Electron microscopy studies of the coronavirus ribonucleoprotein complex

Gui, Miao; Liu, Xing; Guo, Deyin; Zhang, Zhen; Yin, Chengzhu; Chen, Yu; Xiang, Ye

doi:10.1007/s13238-016-0352-8

Cited by 73 publications

(105 citation statements)

References 15 publications

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“…Several models for N-CTD dimer assembly have been proposed for the formation of filamentous RNPs. 33 All of the proposed interfaces between N-CTD dimers occurred on the side-faces of the CTD cuboid perpendicular to the proposed RNA-binding surface ( Figure 3H). Combinatorial use of any region on the side-faces of the CTD dimer cuboid may facilitate manipulation of the RNP length and curvature without obstructing the RNA-binding surface.…”

Section: ■ Resultsmentioning

confidence: 96%

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Structure-Based Stabilization of Non-native Protein–Protein Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug Design

et al. 2020

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Structure-based stabilization of protein−protein interactions (PPIs) is a promising strategy for drug discovery. However, this approach has mainly focused on the stabilization of native PPIs, and non-native PPIs have received little consideration. Here, we identified a non-native interaction interface on the three-dimensional dimeric structure of the N-terminal domain of the MERS-CoV nucleocapsid protein (MERS-CoV N-NTD). The interface formed a conserved hydrophobic cavity suitable for targeted drug screening. By considering the hydrophobic complementarity during the virtual screening step, we identified 5benzyloxygramine as a new N protein PPI orthosteric stabilizer that exhibits both antiviral and N-NTD protein-stabilizing activities. X-ray crystallography and small-angle X-ray scattering showed that 5-benzyloxygramine stabilizes the N-NTD dimers through simultaneous hydrophobic interactions with both partners, resulting in abnormal N protein oligomerization that was further confirmed in the cell. This unique approach based on the identification and stabilization of non-native PPIs of N protein could be applied toward drug discovery against CoV diseases.

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Section: ■ Resultsmentioning

confidence: 96%

“…The conformation of the solution was consistent with structures previously reported for other CoV N proteins. 33 The N-P3 complex formed a compact hexadecamer with a sunburst configuration ( Figure 3E). The CTDs formed a central ring and non-native NTD dimers formed "spikes" protruding from the ring.…”

Section: ■ Resultsmentioning

confidence: 99%

Structure-Based Stabilization of Non-native Protein–Protein Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug Design

et al. 2020

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“…The nsp3 BDs of the N protein are not involved in its self-assembly and oligomerization. We and others recently showed that CoV N proteins form oligomers constitutively (22,23). Therefore, a possible scenario that could not be excluded a priori was that the introduced substitutions in the N protein were affecting its organization into oligomers, which might indirectly impair binding to nsp3.…”

Section: Resultsmentioning

confidence: 99%

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle

Cong

Ulaşlı

Schepers

et al. 2020

J Virol

347

330

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Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo. Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis. IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.

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“…Although structures are currently available for 6 NTDs and 4 CTDs from different viruses, there is as yet no structure for an RNA-bound complex of either of these domains. Likewise, no structure has been solved for an entire N protein, with or without an RNA ligand, but a recent low-resolution cryo-EM reconstruction of MHV N has taken the first step in that direction (Gui et al, 2017). At the next level, we lack a clear picture of the coronavirus nucleocapsid.…”

Section: Future Perspectivesmentioning

confidence: 99%

“…1). Second, coronaviruses have helically symmetric nucleocapsids (Masters, 2006;Neuman et al, 2006;Gui et al, 2017), a feature that is unusual for positive-strand RNA viruses, which generally have icosahedral capsids. Third, the assembly of the nucleocapsid into the virion occurs through interactions between the carboxy termini of multiple monomers of N protein and of M protein (Escors et al, 2001;Hurst et al, 2005;Verma et al, 2006).…”

Section: Coronavirusesmentioning

confidence: 99%

Coronavirus genomic RNA packaging

2019

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RNA viruses carry out selective packaging of their genomes in a variety of ways, many involving a genomic packaging signal. The first coronavirus packaging signal was discovered nearly thirty years ago, but how it functions remains incompletely understood. This review addresses the current state of knowledge of coronavirus genome packaging, which has mainly been studied in two prototype species, mouse hepatitis virus and transmissible gastroenteritis virus. Despite the progress that has been made in the mapping and characterization of some packaging signals, there is conflicting evidence as to whether the viral nucleocapsid protein or the membrane protein plays the primary role in packaging signal recognition. The different models for the mechanism of genomic RNA packaging that have been prompted by these competing views are described. Also discussed is the recent exciting discovery that selective coronavirus genome packaging is critical for in vivo evasion of the host innate immune response.

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Electron microscopy studies of the coronavirus ribonucleoprotein complex

Cited by 73 publications

References 15 publications

Structure-Based Stabilization of Non-native Protein–Protein Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug Design

Structure-Based Stabilization of Non-native Protein–Protein Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug Design

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle

Coronavirus genomic RNA packaging

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