RESUMO
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.
Assuntos
Coronavirus/genética , Vírus Defeituosos/genética , Genoma Viral , Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Animais , Sítios de Ligação , Northern Blotting , Western Blotting , Bovinos , Linhagem Celular , Coronavirus Bovino/genética , Humanos , Camundongos , Vírus da Hepatite Murina/genética , Proteínas do Nucleocapsídeo/biossíntese , Proteínas do Nucleocapsídeo/genética , Óperon , RNA Mensageiro/genética , Transfecção , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
A region of the bovine coronavirus (BCV) genome that functions as a packaging signal has been cloned. The 291-nucleotide clone shares 72% homology with the region of mouse hepatitis coronavirus (MHV) gene 1b that contains the packaging signal. RNA transcripts were packaged into both BCV and MHV virions when the cloned region was appended to a noncoronavirus RNA. This is the first identification of a BCV packaging signal. The data demonstrate that the BCV genome contains a sequence that is conserved at both the sequence and functional levels, thus broadening our insight into coronavirus packaging.
Assuntos
Coronavirus Bovino/genética , Montagem de Vírus/genética , Animais , Sequência de Bases , Northern Blotting , Bovinos , Cloranfenicol O-Acetiltransferase/genética , Clonagem Molecular , Genoma Viral , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , Homologia de Sequência do Ácido NucleicoRESUMO
The coronavirus nucleocapsid protein (N) is involved in encapsidation and packaging of viral RNA. In this study we investigated the ability of the bovine coronavirus (BCV) N protein to interact with RNA. Histidine-tagged BCV N (his-N) protein was expressed in bacteria. A filter binding assay was established to quantitatively measure the binding efficiency of purified his-N to different RNAs. The results indicate that bacterially expressed N bound both BCV and mouse hepatitis coronavirus (MHV) RNAs. Binding to in vitro generated BCV and MHV RNA transcripts was significantly higher than binding to a non-coronavirus RNA. Similar binding efficiencies were measured for a BCV defective genome, pDrep, and a transcript that contained the MHV packaging signal. Interestingly, the entire MHV DI, pMIDI-C, was bound at a higher efficiency than the packaging signal alone. This is one of the first reports to show that N interacts with the MHV packaging signal.