Nsp3-N interactions are critical for SARS-CoV-2 fitness and virulence

Significance SARS-CoV-2, the causative agent of COVID-19, encodes several proteins not present in other coronaviruses. SARS-Unique Domain (SUD), a domain in the nonstructural protein 3 (nsp3) of coronavirus, is present only in Sarbecovirus, a subgenus of Betacoronavirus, including two highly pathogenic human coronaviruses, SARS-CoV and SARS-CoV-2. We report that nsp3-S676T, a single mutation in the SUD, causes SARS-CoV-2 fitness loss through dysregulation of its translational enhancement ability. N-S194L, a compensatory mutation identified in the nucleocapsid (N) protein, reverts viral virulence. Our study reveals a mechanism by which N-nsp3 interactions contribute to SARS-CoV-2 replication and pathogenesis but also identifies an effective antiviral therapeutic target for COVID-19.

[1]  R. Bartenschlager,et al.  SARS-CoV-2 nsp3-4 suffice to form a pore shaping replication organelles , 2022, bioRxiv.

[2]  S. Ovchinnikov,et al.  ColabFold: making protein folding accessible to all , 2022, Nature Methods.

[3]  Kristen Fortney,et al.  Eicosanoid signalling blockade protects middle-aged mice from severe COVID-19 , 2022, Nature.

[4]  A. Swaminathan,et al.  SARS-CoV-2 Mutations and COVID-19 Clinical Outcome: Mutation Global Frequency Dynamics and Structural Modulation Hold the Key , 2022, Frontiers in Cellular and Infection Microbiology.

[5]  M. Blackledge,et al.  The intrinsically disordered SARS-CoV-2 nucleoprotein in dynamic complex with its viral partner nsp3a , 2022, Science advances.

[6]  C. A. Koetzner,et al.  Analysis of a crucial interaction between the coronavirus nucleocapsid protein and the major membrane-bound subunit of the viral replicase-transcriptase complex , 2021, Virology.

[7]  F. Barona-Gómez,et al.  Phylogenomics and population genomics of SARS-CoV-2 in Mexico during the pre-vaccination stage reveals variants of interest B.1.1.28.4 and B.1.1.222 or B.1.1.519 and the nucleocapsid mutation S194L associated with symptoms , 2021, Microbial genomics.

[8]  R. Bartenschlager,et al.  Contribution of autophagy machinery factors to HCV and SARS-CoV-2 replication organelle formation , 2021, Cell Reports.

[9]  J. Lieberman,et al.  Targeting stem-loop 1 of the SARS-CoV-2 5′ UTR to suppress viral translation and Nsp1 evasion , 2021, bioRxiv.

[10]  J. Doudna,et al.  Rapid assessment of SARS-CoV-2–evolved variants using virus-like particles , 2021, bioRxiv.

[11]  Haibo Wu,et al.  Nucleocapsid mutations R203K/G204R increase the infectivity, fitness, and virulence of SARS-CoV-2 , 2021, Cell Host & Microbe.

[12]  H. Leonhardt,et al.  The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation , 2021, The EMBO journal.

[13]  P. Agarwal,et al.  SARS-CoV-2 Genomes From Oklahoma, United States , 2021, Frontiers in Genetics.

[14]  S. Perlman,et al.  SARS-CoV-2-induced immune activation and death of monocyte-derived human macrophages and dendritic cells. , 2020, The Journal of infectious diseases.

[15]  V. Thiel,et al.  Coronavirus biology and replication: implications for SARS-CoV-2 , 2020, Nature Reviews Microbiology.

[16]  N. Ban,et al.  SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation , 2020, bioRxiv.

[17]  D. Agard,et al.  A molecular pore spans the double membrane of the coronavirus replication organelle , 2020, Science.

[18]  Maxwell I. Zimmerman,et al.  The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA , 2020, bioRxiv.

[19]  D. Ruggero,et al.  The Role of Translation Control in Tumorigenesis and Its Therapeutic Implications , 2020, Annual Review of Cancer Biology.

[20]  E. Holmes,et al.  Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding , 2020, The Lancet.

[21]  M. Ulaşlı,et al.  Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle , 2019, Journal of Virology.

[22]  Anthony R. Fehr.  Bacterial Artificial Chromosome-Based Lambda Red Recombination with the I-SceI Homing Endonuclease for Genetic Alteration of MERS-CoV , 2019, Methods in molecular biology.

[23]  S. Koul,et al.  Eukaryotic Translation Initiation Factor 4 Gamma 1 (eIF4G1) is upregulated during Prostate cancer progression and modulates cell growth and metastasis , 2018, Scientific Reports.

[24]  A. Koster,et al.  Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication , 2017, mBio.

[25]  Rolf Hilgenfeld,et al.  Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein , 2017, Antiviral Research.

[26]  Jincun Zhao,et al.  The Conserved Coronavirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome Coronavirus Infection , 2016, mBio.

[27]  Marco Y. Hein,et al.  p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1 , 2016, Proceedings of the National Academy of Sciences.

[28]  Rolf Hilgenfeld,et al.  A G-quadruplex-binding macrodomain within the “SARS-unique domain” is essential for the activity of the SARS-coronavirus replication–transcription complex , 2015, Virology.

[29]  N. Sonenberg,et al.  Targeting the translation machinery in cancer , 2015, Nature Reviews Drug Discovery.

[30]  S. Perlman,et al.  The nsp3 Macrodomain Promotes Virulence in Mice with Coronavirus-Induced Encephalitis , 2014, Journal of Virology.

[31]  B. Neuman,et al.  Severe Acute Respiratory Syndrome Coronavirus Nonstructural Proteins 3, 4, and 6 Induce Double-Membrane Vesicles , 2013, mBio.

[32]  C. A. Koetzner,et al.  Characterization of a Critical Interaction between the Coronavirus Nucleocapsid Protein and Nonstructural Protein 3 of the Viral Replicase-Transcriptase Complex , 2013, Journal of Virology.

[33]  D. Giedroc,et al.  Solution Structure of Mouse Hepatitis Virus (MHV) nsp3a and Determinants of the Interaction with MHV Nucleocapsid (N) Protein , 2013, Journal of Virology.

[34]  P. Pandolfi,et al.  eIF4E phosphorylation promotes tumorigenesis and is associated with prostate cancer progression , 2010, Proceedings of the National Academy of Sciences.

[35]  S. Goebel,et al.  An Interaction between the Nucleocapsid Protein and a Component of the Replicase-Transcriptase Complex Is Crucial for the Infectivity of Coronavirus Genomic RNA , 2010, Journal of Virology.

[36]  Kurt Wüthrich,et al.  SARS Coronavirus Unique Domain: Three-Domain Molecular Architecture in Solution and RNA Binding , 2010, Journal of Molecular Biology.

[37]  I. Sola,et al.  Coronavirus Nucleocapsid Protein Facilitates Template Switching and Is Required for Efficient Transcription , 2009, Journal of Virology.

[38]  Rolf Hilgenfeld,et al.  The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes , 2009, PLoS pathogens.

[39]  Abraham J Koster,et al.  SARS-Coronavirus Replication Is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum , 2008, PLoS biology.

[40]  Margaret A. Johnson,et al.  Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3 , 2008, Journal of Virology.

[41]  S. Baker,et al.  Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles , 2008, Virology.

[42]  Y. Guan,et al.  Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage , 2003, Journal of Molecular Biology.

[43]  M. Welsh,et al.  An in vitro model of differentiated human airway epithelia. Methods for establishing primary cultures. , 2002, Methods in molecular biology.

[44]  S. Tahara,et al.  High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA. , 2000, The Journal of general virology.