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Background: Lung cancers represent the main cause of cancer related-death worldwide. Recently, immunotherapy alone or in combination with chemotherapy has deeply impacted the therapeutic care leading to an improved overall survival. However, relapse will finally occur, with no efficient second line treatment so far. New therapies development based on the comprehension of resistance mechanisms is necessary. However, the difficulties to obtain tumor samples before and after first line treatment hamper to clearly understand the consequence of these molecules on tumor cells and also to identify adapted second line therapies. Methods: To overcome this difficulty, we developed multicellular tumor spheroids (MCTS) using characterized Non-Small Cell Lung Cancer (NSCLC) cell lines, monocytes from healthy donors and fibroblasts. MCTS were treated with carboplatin-paclitaxel or -gemcitabine combinations according to clinical administration schedules. The treatments impact was studied using cell viability assay, histological analyses, 3'RNA sequencing, real-time PCR, flow cytometry and confocal microscopy. Results: We showed that treatments induced a decrease in cell viability and strong modifications in the transcriptomic profile notably at the level of pathways involved in DNA damage repair and cell cycle. Interestingly, we also observed a modification of genes expression considered as hallmarks of response to immune check point inhibitors and immunogenicity, particularly an increase in CD274 gene expression, coding for PD-L1. This result was validated at the protein level and shown to be restricted to tumor cells on MCTS containing fibroblasts and macrophages. This increase was also observed in an additional cell line, expressing low basal CD274 level. Conclusions: This study shows that MCTS are interesting models to study the impact of first line therapies using conditions close to clinical practice and also to identify more adapted second line or concomitant therapies for lung cancer treatment. [ABSTRACT FROM AUTHOR]

Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry. [ABSTRACT FROM AUTHOR]

Attenuated measles virus (MV) exerts its oncolytic activity in malignant pleural mesothelioma (MPM) cells that lack type-I interferon (IFN-I) production or responsiveness. However, other cells in the tumor microenvironment (TME), such as myeloid cells, possess functional antiviral pathways. In this study, we aimed to characterize the interplay between MV and the myeloid cells in human MPM. We cocultured MPM cell lines with monocytes or macrophages and infected them with MV. We analyzed the transcriptome of each cell type and studied their secretion and phenotypes by high-dimensional flow cytometry. We also measured transgene expression using an MV encoding GFP (MV-GFP). We show that MPM cells drive the differentiation of monocytes into M2-like macrophages. These macrophages inhibit GFP expression in tumor cells harboring a defect in IFN-I production and a functional signaling downstream of the IFN-I receptor, while having minimal effects on GFP expression in tumor cells with defect of responsiveness to IFN-I. Interestingly, inhibition of the IFN-I signaling by ruxolitinib restores GFP expression in tumor cells. Upon MV infection, cocultured macrophages express antiviral pro-inflammatory genes and induce the expression of IFN-stimulated genes in tumor cells. MV also increases the expression of HLA and costimulatory molecules on macrophages and their phagocytic activity. Finally, MV induces the secretion of inflammatory cytokines, especially IFN-I, and PD-L1 expression in tumor cells and macrophages. These results show that macrophages reduce viral proteins expression in some MPM cell lines through their IFN-I production and generate a pro-inflammatory interplay that may stimulate the patient's anti-tumor immune response. [ABSTRACT FROM AUTHOR]

Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro -infected hepatocytes and in chronically infected patients. The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription. [Display omitted] • We uncovered a heterogeneous transcriptional termination of HBV transcripts in cellular models and in patients with CHB. • HBV transcription termination is functionally linked to HBV replication. • RNA helicases DDX5 and DDX17 promote HBV transcriptional read-through and viral RNA destabilisation. • DDX5 and DDX17 are host HBV restriction factors and are downregulated in patients with CHB. [ABSTRACT FROM AUTHOR]

