The nsp9 Replicase Protein of SARS-Coronavirus, Structure and Functional Insights

Sutton, Geoff; Fry, Elizabeth E.; Carter, Lester; Sainsbury, Sarah; Walter, T S; Nettleship, Joanne E.; Berrow, Nicholas S.; Owens, Ray; Gilbert, R.J.C.; Davidson, Andrew D.; Siddell, Stuart G.; Poon, Leo L. M.; Diprose, Jonathan M.; Alderton, David; Walsh, M.A.; Grimes, Jonathan M.; Stuart, D.I.

doi:10.1016/j.str.2004.01.016

Cited by 235 publications

(145 citation statements)

References 52 publications

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“…CoV nsp9 has diverse dimerization, while its function of enhancement nuclear binding affinity is conservative (22)(23)(24)(25). We propose that most nsp9 proteins originated from ancestral coronavirus, which have N-finger motifs and GXXXG motifs that both play critical roles in dimerization.…”

Section: Resultsmentioning

confidence: 96%

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Dimerization of Coronavirus nsp9 with Diverse Modes Enhances Its Nucleic Acid Binding Affinity

Zeng

Deng

Shi

et al. 2018

J Virol

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Coronaviruses pose serious health threats to humans and other animals. Understanding the mechanisms of their replication has important implications for global health and economic stability. Nonstructural protein 9 (nsp9) is an essential RNA binding protein for coronavirus replication. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. Here, we report four crystal structures, including wild-type porcine delta coronavirus (PDCoV) nsp9, PDCoV nsp9-ΔN7 (N-terminal 7 amino acids deleted), wild-type porcine epidemic diarrhea virus (PEDV) nsp9, and PEDV nsp9-C59A mutant. These structures reveal the diverse dimerization forms of coronavirus nsp9. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. Interestingly, size exclusion chromatography and analytical ultracentrifugation analyses indicate that the PDCoV nsp9-ΔN7 and PEDV nsp9-C59A mutants are monomeric in solution. In addition, electrophoretic mobility shift assays and microscale thermophoresis analysis indicate that the monomeric forms of PDCoV nsp9 and PEDV nsp9 still have nucleic acid binding affinity, although it is lower than that of the wild type. Our results show that the diverse dimerization forms of coronavirus nsp9 proteins enhance their nucleic acid binding affinity. Coronaviruses cause widespread respiratory, gastrointestinal, and central nervous system diseases in humans and other animals, threatening human health and causing economic loss. Coronavirus nsp9, a member of the replication complex, is an important RNA binding subunit in the RNA-synthesizing machinery of all coronaviruses. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. In this study we determined the nsp9 crystal structures of PDCoV and PEDV. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. This study provides a structural and functional basis for understanding the mechanism of dimerization and shows that the diverse dimerization modes of coronavirus nsp9 proteins enhance their nucleic acid binding affinity. Importantly, these findings may provide a new insight for antiviral drug development.

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Section: Resultsmentioning

confidence: 96%

“…Meanwhile, the dimerization of nsp9 is critical for viral replication (21). For nsp9, there are five crystallographic structures that showed a variety of dimeric interfaces (22)(23)(24)(25). However, the dimerization mechanism of all CoV nsp9s, whether from emerging viruses or not, is not so clear yet.…”

mentioning

confidence: 99%

Dimerization of Coronavirus nsp9 with Diverse Modes Enhances Its Nucleic Acid Binding Affinity

Zeng

Deng

Shi

et al. 2018

J Virol

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“…The nsp7-nsp8-nsp12 complex formed in vitro is able to catalyze de novo synthesis of relatively long RNAs (up to 340 nt) in a processive manner. Also interacting with nsp8 is nsp9, a small protein that binds ssRNA without sequence specificity (130–132). Dimerization of nsp9 is critical for virus replication (133), and its structural and functional features strongly suggest that it could be a component of the RTC catalytic core, stabilizing viral RNAs during RNA synthesis and processing.…”

Section: Cellular and Viral Proteins Of The Coronavirus Replication-tmentioning

confidence: 99%

Continuous and Discontinuous RNA Synthesis in Coronaviruses

et al. 2015

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Replication of the coronavirus genome requires continuous RNA synthesis, whereas transcription is a discontinuous process unique among RNA viruses. Transcription includes a template switch during the synthesis of subgenomic negative-strand RNAs to add a copy of the leader sequence. Coronavirus transcription is regulated by multiple factors, including the extent of base-pairing between transcription-regulating sequences of positive and negative polarity, viral and cell protein–RNA binding, and high-order RNA-RNA interactions. Coronavirus RNA synthesis is performed by a replication-transcription complex that includes viral and cell proteins that recognize cis-acting RNA elements mainly located in the highly structured 5′ and 3′ untranslated regions. In addition to many viral nonstructural proteins, the presence of cell nuclear proteins and the viral nucleocapsid protein increases virus amplification efficacy. Coronavirus RNA synthesis is connected with the formation of double-membrane vesicles and convoluted membranes. Coronaviruses encode proofreading machinery, unique in the RNA virus world, to ensure the maintenance of their large genome size.

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“…In addition to the nsps mentioned above, other CoV nsps are involved in RNA binding (nsp9 and nsp10; [48,49]) or in evasion of the antiviral response of the host (nsp1 and nsp3; [50,51,52,53,54,55,56,57]). The function of nsp2 is not yet known, although this protein was shown not to be essential for virus replication [58,59].…”

Section: Coronavirus Nonstructural Proteinsmentioning

confidence: 99%

Biogenesis and Dynamics of the Coronavirus Replicative Structures

Hagemeijer

Rottier

Haan

2012

Viruses

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Coronaviruses are positive-strand RNA viruses that are important infectious agents of both animals and humans. A common feature among positive-strand RNA viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. Upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in RNA synthesis localize. Double-stranded RNA, presumably functioning as replicative intermediate during viral RNA synthesis, has been detected at the double-membrane vesicle interior. Recent studies have provided new insights into the assembly and functioning of the coronavirus replicative structures. This review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the replication-transcription complexes, and the location of viral RNA synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins.

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The nsp9 Replicase Protein of SARS-Coronavirus, Structure and Functional Insights

Cited by 235 publications

References 52 publications

Dimerization of Coronavirus nsp9 with Diverse Modes Enhances Its Nucleic Acid Binding Affinity

Dimerization of Coronavirus nsp9 with Diverse Modes Enhances Its Nucleic Acid Binding Affinity

Continuous and Discontinuous RNA Synthesis in Coronaviruses

Biogenesis and Dynamics of the Coronavirus Replicative Structures

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