Epigenetic Mechanisms Histone Deacetylase-Dependent Regulate the Glioblastoma Angiogenic Matrisome and Disrupt Endothelial Cell Behavior In Vitro.

Glioblastoma (GBM) is the most aggressive brain tumor and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. Epitranscriptomics has shed light on new druggable Epigenetic therapies specifically designed to modulate GBM biology and behavior such as Histone Deacetylase inhibitors (iHDAC). Although the effects of iHDAC on GBM have been largely explored, there is a lack of information on the underlaying mechanisms HDAC-dependent that modulate the repertoire of GBM secreted molecules focusing on the set of Extracellular Matrix (ECM) associated proteins, the Matrisome, that may impact the surrounding tumor microenvironment. To acquire a better comprehension of the impacts of HDAC activity on the GBM Matrisome, we studied the alterations on the Matrisome-associated ECM regulators, Core Matrisome ECM glycoproteins, ECM-affiliated proteins and Proteoglycans upon HDAC inhibition in vitro as well as their relationship with glioma pathophysiological/clinical features and angiogenesis. For this, U87MG GBM cells were treated for with iHDAC or vehicle (control) and the whole secretome was processed by Mass Spectrometry NANOLC-MS/MS. In silico analyses revealed that proteins associated to the Angiogenic Matrisome (AngioMatrix), including Decorin, ADAM10, ADAM12 and ADAM15 were differentially regulated in iHDAC versus control secretome. Interestingly, genes coding for the Matrisome proteins differentially regulated were found mutated in patients and were correlated to glioma pathophysiological/clinical features. In vitro functional assays, using HBMEC endothelial cells exposed to the secretome of control or iHDAC treated GBM cells, coupled to 2D and 3D GBM cell culture system, showed impaired migratory capacity of endothelial cells and disrupted tubulogenesis in a Fibronectin and VEGF independent fashion. Collectively, our study provides understanding of epigenetic mechanisms HDAC-dependent to key Matrisomal proteins that may contribute to identify new druggable Epigenetic therapies or gliomagenesis biomarkers with relevant implications to improve therapeutic protocols for this malignancy.

Broad-spectrum antiviral activity of two structurally analogous CYP3A inhibitors against pathogenic human coronaviruses in vitro.

Coronaviruses pose a permanent risk of outbreaks, with three highly pathogenic species and strains (SARS-CoV, MERS-CoV, SARS-CoV-2) having emerged in the last twenty years. Limited antiviral therapies are currently available and their efficacy in randomized clinical trials enrolling SARS-CoV-2 patients has not been consistent, highlighting the need for more potent treatments. We previously showed that cobicistat, a clinically approved inhibitor of Cytochrome P450-3A (CYP3A), has direct antiviral activity against early circulating SARS-CoV-2 strains in vitro and in Syrian hamsters. Cobicistat is a derivative of ritonavir, which is co-administered as pharmacoenhancer with the SARS-CoV-2 protease inhibitor nirmatrelvir, to inhibit its metabolization by CPY3A and preserve its antiviral efficacy. Here, we used automated image analysis for a screening and parallel comparison of the anti-coronavirus effects of cobicistat and ritonavir. Our data show that both drugs display antiviral activity at low micromolar concentrations against multiple SARS-CoV-2 variants in vitro, including epidemiologically relevant Omicron subvariants. Despite their close structural similarity, we found that cobicistat is more potent than ritonavir, as shown by significantly lower EC50 values in monotherapy and higher levels of viral suppression when used in combination with nirmatrelvir. Finally, we show that the antiviral activity of both cobicistat and ritonavir is maintained against other human coronaviruses, including HCoV-229E and the highly pathogenic MERS-CoV. Overall, our results demonstrate that cobicistat has more potent anti-coronavirus activity than ritonavir and suggest that dose adjustments could pave the way to the use of both drugs as broad-spectrum antivirals against highly pathogenic human coronaviruses.

Tissue-like environments shape functional interactions of HIV-1 with immature dendritic cells.

Immature dendritic cells (iDCs) migrate in microenvironments with distinct cell and extracellular matrix densities in vivo and contribute to HIV-1 dissemination and mounting of antiviral immune responses. Here, we find that, compared to standard 2D suspension cultures, 3D collagen as tissue-like environment alters iDC properties and their response to HIV-1 infection. iDCs adopt an elongated morphology with increased deformability in 3D collagen at unaltered activation, differentiation, cytokine secretion, or responsiveness to LPS. While 3D collagen reduces HIV-1 particle uptake by iDCs, fusion efficiency is increased to elevate productive infection rates due to elevated cell surface exposure of the HIV-1-binding receptor DC-SIGN. In contrast, 3D collagen reduces HIV transfer to CD4 T cells from iDCs. iDC adaptations to 3D collagen include increased pro-inflammatory cytokine production and reduced antiviral gene expression in response to HIV-1 infection. Adhesion to a 2D collagen matrix is sufficient to increase iDC deformability, DC-SIGN exposure, and permissivity to HIV-1 infection. Thus, mechano-physical cues of 2D and 3D tissue-like collagen environments regulate iDC function and shape divergent roles during HIV-1 infection.