Background & Aims Hepatitis B virus (HBV) persistence and the pathobiology of chronic HBV (CHB) infections result from the interplay between viral replication and host immune responses. We aimed to comprehensively analyse the expression of intrahepatic host genes as well as serum and liver HBV markers in a large cohort of untreated CHB patients. Methods One-hundred and five CHB patients untreated at the time of liver biopsy (34 HBeAg[+] and 71 HBeAg[-]) were analysed for the intrahepatic expression profile of 67 genes belonging to multiple innate immunity pathways. Results were correlated to serological (quantification of HBsAg [qHBsAg] and HBV DNA) and intrahepatic viral markers (total HBV DNA, pre-genomic RNA and covalently closed circular HBV DNA). Results Intrahepatic gene expression profiling revealed a strong downregulation of antiviral effectors, interferon stimulated genes, Toll-like and pathogen recognition receptor pathways in CHB patients as compared to non-infected controls, which was not directly correlated to HBV replication. A subset of genes [CXCL10, GBP1, IFITM1, IFNB1, IL10, IL6, ISG15, TLR3, SOCS1, SOCS3] was more repressed in HBeAg(-) respect to HBeAg(+) patients (median of serum HBV DNA 7.9 x 10³vs. 7.9 x 107 IU/ml, respectively). Notably, HBeAg(-) patients with lower qHBsAg (<5 x 10³ IU/ml) showed a relief of repression of genes belonging to multiple pathways. Conclusions Our results show a strong impairment of innate immune responses in the liver of CHB patients. The association of low levels of qHBsAg with gene repression, if confirmed, might prove useful for the identification of patients who would most benefit from immune-modulators and/or HBsAg targeting agents as strategies to restore immune responsiveness. Lay summary Chronic hepatitis B virus (HBV) infections represent a major public health problem worldwide. Over 200 million people are chronically infected and at risk of developing chronic hepatitis, liver cirrhosis and cancer. Our work aimed to understand the molecular consequences of chronic hepatitis B in the infected liver. It was conducted in a large cohort of untreated chronically infected HBV patients and analysed the expression of immunity and liver disease-related genes in the liver, with respect to markers of viral replication and persistence. Our results indicate that chronic HBV infection has a suppressive effect on immune responses, which was more pronounced with high levels of hepatitis B virus surface antigen (HBsAg). These data provide novel insight into the mechanisms of HBV persistence in the liver and suggest that approaches aimed at reducing HBsAg levels, may restore immune responsiveness against the virus. [ABSTRACT FROM AUTHOR]

HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin- 1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection. [ABSTRACT FROM AUTHOR]

• Hepatitis B virus proteins are observed in liver macrophages from patients. • Hepatitis B virus impairs pro-inflammatory macrophage secretion. • Hepatitis B virus increases anti-inflammatory macrophage secretion. • Impairment of pro-inflammatory secretions favours the establishment of hepatitis B virus infection. • Increase of IL-10 secretion further impairs lymphocyte activation. Liver macrophages can be involved in both pathogen clearance and/or pathogenesis. To get further insight on their role during chronic hepatitis B virus (HBV) infections, our aim was to phenotypically and functionally characterize in vivo and ex vivo the interplay between HBV, primary human liver macrophages (PLMs) and primary blood monocytes differentiated into pro-inflammatory or anti-inflammatory macrophages (M1-MDMs or M2-MDMs, respectively). PLMs or primary blood monocytes, either ex vivo differentiated into M1-MDMs or M2-MDMs, were exposed to HBV and their activation followed by ELISA or quantitative reverse transcription PCR (RT-qPCR). Liver biopsies from HBV-infected patients were analysed by RT-qPCR or immunohistochemistry. Viral parameters in HBV-infected primary human hepatocytes and differentiated HepaRG cells were followed by ELISA, qPCR and RT-qPCR analyses. HBc protein was present within the macrophages of liver biopsies taken from HBV-infected patients. Macrophages from HBV-infected patients also expressed higher levels of anti-inflammatory macrophage markers than those from non-infected patients. Ex vivo exposure of naive PLMs to HBV led to reduced secretion of pro-inflammatory cytokines. Upon exposure to HBV or HBV-producing cells during differentiation and activation, M1-MDMs secreted less IL-6 and IL-1β, whereas M2-MDMs secreted more IL-10 when exposed to HBV during activation. Finally, cytokines produced by M1-MDMs, but not those produced by HBV-exposed M1-MDMs, decreased HBV infection of hepatocytes. Altogether, our data strongly suggest that HBV modulates liver macrophage functions to favour the establishment of infection. Hepatitis B virus modulates liver macrophage function in order to favour the establishment and likely maintenance of infection. It impairs the production of the antiviral cytokine IL-1β, while promoting that of IL-10 in the microenvironment. This phenotype can be recapitulated in naive liver macrophages or monocyte-derived-macrophages ex vivo by short exposure to the virus or cells replicating the virus, thus suggesting an "easy to implement" mechanism of inhibition. [ABSTRACT FROM AUTHOR]

