Coronavirus Nucleocapsid Protein

Cologna, Raymond; Hogue, Brenda G.

doi:10.1007/978-1-4615-5331-1_46

Cited by 11 publications

(13 citation statements)

References 11 publications

(1 reference statement)

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“…5A, lane 5). However, less protein bound the pGEM RNA than the coronavirus-specific RNA transcripts, consistent with the earlier preliminary filter-binding results, suggesting that N binds coronavirus RNAs better than it binds heterologous RNAs (Cologna and Hogue, 1998).…”

Section: Mapping N-binding Sites By Filter Bindingsupporting

confidence: 89%

“…The reaction conditions were initially established using purified histidine (his)-tagged BCV N protein that had been expressed in bacteria. We previously reported our preliminary results using the his-tagged N, which demonstrated that N binds Drep, MHV DI MIDI-C, and a transcript that contained the MHV packaging signal more efficiently than a noncoronavirus RNA (Cologna and Hogue, 1998).…”

Section: Mapping N-binding Sites By Filter Bindingmentioning

confidence: 96%

“…A histidine-tagged BCV N fusion protein was expressed and purified from bacteria as previously described (Cologna and Hogue, 1998). Six histidine residues replaced the first 17 amino acids at the aminoterminus of the BCV N protein.…”

Section: Generation Of N-specific Polyclonal Antibodiesmentioning

confidence: 99%

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Identification of Nucleocapsid Binding Sites within Coronavirus-Defective Genomes

2000

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The coronavirus nucleocapsid (N) protein is a major structural component of virions that associates with the genomic RNA to form a helical nucleocapsid. N appears to be a multifunctional protein since data also suggest that the protein may be involved in viral RNA replication and translation. All of these functions presumably involve interactions between N and viral RNAs. As a step toward understanding how N interacts with viral RNAs, we mapped high-efficiency N-binding sites within BCV- and MHV-defective genomes. Both in vivo and in vitro assays were used to study binding of BCV and MHV N proteins to viral and nonviral RNAs. N-viral RNA complexes were detected in bovine coronavirus (BCV)-infected cells and in cells transiently expressing the N protein. Filter binding was used to map N-binding sites within Drep, a BCV-defective genome that is replicated and packaged in the presence of helper virus. One high-efficiency N-binding site was identified between nucleotides 1441 and 1875 at the 3' end of the N ORF within Drep. For comparative purposes N-binding sites were also mapped for the mouse hepatitis coronavirus (MHV)-defective interfering (DI) RNA MIDI-C. Binding efficiencies similar to those for Drep were measured for RNA transcripts of a region encompassing the MHV packaging signal (nts 3949-4524), as well as a region at the 3' end of the MHV N ORF (nts 4837-5197) within MIDI-C. Binding to the full-length MIDI-C transcript (approximately 5500 nts) and to an approximately 1-kb transcript from the gene 1a region (nts 935-1986) of MIDI-C that excluded the packaging signal were both significantly higher than that measured for the smaller transcripts. This is the first identification of N-binding sequences for BCV. It is also the first report to demonstrate that N interacts in vitro with sequences other than the packaging signal and leader within the MHV genome. The data clearly demonstrate that N binds coronavirus RNAs more efficiently than nonviral RNAs. The results have implications with regard to the multifunctional role of N.

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Section: Mapping N-binding Sites By Filter Bindingsupporting

confidence: 89%

Section: Mapping N-binding Sites By Filter Bindingmentioning

confidence: 96%

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Identification of Nucleocapsid Binding Sites within Coronavirus-Defective Genomes

2000

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“…The interactions of the MHV and BCV N proteins with packaging signals, which are located on the end of the polymerase gene, have been observed with in vitro assays (Van der Most et al, 1991;Cologna and Hogue, 1997). However, subgenomic RNAs, lacking the described packaging signals, have been detected in the packaged IBV particles (Zhao et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

The amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3′ genomic RNA

Zhou

Collisson

2000

Virus Research

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Previous studies indicated that the nucleocapsid (N) protein of infectious bronchitis virus (IBV) interacted with specific sequences in the 3' non-coding region of IBV RNA. In order to identify domains in the N protein that bind to RNA, the whole protein (409 amino acids) and six overlapping fragments were expressed as fusion polypeptides with six histidine-tags. Using gel shift assays, the intact N protein and amino polypeptides, from residues 1 to 171 and residues 1 to 274, and carboxyl polypeptides, extending from residues 203 to 409 and residues 268 to 407, were found to interact with positive-stranded IBV RNA representing the 3' end of the genome. The two 32P-labeled probes that interacted with N and the amino and carboxyl fragments of N were RNA consisting of the IBV N gene and adjacent 3' non-coding terminus, and RNA consisting of the 155-nucleotide sequences at the 3' end of the 504-nt 3' untranslated region. In contrast, the polypeptide fragment from the middle region, residues 101-283, did not interact with these 3' IBV RNAs. The binding site in the amino region of N was either not present or only partially present in the first 91 residues because no interaction with RNA was observed with the polypeptide incorporating these residues. Cache Valley virus N expressed with a histidine tag, bovine serum albumin, and the basic lysozyme protein did not shift the IBV RNA. The lower molarities of the carboxyl fragment compared with residue 1-274 fragment needed for equivalent shifts suggested that the binding avidity for RNA at the carboxyl domain was greater than the amino domain.

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“…As an important structural protein of TGEV particles, the M protein is located in the lipid envelop and associates with the Golgi complex in the cell, suggesting that the M protein plays a pivotal role in virus assembly and budding. Additional evidence indicates that the M protein is an indispensable part for the replication of virus particles in host cells, which may also be used for virus-specific diagnostics and treatment (Cologna and Hogue, 1998;De Haan et al, 1998;Zou et al, 2013). However, there have been few reports regarding the interaction between the TGEV M protein and intestinal cells.…”

Section: Introductionmentioning

confidence: 99%

EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication

Song

Yang

Wang

et al. 2019

Research in Veterinary Science

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Transmissible gastroenteritis coronavirus (TGEV) is enteropathogenic coronavirus that causes diarrhea in pigs, and is associated with high morbidity and mortality in sucking piglets. The TGEV membrane (M) protein is a decisive protein for the proliferation of viral proteins, and is associated with virus assembly and budding. To identify the cellular proteins that interact with the TGEV M protein, yeast two-hybrid screening was employed, and seven cellular proteins were identified M-binding partners. Using the GST pull-down approach and a CO-IP assay, the M protein was found to interact with porcine intestinal cells via eukaryotic translation initiation factor 4-alpha (EIF4A2), an essential component of the cellular translational machinery. Additionally, confocal microscopy revealed that EIF4A2 and M were colocalized in the cytoplasm. Furthermore, the function of EIF4A2 in intestinal cells during TGEV infection was examined. A knockdown of EIF4A2 by siRNA markedly decreased M protein proliferation and TGEV replication in target cells. Thus demonstrating that EIF4A2 plays a significant role in TGEV replication. The present study provides mechanistic insight into the interaction between the TGEV M protein and intestinal cells which contributes to the understanding of coronavirus replication and may be useful for the development of novel therapeutic strategies for TGEV infection.

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Coronavirus Nucleocapsid Protein

Cited by 11 publications

References 11 publications

Identification of Nucleocapsid Binding Sites within Coronavirus-Defective Genomes

Identification of Nucleocapsid Binding Sites within Coronavirus-Defective Genomes

The amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3′ genomic RNA

EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication

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