The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters.

Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Two independent cell-to-cell membrane fusion assays showed that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low-micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19. IMPORTANCE The lack of effective antiviral treatments against SARS-CoV-2 is a significant limitation in the fight against the COVID-19 pandemic. Single-drug regimens have so far yielded limited results, indicating that combinations of antivirals might be required, as previously seen for other RNA viruses. Our work introduces the drug booster cobicistat, which is approved by the FDA and typically used to potentiate the effect of anti-HIV protease inhibitors, as a candidate inhibitor of SARS-CoV-2 replication. Beyond its direct activity as an antiviral, we show that cobicistat can enhance the effect of remdesivir, which was one of the first drugs proposed for treatment of SARS-CoV-2. Overall, the dual action of cobicistat as a direct antiviral and a drug booster can provide a new approach to design combination therapies and rescue the activity of compounds that are only partially effective in monotherapy.

SARS-CoV-2 variants of concern display enhanced intrinsic pathogenic properties and expanded organ tropism in mouse models.

SARS-CoV-2 variants of concern (VOCs) display enhanced transmissibility and resistance to antibody neutralization. Comparing the early 2020 isolate EU-1 to the VOCs Alpha, Beta, and Gamma in mice transgenic for human ACE2 reveals that VOCs induce a broadened scope of symptoms, expand systemic infection to the gastrointestinal tract, elicit the depletion of natural killer cells, and trigger variant-specific cytokine production patterns. Gamma infections result in accelerated disease progression associated with increased immune activation and inflammation. All four SARS-CoV-2 variants induce pDC depletion in the lungs, paralleled by reduced interferon responses. Remarkably, VOCs also use the murine ACE2 receptor for infection to replicate in the lungs of wild-type animals, which induce cellular and innate immune responses that apparently curtail the spread of overt disease. VOCs thus display distinct intrinsic pathogenic properties with broadened tissue and host range. The enhanced pathogenicity of VOCs and their potential for reverse zoonotic transmission pose challenges to clinical and pandemic management.

SERINC5 Can Enhance Proinflammatory Cytokine Production by Primary Human Myeloid Cells in Response to Challenge with HIV-1 Particles.

HIV-1 has to overcome physical barriers posed by host cell restriction factors (RFs) for efficient replication. Some RFs, including Trim5α and tetherin, trigger antiviral signaling in addition to directly impairing HIV replication. SERINC5 (S5) is an RF that is incorporated into HIV-1 particles to potently impair their infectivity and is efficiently antagonized by the viral pathogenesis factor Nef. Since effects of S5 on HIV-1 infectivity were mostly studied in reporter cell lines, we analyzed the effects of S5 during infection of primary HIV-1 target cells. In activated CD4+ T lymphocytes, virion incorporation of S5 only moderately impaired virion infectivity and was not associated with altered innate immune recognition. In contrast, in monocyte-derived macrophages, S5 virion incorporation potentiated the production of proinflammatory cytokines with very potent but donor-dependent effects on virion infectivity. Nef counteracted effects of S5 on both cytokine production and virion infectivity. Similar S5-induced cytokine production was observed in immature monocyte-derived dendritic cells. Notably, S5-mediated enhancement of cytokine production was not linked to the efficacy of productive infection and could be overcome by using vesicular stomatitis virus glycoprotein (VSV-G) but not infectivity restriction-insensitive HIV-1 Env for cell entry. Moreover, inhibiting entry of S5-negative HIV-1 ΔNef particles increased proinflammatory cytokine production comparably to virion incorporation of S5. Together, these results describe the sensitization of noninfectious HIV-1 particles to proinflammatory cytokine production by myeloid target cells as an additional and Nef-sensitive activity of S5. Moreover, the study reveals important cell-type and donor-dependent differences in the sensitivity of HIV target cells for antiviral effects of S5.IMPORTANCE SERINC5 (S5) is a host cell restriction factor (RF) that impairs the infectivity of HIV-1 particles in target cell lines. To assess the potential physiological relevance of this restriction, we assessed the effects of S5 on HIV-1 infection of relevant primary human target cells. We found that effects of S5 on infection of CD4+ T lymphocytes were negligible. In myeloid target cells, however, virion incorporation of S5 potently suppressed infectivity and promoted innate immune recognition of HIV-1 particles characterized by proinflammatory cytokine production. Both effects were not observed in cells of all donors analyzed, were exerted independently of one another, and were counteracted by the HIV-1 pathogenesis factor Nef. These results identify the sensitization of HIV-1 particles for innate immune recognition by myeloid target cells as a novel activity of S5 and emphasize the need to study RF function in the context of primary target cells and taking donor variabilities into account.