In HBV-infected patients, therapies with nucleoside analogues or IFNα remain ineffective in eradicating the infection. Our aim was to re-analyze the anti-HBV activity of a large panel of IFNs and cytokines in vitro using non-transformed cultured hepatocytes infected with HBV, to identify new immune-therapeutic options. HepaRG cells and primary human hepatocytes were infected with HBV and, when infection was established, treated with various concentrations of different IFNs or inflammatory cytokines. Viral parameters were evaluated by quantifying HBV nucleic acids by qPCR and Southern Blot, and secreted HBV antigens were evaluated using ELISA. The cytokines tested were type-I IFNs, IFNγ, type-III IFNs, TNFα, IL-6, IL-1β, IL-18 as well as nucleos(t)ide analogues tenofovir and ribavirin. Cytokines and drugs, with the exception of IL-18 and ribavirin, exhibited a suppressive effect on HBV replication at least as strong as, but often stronger than, IFNα. The cytokine presenting the highest effect on HBV DNA was IL-1β, which exerted its inhibition within picomolar range. Importantly, we noticed differential effects on other parameters (HBV RNA, HBeAg, HBsAg) between both IFNs and inflammatory cytokines, thus suggesting different mechanisms of action. The combination of IL-1β and already used therapies, i.e. IFNα or tenofovir, demonstrated a stronger or similar anti-HBV activity. IL-1β was found to have a very potent antiviral effect against HBV in vitro . HBV was previously shown to promptly inhibit IL-1β production in Kupffer cells. Strategies aiming at unlocking this inhibition and restoring local production of IL-1β may help to further inhibit HBV replication in vivo . [ABSTRACT FROM AUTHOR]

Objectives Caveats in the understanding of ribavirin (RBV) mechanisms of action has somehow prevented the development of better analogues able to further improve its therapeutic contribution in interferon (IFN)-based and direct antiviral agent-based regimens for chronic HCV or other indications. Here, we describe a new mechanism by which RBV modulates IFN-stimulated genes (ISGs) and contributes to restore hepatic immune responsiveness. Design RBV effect on ISG expression was monitored in vitro and in vivo, that is, in non-transformed hepatocytes and in the liver of RBV mono-treated patients, respectively. Modulation of histone modifications and recruitment of histone-modifying enzymes at target promoters was analysed by chromatin immunoprecipitation in RBV-treated primary human hepatocytes and in patients' liver biopsies. Results RBV decreases the mRNA levels of several abnormally preactivated ISGs in patients with HCV, who are non-responders to IFN therapy. RBV increases G9a histone methyltransferase recruitment and histone-H3 lysine-9 dimethylation/trimethylation at selected ISG promoters in vitro and in vivo. G9a pharmacological blockade abolishes RBV-induced ISG downregulation and severely impairs RBV ability to potentiate IFN antiviral action and induction of ISGs following HCV infection of primary human hepatocytes. Conclusions RBV-induced epigenetic changes, leading to decreased ISG expression, restore an IFN-responsive hepatic environment in patients with HCV, which may also prove useful in IFN-free regimens. [ABSTRACT FROM AUTHOR]

Hepatitis Cvirus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCVJFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation ofHCVviral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) covalently closed circular (ccc)DNA is the key genomic form responsible for viral persistence and virological relapse after treatment withdrawal. The assessment of residual intrahepatic cccDNA levels and activity after long-term nucleos(t)ide analogues therapy still represents a technical challenge. Quantitative (q)PCR, rolling circle amplification (RCA) and droplet digital (dd)PCR assays were used to quantify residual intrahepatic cccDNA in liver biopsies from 56 chronically HBV infected patients after 3 to 5 years of telbivudine treatment. Activity of residual cccDNA was evaluated by quantifying 3.5 kB HBV RNA (preC/pgRNA) and by assessing cccDNA-associated histone tails post-transcriptional modifications (PTMs) by micro-chromatin immunoprecipitation. Long-term telbivudine treatment resulted in serum HBV DNA suppression, with most of the patients reaching undetectable levels. Despite 38 out of 56 patients had undetectable cccDNA when assessed by qPCR, RCA and ddPCR assays detected cccDNA in all-but-one negative samples. Low preC/pgRNA level in telbivudine-treated samples was associated with enrichment for cccDNA histone PTMs related to repressed transcription. No difference in cccDNA levels was found according to serum viral markers evolution. This panel of cccDNA evaluation techniques should provide an added value for the new proof-of-concept clinical trials aiming at a functional cure of chronic hepatitis B. [ABSTRACT FROM AUTHOR]

Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH. [ABSTRACT FROM AUTHOR]