Identification of nucleocapsid binding sites within coronavirus-defective genomes.
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.
Cologna
R
R
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Spagnolo
J F
JF
Hogue
B G
BG
D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D017934:Coronavirus / Q000235:genetics*; D017938:Coronavirus, Bovine / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics*; D016679:Genome, Viral; D006801:Humans; D051379:Mice; D006517:Murine hepatitis virus / Q000235:genetics; D019251:Nucleocapsid / Q000378:metabolism*; D019590:Nucleocapsid Proteins / Q000096:biosynthesis / Q000235:genetics; D009876:Operon; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism*; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism
D019590:Nucleocapsid Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D054334:Viral Regulatory and Accessory Proteins