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Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes.

ABSTRACT: The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2'-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.

SUBMITTER: Bouvet M

PROVIDER: S-EPMC4162180 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes.

Bouvet Mickaël M Lugari Adrien A Posthuma Clara C CC Zevenhoven Jessika C JC Bernard Stéphanie S Betzi Stéphane S Imbert Isabelle I Canard Bruno B Guillemot Jean-Claude JC Lécine Patrick P Pfefferle Susanne S Drosten Christian C Snijder Eric J EJ Decroly Etienne E Morelli Xavier X

The Journal of biological chemistry 20140729 37

The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, ...[more]

PMID: 25074927

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Project description:The SARS coronavirus (SARS-CoV) replicase gene encodes 16 nonstructural proteins (nsps) with multiple enzymatic activities. Several of these enzymes are common components of replication complexes of other plus-strand RNA viruses, such as picornavirus 3C-like protease, papain-like protease, RNA-dependent RNA polymerase, RNA helicase, and ribose 2′-O-methyltransferase activities, while others such as exoribonuclease, endoribonuclease, and adenosine diphosphate-ribose 1″-phosphatase activities, are rarely or not conserved in viruses outside the order Nidovirales. The latter enzymes are believed to be involved in unique metabolic pathways used by coronaviruses to (1) replicate and transcribe their extremely large RNA genomes, and (2) interfere with cellular functions and antiviral host responses. Since the global outbreak of SARS in 2003, major efforts have been made to elucidate the structures of the protein components of the SARS-CoV replication/transcription complex. Thus, in less than 5 years, the structures of as many as 16 SARS-CoV proteins or functional domains have been determined. Remarkably, eight of these 16 structures had novel folds, illustrating the uniqueness of the coronavirus replicative machinery. Furthermore, several new protein functions and potential drug targets have been identified in these studies. Current structural studies mainly focus on the few remaining proteins for which no structural information is available and larger protein complexes comprised of different nsps. The studies aim at obtaining detailed information on the functions and macromolecular assembly of the coronavirus replication/transcription machinery which, over a long period of time, may be used to develop selective antiviral drugs. This chapter reviews structural information on the SARS-CoV macro domain (ADRP) as well as nsps 7, 8, 9, and 15 and summarizes our current knowledge of active-site residues and intermolecular interactions of these proteins.

Project description:UnlabelledThe 5' cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases (2'-O-MTase) to autonomously modify their mRNAs and carry a cap-1 structure (m7GpppNm) at the 5' end, thereby facilitating viral replication and escaping innate immune recognition in host cells. Previous studies showed that the 2'-O-MTase activity of severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 16 (nsp16) needs to be activated by nsp10, whereas nsp16 of feline coronavirus (FCoV) alone possesses 2'-O-MTase activity (E. Decroly et al., J Virol 82:8071-8084, 2008, http://dx.doi.org/10.1128/JVI.00407-08; M. Bouvet et al., PLoS Pathog 6:e1000863, 2010, http://dx.doi.org/10.1371/journal.ppat.1000863; E. Decroly et al., PLoS Pathog 7:e1002059, 2011, http://dx.doi.org/10.1371/journal.ppat.1002059; Y. Chen et al., PLoS Pathog 7:e1002294, 2011, http://dx.doi.org/10.1371/journal.ppat.1002294) . In this study, we demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a universal and conserved mechanism in coronaviruses, including FCoV, and that nsp10 is functionally interchangeable in the stimulation of nsp16 of different coronaviruses. Based on our current and previous studies, we designed a peptide (TP29) from the sequence of the interaction interface of mouse hepatitis virus (MHV) nsp10 and demonstrated that the peptide inhibits the 2'-O-MTase activity of different coronaviruses in biochemical assays and the viral replication in MHV infection and SARS-CoV replicon models. Interestingly, the peptide TP29 exerted robust inhibitory effects in vivo in MHV-infected mice by impairing MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production at an early stage of infection. Therefore, as a proof of principle, the current results indicate that coronavirus 2'-O-MTase activity can be targeted in vitro and in vivo.ImportanceCoronaviruses are important pathogens of animals and human with high zoonotic potential. SARS-CoV encodes the 2'-O-MTase that is composed of the catalytic subunit nsp16 and the stimulatory subunit nsp10 and plays an important role in virus genome replication and evasion from innate immunity. Our current results demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a common mechanism for coronaviruses, and nsp10 is functionally interchangeable in the stimulation of nsp16 among different coronaviruses, which underlies the rationale for developing inhibitory peptides. We demonstrate that a peptide derived from the nsp16-interacting domain of MHV nsp10 could inhibit 2'-O-MTase activity of different coronaviruses in vitro and viral replication of MHV and SARS-CoV replicon in cell culture, and it could strongly inhibit virus replication and pathogenesis in MHV-infected mice. This work makes it possible to develop broad-spectrum peptide inhibitors by targeting the nsp16/nsp10 2'-O-MTase of coronaviruses.

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Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes.

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Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes.

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