In vitro assembly of Ebola virus nucleocapsid-like complex expressed in E. coli

Peng, Ruchao; Zhu, Tengfei; Oladejo, Babayemi Olawale; Musyoki, Abednego Moki; Cui, Yingzi; Shi, Yi; Wang, Peiyi; Gao, George F.

doi:10.1007/s13238-016-0314-1

Cited by 9 publications

(3 citation statements)

References 34 publications

(72 reference statements)

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“…Although all truncated constructs could form higher-order structures to some extent under 2 M NaCl condition (Figs. 2 and 3 ), indicative of the universal effect of salt on protein assembly [ 54 , 55 ], only those constructs that contained CTD but not NTD formed higher-order assembly in the presence of RNAs under physiological relevant salt condition (Fig. 4 ), consistent with previous studies that CTD domain of N was prone to oligomerization [ 30 , 55 – 57 ].…”

Section: Resultssupporting

confidence: 89%

Modular characterization of SARS-CoV-2 nucleocapsid protein domain functions in nucleocapsid-like assembly

et al. 2023

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SARS-CoV-2 and its variants, with the Omicron subvariant XBB currently prevailing the global infections, continue to pose threats on public health worldwide. This non-segmented positive-stranded RNA virus encodes the multi-functional nucleocapsid protein (N) that plays key roles in viral infection, replication, genome packaging and budding. N protein consists of two structural domains, NTD and CTD, and three intrinsically disordered regions (IDRs) including the NIDR, the serine/arginine rich motif (SRIDR), and the CIDR. Previous studies revealed functions of N protein in RNA binding, oligomerization, and liquid–liquid phase separation (LLPS), however, characterizations of individual domains and their dissected contributions to N protein functions remain incomplete. In particular, little is known about N protein assembly that may play essential roles in viral replication and genome packing. Here, we present a modular approach to dissect functional roles of individual domains in SARS-CoV-2 N protein that reveals inhibitory or augmented modulations of protein assembly and LLPS in the presence of viral RNAs. Intriguingly, full-length N protein (NFL) assembles into ring-like architecture whereas the truncated SRIDR-CTD-CIDR (N182-419) promotes filamentous assembly. Moreover, LLPS droplets of NFL and N182-419 are significantly enlarged in the presence of viral RNAs, and we observed filamentous structures in the N182-419 droplets using correlative light and electron microscopy (CLEM), suggesting that the formation of LLPS droplets may promote higher-order assembly of N protein for transcription, replication and packaging. Together this study expands our understanding of the multiple functions of N protein in SARS-CoV-2.

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Section: Resultssupporting

confidence: 89%

Modular characterization of SARS-CoV-2 nucleocapsid protein domain functions in nucleocapsid-like assembly

et al. 2023

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“…Following previous reports (Guryanov et al, 2015; Peng et al, 2016), the NDV N was expressed in an Escherichia coli system and pure protein was obtained after tandem affinity and gel-filtration chromatography. The N was found to be of high purity in SDS-PAGE, with an absorbance of A260/A280 of ∼1.1, suggesting the presence of RNA-bound N (Figure 1—figure supplement 1A).…”

Section: Resultsmentioning

confidence: 99%

Self-capping of nucleoprotein filaments protects the Newcastle disease virus genome

Song

Hong

Zhu

et al. 2019

eLife

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Non-segmented negative-strand RNA viruses, such as measles, ebola and Newcastle disease viruses (NDV), encapsidate viral genomic RNAs into helical nucleocapsids, which serve as the template for viral replication and transcription. Here, the clam-shaped nucleocapsid structure, where the NDV viral genome is sequestered, was determined at 4.8 Å resolution by cryo-electron microscopy. The clam-shaped structure is composed of two single-turn spirals packed in a back-to-back mode. This tightly packed structure functions as a seed for the assembly of a nucleocapsid from both directions, facilitating the growth of double-headed filaments with two separate RNA strings inside. Disruption of this structure by mutations in its loop interface yielded a single-headed unfunctional filament.

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“…As an indispensable component of the viral replication machinery (5), NP interacts with other viral proteins, including VP24 (6), VP40 (7), VP35, and VP30 (8), to organize the replication process. It also assembles into a helical tubular structure as the scaffold of nucleocapsid formation (9)(10)(11)(12). Sequence homology (13) shows that NP contains a conserved N-terminal region that is sufficient for self-assembly and single-stranded RNA (ssRNA) packaging (9), as well as a largely unstructured C-terminal region (14) that contains a domain required for virion budding (7).…”

mentioning

confidence: 99%

Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus

Zhu

Song

Peng

et al. 2017

J Virol

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Filovirus nucleoprotein (NP), viral protein 35 (VP35), and polymerase L are essential for viral replication and nucleocapsid formation. Here, we identify a 28-residue peptide (NP binding peptide [NPBP]) from Marburg virus (MARV) VP35 through sequence alignment with previously identified Ebola virus (EBOV) NPBP, which bound to the core region (residues 18 to 344) of the N-terminal portion of MARV NP with high affinity. The crystal structure of the MARV NP core/NPBP complex at a resolution of 2.6 Å revealed that NPBP binds to the C-terminal region of the NP core via electrostatic and nonpolar interactions. Further structural analysis revealed that the MARV and EBOV NP cores hold a conserved binding pocket for NPBP, and this pocket could serve as a promising target for the design of universal drugs against filovirus infection. In addition, cross-binding assays confirmed that the NP core of MARV or EBOV can bind the NPBP from the other virus, although with moderately reduced binding affinities that result from termini that are distinct between the MARV and EBOV NPBPs. Historically, Marburg virus (MARV) has caused severe disease with up to 90% lethality. Among the viral proteins produced by MARV, NP and VP35 are both multifunctional proteins that are essential for viral replication. In its relative, Ebola virus (EBOV), an N-terminal peptide from VP35 binds to the NP N-terminal region with high affinity. Whether this is a common mechanism among filoviruses is an unsolved question. Here, we present the crystal structure of a complex that consists of the core domain of MARV NP and the NPBP peptide from VP35. As we compared MARV NPBP with EBOV NPBP, several different features at the termini were identified. Although these differences reduce the affinity of the NP core for NPBPs across genera, a conserved pocket in the C-terminal region of the NP core makes cross-species binding possible. Our results expand our knowledge of filovirus NP-VP35 interactions and provide more details for therapeutic intervention.

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In vitro assembly of Ebola virus nucleocapsid-like complex expressed in E. coli

Cited by 9 publications

References 34 publications

Modular characterization of SARS-CoV-2 nucleocapsid protein domain functions in nucleocapsid-like assembly

Modular characterization of SARS-CoV-2 nucleocapsid protein domain functions in nucleocapsid-like assembly

Self-capping of nucleoprotein filaments protects the Newcastle disease virus genome

Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus

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