible therapeutic use of new natural compounds that may reduce the clinical severity of future pandemics.

Epstein-Barr virus in breast carcinoma and in triple negative cases impact on clinical outcomes.

BACKGROUND: The contribution of viral infection in tumors pathogenesis has currently attracted attention. Epstein-Barr virus is an infectious agent involved in numerous human malignancies, including breast cancer. Although, their prognostic impact in breast tumor is rarely investigated. Therefore, we sought in our study to evaluate the prevalence of EBV in Tunisian breast carcinoma and to examine their potential association with clinicopathological features and overall survival. METHODS: Our retrospective study included 100 formalin fixed paraffin embedded samples from Tunisian breast carcinoma. EBV infection was evaluated by immunohistochemical analysis, using monoclonal antibody against latent membrane protein 1 (LMP-1) and polymerase chain reaction. A subset of PCR positive specimens was subjected to in situ hybridization for the detection of EBER expression. Biomarker's expression was evaluated by immunohistochemistry method. Statistical analysis was also explored. RESULTS: The expression status of ER, PR and HER2 was 81%, 71.4% and 33.7% respectively. The triple negative profile was present in 10.84% of cases. LMP-1 expression was negative in all breast cancer specimens. PCR assay showed that 44% of patients were positive for EBV genome. None of the 15 PCR positive cases showed positive results for EBV by ISH. According to the molecular phenotype, there was a statistically significant difference in EBV DNA prevalence between breast cancer subgroups including TN (67%), Lum B (64%), HER2 + (50%) and Lum A (30%). Bivariate analysis showed that EBV DNA was significantly associated with HER2 + (p = 0.035), tumor size (p = 0.018) and high SBR grade (p = 0.009). Multiple logistic regression analysis confirms the positive correlation of EBV with tumor size (p = 0.048) and SBR grade (p = 0.042). Kaplan-Meier analysis showed that patients with EBV+ had significantly shorter overall survival than those with EBV- (p = 0.032). CONCLUSIONS: Our study demonstrated the presence of EBV DNA in Tunisian breast carcinoma. EBV DNA was associated with aggressive features and poor overall survival. Further investigations will be required in large samples size to clarify the potential role of EBV in breast tumor progression.