Insights on TAM Formation from a Boolean Model of Macrophage Polarization Based on In Vitro Studies

International audienceThe tumour microenvironment is the surrounding of a tumour, including blood vessels, fibroblasts, signaling molecules, the extracellular matrix and immune cells, especially neutrophils and monocyte-derived macrophages. In a tumour setting, macrophages encompass a spectrum between a tumour-suppressive (M1) or tumour-promoting (M2) state. The biology of macrophages found in tumours (Tumour Associated Macrophages) remains unclear, but understanding their impact on tumour progression is highly important. In this paper, we perform a comprehensive analysis of a macrophage polarization network, following two lines of enquiry: (i) we reconstruct the macrophage polarization network based on literature, extending it to include important stimuli in a tumour setting, and (ii) we build a dynamical model able to reproduce macrophage polarization in the presence of different stimuli, including the contact with cancer cells. Our simulations recapitulate the documented macrophage phenotypes and their dependencies on specific receptors and transcription factors, while also unravelling the formation of a special type of tumour associated macrophages in an in vitro model of chronic lymphocytic leukaemia. This model constitutes the first step towards elucidating the cross-talk between immune and cancer cells inside tumours, with the ultimate goal of identifying new therapeutic targets that could control the formation of tumour associated macrophages in patients

The methyltransferase domain of the Respiratory Syncytial Virus L protein catalyzes cap N7 and 2’-O-methylation

International audienceRespiratory syncytial virus (RSV) is a negative sense single-stranded RNA virus and one of the main causes of severe lower respiratory tract infections in infants and young children. RSV RNA replication/transcription and capping are ensured by the viral Large (L) protein. The L protein contains a polymerase domain associated with a polyribonucleotidyl transferase domain in its N-terminus, and a methyltransferase (MTase) domain followed by the Cterminal domain (CTD) enriched in basic amino acids at its C-terminus. The MTase-CTD of Mononegavirales forms a clamp to accommodate RNA that is subsequently methylated on the cap structure and depending on the virus, on internal positions. These enzymatic activities are essential for efficient viral mRNA translation into proteins, and to prevent the recognition of uncapped viral RNA by innate immunity sensors. In this work, we demonstrated that the MTase-CTD of RSV, as well as the full-length L protein in complex with phosphoprotein (P), catalyzes the N7-and 2'-O-methylation of the cap structure of a short RNA sequence that corresponds to the 5' end of viral mRNA. Using different experimental systems, we showed that the RSV MTase-CTD methylates the cap structure with a preference for N7-methylation as first reaction. However, we did not observe cap-independent internal methylation, as recently evidenced for the Ebola virus MTase. We also found that at μM concentrations, sinefungin, a S-adenosylmethionine analogue, inhibits the MTase activity of the RSV L protein and of the MTase-CTD domain. Altogether, these results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion

Structure of the Respiratory Syncytial Virus Polymerase Complex

International audienceGraphical Abstract Highlights d Cryo-EM structure of RSV L bound by tetrameric RSV P solved to 3.2 Å d P tetramer adopts an asymmetric tentacular arrangement when bound to L d L priming loop adopts elongation-compatible state without PRNTase-RdRp separation d Structure rationalizes escape from small-molecule antivirals In Brief Respiratory syncytial virus (RSV) remains a leading cause of bronchiolitis and hospitalization, especially of infants. Gilman et al. present a 3.2-Å cryo-EM structure of the RSV L polymerase in complex with the P phosphoprotein-components of the core viral replication machinery that represent an attractive target for the development of therapeutic agents. Data Resources 6PZ

Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor

The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs

Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor

Abstract: The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 angstrom resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs

JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase

Abstract: Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology

JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase

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