HPV DNA Associates With Breast Cancer Malignancy and It Is Transferred to Breast Cancer Stromal Cells by Extracellular Vesicles.

A causal link between Human Papillomavirus (HPV) and breast cancer (BC) remains controversial. In spite of this, the observation that HPV DNA is over-represented in the Triple Negative (TN) BC has been reported. Here we remark the high prevalence of HPV DNA (44.4%) in aggressive BC subtypes (TN and HER2+) in a population of 273 Italian women and we convey the presence of HPV DNA in the epithelial and stromal compartments by in situ hybridization. As previously reported, we also found that serum derived-extracellular vesicles (EVs) from BC affected patients contain HPV DNA. Interestingly, in one TNBC patient, the same HPV DNA type was detected in the serum-derived EVs, cervical and BC tissue samples. Then, we report that HPV DNA can be transferred by EVs to recipient BC stromal cells that show an activated phenotype (e.g., CD44, IL6 expression) and an enhanced capability to sustain mammospheres (MS) formation. These data suggest that HPV DNA vehiculated by EVs is a potential trigger for BC niche aggressiveness.

The chaperone dynein LL1 mediates cytoplasmic transport of empty and mature hepatitis B virus capsids.

BACKGROUND & AIMS: Hepatitis B virus (HBV) has a DNA genome but replicates within the nucleus by reverse transcription of an RNA pregenome, which is converted to DNA in cytoplasmic capsids. Capsids in this compartment are correlated with inflammation and epitopes of the capsid protein core (Cp) are a major target for T cell-mediated immune responses. We investigated the mechanism of cytoplasmic capsid transport, which is important for infection but also for cytosolic capsid removal. METHODS: We used virion-derived capsids containing mature rcDNA (matC) and empty capsids (empC). RNA-containing capsids (rnaC) were used as a control. The investigations comprised pull-down assays for identification of cellular interaction partners, immune fluorescence microscopy for their colocalization and electron microscopy after microinjection to determine their biological significance. RESULTS: matC and empC underwent active transport through the cytoplasm towards the nucleus, while rnaC was poorly transported. We identified the dynein light chain LL1 as a functional interaction partner linking capsids to the dynein motor complex and showed that there is no compensatory transport pathway. Using capsid and dynein LL1 mutants we characterized the required domains on the capsid and LL1. CONCLUSIONS: This is the first investigation on the detailed molecular mechanism of how matC pass the cytoplasm upon infection and how empC can be actively removed from the cytoplasm into the nucleus. Considering that hepatocytes with cytoplasmic capsids are better recognized by the T cells, we hypothesize that targeting capsid DynLL1-interaction will not only block HBV infection but also stimulate elimination of infected cells. LAY SUMMARY: In this study, we identified the molecular details of HBV translocation through the cytoplasm. Our evidence offers a new drug target which could not only inhibit infection but also stimulate immune clearance of HBV infected cells.

Nuclear Import of Hepatitis B Virus Capsids and Genome.

Hepatitis B virus (HBV) is an enveloped pararetrovirus with a DNA genome, which is found in an up to 36 nm-measuring capsid. Replication of the genome occurs via an RNA intermediate, which is synthesized in the nucleus. The virus must have thus ways of transporting its DNA genome into this compartment. This review summarizes the data on hepatitis B virus genome transport and correlates the finding to those from other viruses.

Importin ß Can Bind Hepatitis B Virus Core Protein and Empty Core-Like Particles and Induce Structural Changes.

Hepatitis B virus (HBV) capsids are found in many forms: immature single-stranded RNA-filled cores, single-stranded DNA-filled replication intermediates, mature cores with relaxed circular double-stranded DNA, and empty capsids. A capsid, the protein shell of the core, is a complex of 240 copies of core protein. Mature cores are transported to the nucleus by a complex that includes both importin α and importin ß (Impα and Impß), which bind to the core protein's C-terminal domains (CTDs). Here we have investigated the interactions of HBV core protein with importins in vitro. Strikingly, empty capsids and free core protein can bind Impß without Impα. Cryo-EM image reconstructions show that the CTDs, which are located inside the capsid, can extrude through the capsid to be bound by Impß. Impß density localized on the capsid exterior near the quasi-sixfold vertices, suggested a maximum of 30 Impß per capsid. However, examination of complexes using single molecule charge-detection mass spectrometry indicate that some complexes include over 90 Impß molecules. Cryo-EM of capsids incubated with excess Impß shows a population of damaged particles and a population of "dark" particles with internal density, suggesting that Impß is effectively swallowed by the capsids, which implies that the capsids transiently open and close and can be destabilized by Impß. Though the in vitro complexes with great excess of Impß are not biological, these results have implications for trafficking of empty capsids and free core protein; activities that affect the basis of chronic HBV infection.