Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases

La phosphoprotéine P du virus de la rage inhibe la protéine kinase TBK1 et interfère avec des condensats liés à l’immunité innée

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Insights into herpesvirus tegument organization from structural analyses of the 970 central residues of HSV-1 UL36 protein.

International audienceThe tegument of all herpesviruses contains a capsid-bound large protein that is essential for multiple viral processes, including capsid transport, decapsidation at the nuclear pore complex, particle assembly, and secondary envelopment, through mechanisms that are still incompletely understood. We report here a structural characterization of the central 970 residues of this protein for herpes simplex virus type 1 (HSV-1 UL36, 3164 residues). This large fragment is essentially a 34-nm-long monomeric fiber. The crystal structure of its C terminus shows an elongated domain-swapped dimer. Modeling and molecular dynamics simulations give a likely molecular organization for the monomeric form and extend our findings to alphaherpesvirinae. Hence, we propose that an essential feature of UL36 is the existence in its central region of a stalk capable of connecting capsid and membrane across the tegument and that the ability to switch between monomeric and dimeric forms may help UL36 fulfill its multiple functions

Negri bodies are viral factories with properties of liquid organelles

Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.Negative strand RNA viruses, such as rabies virus, induce formation of cytoplasmic inclusions for genome replication. Here, Nikolic et al. show that these so-called Negri bodies (NBs) have characteristics of liquid organelles and they identify the minimal protein domains required for NB formation

Properties of rabies virus phosphoprotein and nucleoprotein biocondensates formed in vitro and in cellulo.

Rabies virus (RABV) transcription and replication take place within viral factories having liquid properties, called Negri bodies (NBs), that are formed by liquid-liquid phase separation (LLPS). The co-expression of RABV nucleoprotein (N) and phosphoprotein (P) in mammalian cells is sufficient to induce the formation of cytoplasmic biocondensates having properties that are like those of NBs. This cellular minimal system was previously used to identify P domains that are essential for biocondensates formation. Here, we constructed fluorescent versions of N and analyzed by FRAP their dynamics inside the biocondensates formed in this minimal system as well as in NBs of RABV-infected cells using FRAP. The behavior of N appears to be different of P as there was no fluorescence recovery of N proteins after photobleaching. We also identified arginine residues as well as two exposed loops of N involved in condensates formation. Corresponding N mutants exhibited distinct phenotypes in infected cells ranging from co-localization with NBs to exclusion from them associated with a dominant-negative effect on infection. We also demonstrated that in vitro, in crowded environments, purified P as well as purified N0-P complex (in which N is RNA-free) form liquid condensates. We identified P domains required for LLPS in this acellular system. P condensates were shown to associate with liposomes, concentrate RNA, and undergo a liquid-gel transition upon ageing. Conversely, N0-P droplets were disrupted upon incubation with RNA. Taken together, our data emphasize the central role of P in NBs formation and reveal some physicochemical features of P and N0-P droplets relevant for explaining NBs properties such as their envelopment by cellular membranes at late stages of infection and nucleocapsids ejections from the viral factories

Rabies virus P protein binds to TBK1 and interferes with the formation of innate immunity-related liquid condensates

Summary: Viruses must overcome the interferon-mediated antiviral response to replicate and propagate into their host. Rabies virus (RABV) phosphoprotein P is known to inhibit interferon induction. Here, using a global mass spectrometry approach, we show that RABV P binds to TBK1, a kinase located at the crossroads of many interferon induction pathways, resulting in innate immunity inhibition. Mutations of TBK1 phosphorylation sites abolish P binding. Importantly, we demonstrate that upon RABV infection or detection of dsRNA by innate immunity sensors, TBK1 and its adaptor proteins NAP1 and SINTBAD form dynamic cytoplasmic condensates that have liquid properties. These condensates can form larger aggregates having ring-like structures in which NAP1 and TBK1 exhibit locally restricted movement. P binding to TBK1 interferes with the formation of these structures. This work demonstrates that proteins of the signaling pathway leading to interferon induction transiently form liquid organelles that can be targeted by viruses

Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein

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Biologically relevant properties of RABV P droplets.

A) Segregation of RNA-Cy5 (10- or 40-A nucleotides) at the concentration of 100 nM into non-fluorescent P droplets (P at 5 μM in presence of 5% PEG 8000 in 125 mM NaCl, 20 mM Tris-HCl pH 7.5) and exclusion of BSA-FITC at the concentration of 250 μg/ml (3.8 μM) from P droplets. Fluorescent RNA and BSA were added when LLPS was induced. Analysis was performed by fluorescence microscopy. Exclusion of BSA-FITC from P droplets was validated by confocal microscopy (right panel). Scale bars: 10 μm. B) Droplets formed by non-fluorescent P at the concentration of 20 μM in the presence of 5% PEG 8000 and 100 nM Cy5-RNAs in 125 mM NaCl, 20 mM Tris-HCl pH 7.5 were photobleached at different time-points post-mixing (less than 1 h or more than 4 h). Whole droplets as well as droplets subvolumes were photobleached. Images were acquired on a spinning disk confocal microscope. Scale bars: 5 μm. For the plots on the right, FRAP data were corrected for background, normalized to the minimum and maximum intensity. The mean is shown with error bars representing the SD. C) P at 5 μM (25 nM of fluorescent P protein) in 50 mM NaCl, 20 mM Tris-HCl pH 7.5 was incubated with either 5% PEG 8000, or 80 ng/μL (6μM) of synthetic 40-A nucleotides RNA, or 80 ng/μL of total RNAs extracted from BSR-T7/5 cells. Presence of droplets was assessed by fluorescence microscopy. Scale bar: 10 μm. D) Fluorescent liposomes (PE/PC/PS = 2/2/1 w/w, 100 μg/ml i.e. ~130μM of lipids containing 0.25% of BODIPY 500/510 C12-HPC) were incubated or not with P at 20 μM concentration (50 nM of fluorescent P protein) in 5% PEG 8000, 125 mM NaCl, 20 mM Tris-HCl pH 7.5 before analysis by confocal microscopy. Scale bars: 10 μm.</p

Fusion events of P droplets formed in the same conditions as in S1 Movie.

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