A viral deubiquitylating enzyme targets viral RNA-dependent RNA polymerase and affects viral infectivity

Selective protein degradation via the ubiquitin-proteasome system (UPS) plays an essential role in many major cellular processes, including host-pathogen interactions. We previously reported that the tightly regulated viral RNA-dependent RNA polymerase (RdRp) of the positive-strand RNA virus Turnip yellow mosaic virus (TYMV) is degraded by the UPS in infected cells, a process that affects viral infectivity. Here, we show that the TYMV 98K replication protein can counteract this degradation process thanks to its proteinase domain. In-vitro assays revealed that the recombinant proteinase domain is a functional ovarian tumour (OTU)-like deubiquitylating enzyme (DUB), as is the 98K produced during viral infection. We also demonstrate that 98K mediates in-vivo deubiquitylation of TYMV RdRp protein--its binding partner within replication complexes--leading to its stabilization. Finally, we show that this DUB activity contributes to viral infectivity in plant cells. The identification of viral RdRp as a specific substrate of the viral DUB enzyme thus reveals the intricate interplay between ubiquitylation, deubiquitylation and the interaction between viral proteins in controlling levels of RdRp and viral infectivity.     

The RNA silencing enzyme RNA polymerase v is required for plant immunity

RNA-directed DNA methylation (RdDM) is an epigenetic control mechanism driven by small interfering RNAs (siRNAs) that influence gene function. In plants, little is known of the involvement of the RdDM pathway in regulating traits related to immune responses. In a genetic screen designed to reveal factors regulating immunity in Arabidopsis thaliana, we identified NRPD2 as the OVEREXPRESSOR OF CATIONIC PEROXIDASE 1 (OCP1). NRPD2 encodes the second largest subunit of the plant-specific RNA Polymerases IV and V (Pol IV and Pol V), which are crucial for the RdDM pathway. The ocp1 and nrpd2 mutants showed increases in disease susceptibility when confronted with the necrotrophic fungal pathogens Botrytis cinerea and Plectosphaerella cucumerina. Studies were extended to other mutants affected in different steps of the RdDM pathway, such as nrpd1, nrpe1, ago4, drd1, rdr2, and drm1drm2 mutants. Our results indicate that all the mutants studied, with the exception of nrpd1, phenocopy the nrpd2 mutants; and they suggest that, while Pol V complex is required for plant immunity, Pol IV appears dispensable. Moreover, Pol V defective mutants, but not Pol IV mutants, show enhanced disease resistance towards the bacterial pathogen Pseudomonas syringae DC3000. Interestingly, salicylic acid (SA)-mediated defenses effective against PsDC3000 are enhanced in Pol V defective mutants, whereas jasmonic acid (JA)-mediated defenses that protect against fungi are reduced. Chromatin immunoprecipitation analysis revealed that, through differential histone modifications, SA-related defense genes are poised for enhanced activation in Pol V defective mutants and provide clues for understanding the regulation of gene priming during defense. Our results highlight the importance of epigenetic control as an additional layer of complexity in the regulation of plant immunity and point towards multiple components of the RdDM pathway being involved in plant immunity based on genetic evidence, but whether this is a direct or indirect effect on disease-related genes is unclear.     

Active site localization in a viral mRNA capping enzyme

Capping of reovirus mRNAs is catalyzed by a guanylyltransferase that corresponds to virion structural polypeptide lambda 2. It forms a phosphoamide linked enzyme-pG covalent complex as an intermediate in the capping reaction. The nucleotide attachment site on lambda 2 was localized to a region between amino acids 213 and 269 by incubating virus particles with [alpha-32P]GTP followed by proteolytic cleavage and analysis of the resulting fragments using sequence-directed antibodies as probes. The 213-269 region contains as potential GMP acceptors a single lysine, 1 arginine, and 4 histidine residues, as deduced from the nucleotide sequence of the L2 gene encoding lambda 2. Digestion of 32P-labeled capping intermediate with alkali after oxidation and beta-elimination yielded phospholysine as the only phosphoamino acid, localizing the active site to a region in lambda 2 that includes the lysine at position 226.     

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that drives interactions between N proteins in condensates, and deletion of this region disrupts phase separation. We also identified small molecules that alter the size and shape of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.

The packaging of the SARS-CoV-2 genome is mediated by the nucleocapsid (N) protein; this study shows that the N protein forms liquid condensates with viral genomic RNA and identifies small molecules that alter these condensates.     

The SARS-CoV-2 RNA interactome

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Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping

Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene XRN1, encoding the major exonuclease that degrades uncapped mRNAs. XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants showed no complementation. Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.     

A mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analog inhibitors

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The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases

ATP- and NAD(+)-dependent DNA ligases, ATP-dependent RNA ligases and GTP-dependent mRNA capping enzymes comprise a superfamily of proteins that catalyze nucleotidyl transfer to polynucleotide 5' ends via covalent enzyme-(lysyl-N)-NMP intermediates. The superfamily is defined by five peptide motifs that line the nucleotide-binding pocket and contribute amino acid sidechains essential for catalysis. Early crystal structures revealed a shared core tertiary structure for DNA ligases and capping enzymes, which are composed minimally of a nucleotidyltransferase domain fused to a distal OB-fold domain. Recent structures of viral and bacterial DNA ligases, and a fungal mRNA capping enzyme underscore how the substrate-binding and chemical steps of the ligation and capping pathways are coordinated with large rearrangements of the component protein domains and with remodeling of the atomic contacts between the enzyme and the nucleotide at the active site. The first crystal structure of an RNA ligase suggests that contemporary DNA ligases, RNA ligases and RNA capping enzymes evolved by fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.     

Conversion of a DNA ligase into an RNA capping enzyme.

In eukaryotes, newly synthesised mRNA is 'capped' by the addition of GMP to the 5" end by RNA capping enzymes. Recent structural studies have shown that RNA capping enzymes and DNA ligases have similar protein folds, suggesting a conserved catalytic mechanism. To explore these similarities we have produced a chimeric enzyme comprising the N-terminal domain 1 of a DNA ligase fused to the C-terminal domain 2 of a mRNA capping enzyme. This report shows that this hybrid enzyme retains adenylation activity, characteristic of DNA ligases but, remarkably, the chimera has ATP-dependent mRNA capping activity. This is the first observation of ATP-dependent RNA capping. These results suggest that nucleotidyltransferases may have evolved from a common ancestral gene.     

SARS-CoV-2-encoded nucleocapsid protein acts as a viral suppressor of RNA interference in cells

正Dear Editor,Coronaviruses (Co Vs) are large enveloped non-segmented positive-strand RNA viruses that broadly distribute among humans and other animal species, including bats, mice and birds. SARS-Co V-2 infections can cause diseases, named the2019 novel coronavirus disease (COVID-19). The symptoms of COVID-19 range from mild symptoms to severe respiratory syndromes, including pneumonia, and even death(Chen et al., 2020b; Jiang and Shi, 2020; Xia et al., 2020).