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.

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.

Protein release factor eRF1 in Saccharomyces cerevisiae, in complex with eRF3 and GTP, is methylated on a functionally crucial Gln residue by the S-adenosylmethionine-dependent methyltransferase Ydr140w. Here we show that eRF1 methylation, in addition to these previously characterized components, requires a 15-kDa zinc-binding protein, Ynr046w. Co-expression in Escherichia coli of Ynr046w and Ydr140w allows the latter to be recovered in soluble form rather than as inclusion bodies, and the two proteins co-purify on nickel-nitrilotriacetic acid chromatography when Ydr140w alone carries a His tag. The crystal structure of Ynr046w has been determined to 1.7 Å resolution. It comprises a zinc-binding domain built from both the N-and C-terminal sequences and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three ␣-helices. The active methyltransferase is the heterodimer Ydr140w⅐Ynr046w, but when alone, both in solution and in crystals, Ynr046w appears to be a homodimer. The Ynr046w eRF1 methyltransferase subunit is shared by the tRNA methyltransferase Trm11p and probably by two other enzymes containing a Rossman fold.Termination codons in mRNA are recognized on the ribosome by class I protein termination factors (or release factors (RFs)) 5 in eubacteria, archaea, and eukaryotes (1-3). Three codons are used as stop signals in most organisms: UAA, UGA, and UAG. In bacteria, two class I RFs are required for termination: RF1, which recognizes UAA and UAG codons, and RF2, which recognizes UAA and UGA codons. In contrast, a single RF, eRF1 or aRF1, is sufficient for termination at all three stop codons in eukaryotes and archaea, respectively. eRF1 and aRF1 form closely related protein families but are evolutionarily distinct from the eubacterial RFs (4). Thus, despite a similar function, the only sequence element common to all RFs is a tripeptide sequence, GGQ. Structural and mutational analysis shows that this motif is essential for RF activity and is required to interact with the peptidyl transferase center of the large ribosomal subunit and trigger hydrolysis of the ester bond in peptidyl-tRNA (5, 6). The Gln residue of the GGQ motif is methylated in both bacteria (7) and Saccharomyces cerevisiae (8, 9), and probably in mammals. Bacterial RF methylation depends on the PrmC methyltransferase (MTase) (10, 11), the product of the gene prmC (previously named hemK) situated in Escherichia coli, and most other bacteria immediately downstream of the gene prfA encoding RF1. RF methylation in E. coli strongly stimulates the activity of the factors (7, 11). 6 It is remarkable that the modification of Gln in the GGQ motif is conserved from bacteria to eukaryotes despite the different evolutionary origin of the class I RFs themselves. The S. cerevisiae genome encodes two proteins, Ydr140w and Ynl063w, with significant similarity to bacterial PrmC that goes beyond the motifs known to be involved in AdoMet binding (7). Inactivation of Ydr140w was shown to lead to a loss of eRF1 methylation ...

Class I release factors bind to ribosomes in response to stop codons and trigger peptidyl-tRNA hydrolysis at the P site. Prokaryotic and eukaryotic RFs share one motif: a GGQ tripeptide positioned in a loop at the end of a stem region that interacts with the ribosomal peptidyl transferase center. The glutamine side chain of this motif is specifically methylated in both prokaryotes and eukaryotes. Methylation in E. coli is due to PrmC and results in strong stimulation of peptide chain release. We have solved the crystal structure of the complex between E. coli RF1 and PrmC bound to the methyl donor product AdoHCy. Both the GGQ domain (domain 3) and the central region (domains 2 and 4) of RF1 interact with PrmC. Structural and mutagenic data indicate a compact conformation of RF1 that is unlike its conformation when it is bound to the ribosome but is similar to the crystal structure of the protein alone.

The ubiquitous tripeptide Gly-Gly-Gln in class 1 polypeptide release factors triggers polypeptide release on ribosomes. The Gln residue in both bacterial and yeast release factors is N5-methylated, despite their distinct evolutionary origin. Methylation of eRF1 in yeast is performed by the heterodimeric methyltransferase (MTase) Mtq2p/Trm112p, and requires eRF3 and GTP. Homologues of yeast Mtq2p and Trm112p are found in man, annotated as an N6-DNA-methyltransferase and of unknown function. Here we show that the human proteins methylate human and yeast eRF1.eRF3.GTP in vitro, and that the MTase catalytic subunit can complement the growth defect of yeast strains deleted for mtq2. Structured summary:MINT-6571489: HemK2a (uniprotkb:Q9Y5N5) binds (MI:0407) to hTrm112 (uniprotkb:Q9UI30) by pull down (MI:0096)

