Identification of nucleocapsid binding sites within coronavirus-defective genomes

R. Cologna, J. F. Spagnolo, Brenda Hogue

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

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 (~5500 nts) and to an ~1-kb transcript from the gene 1 a 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. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)235-249
Number of pages15
JournalVirology
Volume277
Issue number2
DOIs
StatePublished - Nov 25 2000
Externally publishedYes

Fingerprint

Bovine Coronavirus
Nucleocapsid
Coronavirus
Hepatitis
Viral RNA
Binding Sites
Genome
RNA
Product Packaging
Open Reading Frames
Helper Viruses
Proteins
Virion
Nucleotides
Genes
Coronavirus nucleocapsid protein

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Identification of nucleocapsid binding sites within coronavirus-defective genomes. / Cologna, R.; Spagnolo, J. F.; Hogue, Brenda.

In: Virology, Vol. 277, No. 2, 25.11.2000, p. 235-249.

Research output: Contribution to journalArticle

Cologna, R. ; Spagnolo, J. F. ; Hogue, Brenda. / Identification of nucleocapsid binding sites within coronavirus-defective genomes. In: Virology. 2000 ; Vol. 277, No. 2. pp. 235-249.
@article{5c51ac0dab7e407fa10320a6a40ec4c0,
title = "Identification of nucleocapsid binding sites within coronavirus-defective genomes",
abstract = "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 (~5500 nts) and to an ~1-kb transcript from the gene 1 a 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. (C) 2000 Academic Press.",
author = "R. Cologna and Spagnolo, {J. F.} and Brenda Hogue",
year = "2000",
month = "11",
day = "25",
doi = "10.1006/viro.2000.0611",
language = "English (US)",
volume = "277",
pages = "235--249",
journal = "Virology",
issn = "0042-6822",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Identification of nucleocapsid binding sites within coronavirus-defective genomes

AU - Cologna, R.

AU - Spagnolo, J. F.

AU - Hogue, Brenda

PY - 2000/11/25

Y1 - 2000/11/25

N2 - 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 (~5500 nts) and to an ~1-kb transcript from the gene 1 a 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. (C) 2000 Academic Press.

AB - 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 (~5500 nts) and to an ~1-kb transcript from the gene 1 a 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. (C) 2000 Academic Press.

UR - http://www.scopus.com/inward/record.url?scp=0034715821&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034715821&partnerID=8YFLogxK

U2 - 10.1006/viro.2000.0611

DO - 10.1006/viro.2000.0611

M3 - Article

VL - 277

SP - 235

EP - 249

JO - Virology

JF - Virology

SN - 0042-6822

IS - 2

ER -