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Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit.
Slanina, Heiko; Madhugiri, Ramakanth; Bylapudi, Ganesh; Schultheiß, Karin; Karl, Nadja; Gulyaeva, Anastasia; Gorbalenya, Alexander E; Linne, Uwe; Ziebuhr, John.
  • Slanina H; Institute of Medical Virology, Justus Liebig University Giessen, 35392 Giessen, Germany.
  • Madhugiri R; Institute of Medical Virology, Justus Liebig University Giessen, 35392 Giessen, Germany.
  • Bylapudi G; Institute of Medical Virology, Justus Liebig University Giessen, 35392 Giessen, Germany.
  • Schultheiß K; Institute of Medical Virology, Justus Liebig University Giessen, 35392 Giessen, Germany.
  • Karl N; Institute of Medical Virology, Justus Liebig University Giessen, 35392 Giessen, Germany.
  • Gulyaeva A; Department of Medical Microbiology, Leiden University Medical Center, Leiden 2300 RC, The Netherlands.
  • Gorbalenya AE; Department of Medical Microbiology, Leiden University Medical Center, Leiden 2300 RC, The Netherlands.
  • Linne U; Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden 2300 RC, The Netherlands.
  • Ziebuhr J; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119991 Moscow, Russia.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Article in English | MEDLINE | ID: covidwho-1039676
Semantic information from SemMedBD (by NLM)
1. Biopolymers PART_OF Cells
Subject
Biopolymers
Predicate
PART_OF
Object
Cells
2. Zinc Finger FYVE Domain-Containing Protein PART_OF C0230171
Subject
Zinc Finger FYVE Domain-Containing Protein
Predicate
PART_OF
Object
C0230171
3. Virus LOCATION_OF Homologous Gene
Subject
Virus
Predicate
LOCATION_OF
Object
Homologous Gene
4. Biopolymers PART_OF Cells
Subject
Biopolymers
Predicate
PART_OF
Object
Cells
5. Zinc Finger FYVE Domain-Containing Protein 9, human PART_OF Flank (surface region)
Subject
Zinc Finger FYVE Domain-Containing Protein 9, human
Predicate
PART_OF
Object
Flank (surface region)
6. Virus LOCATION_OF Homologous Gene
Subject
Virus
Predicate
LOCATION_OF
Object
Homologous Gene
ABSTRACT
RNA-dependent RNA polymerases (RdRps) of the Nidovirales (Coronaviridae, Arteriviridae, and 12 other families) are linked to an amino-terminal (N-terminal) domain, called NiRAN, in a nonstructural protein (nsp) that is released from polyprotein 1ab by the viral main protease (Mpro). Previously, self-GMPylation/UMPylation activities were reported for an arterivirus NiRAN-RdRp nsp and suggested to generate a transient state primed for transferring nucleoside monophosphate (NMP) to (currently unknown) viral and/or cellular biopolymers. Here, we show that the coronavirus (human coronavirus [HCoV]-229E and severe acute respiratory syndrome coronavirus 2) nsp12 (NiRAN-RdRp) has Mn2+-dependent NMPylation activity that catalyzes the transfer of a single NMP to the cognate nsp9 by forming a phosphoramidate bond with the primary amine at the nsp9 N terminus (N3825) following Mpro-mediated proteolytic release of nsp9 from N-terminally flanking nsps. Uridine triphosphate was the preferred nucleotide in this reaction, but also adenosine triphosphate, guanosine triphosphate, and cytidine triphosphate were suitable cosubstrates. Mutational studies using recombinant coronavirus nsp9 and nsp12 proteins and genetically engineered HCoV-229E mutants identified residues essential for NiRAN-mediated nsp9 NMPylation and virus replication in cell culture. The data corroborate predictions on NiRAN active-site residues and establish an essential role for the nsp9 N3826 residue in both nsp9 NMPylation in vitro and virus replication. This residue is part of a conserved N-terminal NNE tripeptide sequence and shown to be the only invariant residue in nsp9 and its homologs in viruses of the family Coronaviridae The study provides a solid basis for functional studies of other nidovirus NMPylation activities and suggests a possible target for antiviral drug development.
Subject(s)
Keywords

Full text: Available Collection: International databases Database: MEDLINE Main subject: Virus Replication / RNA-Binding Proteins / Viral Nonstructural Proteins / Coronavirus 229E, Human / SARS-CoV-2 Limits: Humans Language: English Year: 2021 Document Type: Article Affiliation country: Pnas.2022310118

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Virus Replication / RNA-Binding Proteins / Viral Nonstructural Proteins / Coronavirus 229E, Human / SARS-CoV-2 Limits: Humans Language: English Year: 2021 Document Type: Article Affiliation country: Pnas.2022310118