The hepatitis C virus (HCV) genotype 2a isolate JFH1 represents the only cloned HCV wild-type sequence capable of efficient replication in cell culture as well as in vivo. Previous reports have pointed to NS5B, the viral RNA-dependent RNA polymerase (RdRp), as a major determinant for efficient replication of this isolate. To understand the contribution of the JFH1 NS5B gene at the molecular level, we aimed at conferring JFH1 properties to NS5B from the closely related J6 isolate. We created intragenotypic chimeras in the NS5B regions of JFH1 and J6 and compared replication efficiency in cell culture and RdRp activity of the purified proteins in vitro, revealing more than three independent mechanisms conferring the role of JFH1 NS5B in efficient RNA replication. Most critical was residue I405 in the thumb domain of the polymerase, which strongly stimulated replication in cell culture by enhancing overall de novo RNA synthesis. A structural comparison of JFH1 and J6 at high resolution indicated a clear correlation of a closed-thumb conformation of the RdRp and the efficiency of the enzyme at de novo RNA synthesis, in accordance with the proposal that I405 enhances de novo initiation. In addition, we identified several residues enhancing replication independent of RdRp activity in vitro. The functional properties of JFH1 NS5B could be restored by a few single-nucleotide substitutions to the J6 isolate. Finally, we were able to enhance the replication efficiency of a genotype 1b isolate with the I405 mutation, indicating that this mechanism of action is conserved across genotypes.The hepatitis C virus (HCV) is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae (47). The genome of HCV encompasses a single ϳ9,600-nucleotide (nt)-long RNA molecule carrying one large open reading frame (ORF), flanked by nontranslated regions (NTRs), that is translated primarily into one polyprotein. The polyprotein precursor is cleaved by cellular and viral proteases into at least 10 different products (for a review, see reference 5). The nonstructural proteins NS3 to NS5B are necessary and sufficient for autonomous RNA replication. They form a membrane-associated replication complex, in which NS5B is the RNA-dependent RNA polymerase (RdRp), the key enzyme of viral RNA replication. Purified NS5B can initiate RNA synthesis in vitro by a primer-dependent mechanism or de novo (7,28,30,55). De novo initiation at the 3Ј end of the viral positiveand negative-strand RNA is likely to be the physiological mode of initiation of RNA synthesis in infected cells. The crystal structures of several viral RdRps that initiate RNA synthesis de novo have been reported, including that of HCV NS5B (3,13,25), the first such structure to be solved, and more recently those of other Flaviviridae polymerases (15, 31, 51). All of these enzymes are homologous and for all of them the "fingers" and "thumb" subdomains are connected (through the so-called "fingertips") and cluster around the central, catalytic "palm" s...

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5B initiates RNA synthesis by a de novo mechanism and then processively copies the whole RNA template. Dissections of de novo RNA synthesis by genotype 1 NS5B proteins previously established that there are two successive crucial steps in de novo initiation. The first is dinucleotide formation, which requires a closed conformation, and the second is the transition to elongation, which requires an opening of NS5B. We also recently published a combined structural and functional analysis of genotype 2 HCV-NS5B proteins (of strains JFH1 and J6) that established residue 405 as a key element in de novo RNA synthesis ( T he hepatitis C virus (HCV) is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae (28). The genome of HCV is a 9,600-nucleotide (nt)-long RNA molecule encompassing a single open reading frame (ORF) that is translated primarily into one polyprotein and flanked by nontranslated regions (NTRs). The NTRs are the most conserved parts of the viral genome and play important roles in viral translation and replication. The polyprotein precursor is cleaved by cellular and viral proteases into at least 10 different mature proteins. The nonstructural proteins NS3 to NS5B are associated within a membrane replication complex in which NS5B is the RNA-dependent RNA polymerase (RdRp). NS5B copies the RNA genome into a complementary negative strand and subsequently uses this negative strand as the template for the synthesis of new HCV genomes (for a review, see reference 21).In vitro, recombinant NS5B is a highly processive RdRp that copies RNA templates up to the size of the HCV genome (4). C-terminal deletions of the 21-residue transmembrane helix (NS5B⌬C21) are fully active and much easier to produce and purify than the full-length enzyme (37), so that these deletions are used almost exclusively for structural and functional characterization of NS5B. These studies have shown that NS5B can initiate RNA synthesis by a de novo mechanism (19) allowing full copy of the template in a single polymerization round (14,29). De novo initiation from the 3= end of the viral positive-and negative-strand RNA is likely to be the physiological mode of initiation of RNA synthesis in infected cells (8). Recombinant NS5B is very poorly active overall despite its high processivity and a turnover number comparable to that of other polymerases (7). Dissections of the early steps of RNA synthesis in vitro (12,14,29) have shown that this is due to two inefficient steps in de novo RNA synthesis by NS5B; the first step is de novo initiation proper, i.e., formation of the first dinucleotide complementary to the last two 3= bases of the template, and the second step is use of this dinucleotide as a primer for further RNA synthesis.Structural analysis of NS5B led us to propose that these two steps are closely linked to two conformational st...

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