Identification of a Bovine Coronavirus Packaging Signal

Abstract

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.The coronavirus genome is a single-stranded, positive-sense, 27-to 31-kb RNA molecule, the largest among all RNA viruses. The genome is encapsidated by multiple copies of the nucleocapsid (N) protein and is packaged as a helical nucleocapsid in the mature enveloped virion (for reviews, see references 14 and 25). During infection, coronaviruses synthesize six or seven subgenomic mRNAs which share 5Ј and 3Ј ends that are identical to the genomic RNA (for a review, see reference 29).How coronaviruses recognize the viral RNA(s) to ensure specific encapsidation and packaging is only beginning to be understood. Thus far, a packaging signal has been identified only for mouse hepatitis coronavirus (MHV). By using MHV defective interfering (DI) RNAs, a 69-nucleotide (nt) packaging signal that maps approximately 20 kb from the 5Ј end of the genome within the gene 1b open reading frame was identified ( Fig. 1) (8,16,28). Inclusion of the packaging signal in a non-MHV RNA is sufficient to allow the RNA to be packaged into MHV virions (30). Mutagenesis of the predicted bulged stem-loop structure of the MHV packaging signal disrupts the ability of the sequence to function as a packaging signal (8). It was also shown that an MHV strain A59 subgenomic DI RNA is packaged into virions when it contains the DI packaging signal, but the subgenomic RNA was reported to be packaged less efficiently than its DI genomic RNA (4).Sequences that are required for RNAs to be packaged by bovine coronavirus (BCV) have not been determined. BCV belongs to the same antigenic subgroup as MHV, and data based on the 3Ј one-third of the genome that has been cloned and sequenced indicate that the two viruses are closely related (1, 2, 13, 15). However, multiple pieces of data suggest that BCV is different from MHV with regard to RNA packaging. The first is that BCV packages subgenomic RNAs in addition to genomic RNA, whereas MHV packages very little, if any, of its subgenomics. BCV N and M mRNAs were shown to be more abundant in virions, on a molar basis, than was genome (11). The second difference is that the BCV defective RNA Drep (Fig. 1), which is replicated and packaged by BCV, does not contain any gene 1b sequence (6).To gain further insight into the requirements for coronavirus packaging, we asked whether the BCV genome contains sequence information equivalent to the MHV packaging signal. Reverse transcription followed by PCR (RT-PCR) was carried out by ...

Coronavirus cis-Acting RNA Elements

“…More recently, a PS that is conserved in lineage A betacoronaviruses and a novel 95-nt BSL were predicted and supported by chemical and enzymatic probing experiments . The conservation of this PS among lineage A coronaviruses is consistent with earlier observations that the BCoV PS is functionally replaceable with its MHV counterpart (Cologna and Hogue, 2000). Remarkably, this structurally and functionally conserved PS of lineage A betacoronaviruses is not conserved in other lineages of betacoronaviruses and other coronavirus genera (Kuo and Masters, 2013), suggesting differential requirements for genome packaging among closely related coronaviruses.…”

“…MHV DI RNA studies revealed a 69-nt SL structure that was (i) located in the 3 0 region of ORF1b, (ii) confirmed to be required for DI RNA packaging, and (iii) shown to interact with the viral N protein (Fosmire et al, 1992;Molenkamp and Spaan, 1997;Woo et al, 1997). Subsequent studies indicated that a larger PS element and, possibly, additional factors are required for optimal packaging efficiency (Bos et al, 1997;Cologna and Hogue, 2000;Narayanan and Makino, 2001). More recently, a PS that is conserved in lineage A betacoronaviruses and a novel 95-nt BSL were predicted and supported by chemical and enzymatic probing experiments .…”

Characterization of the Coronavirus M Protein and Nucleocapsid Interaction in Infected Cells

“…The secondary structure of the packaging signal is important for its biological function (11), and the presence of the packaging signal in non-MHV RNA transcripts allows the packaging of these RNA transcripts into MHV particles (50). Recent studies of the MHV packaging signal and bovine coronavirus packaging signal confirmed the previous studies on the MHV packaging signal (3,8,33). M protein may directly interact with mRNA 1, through the packaging signal, to initiate the M protein-nucleocapsid interaction.…”

The Nucleocapsid Protein of Coronavirus Infectious Bronchitis Virus: Crystal Structure of Its N-Terminal Domain and Multimerization Properties

“…Specific packaging of viral genetic material is usually performed via the recognition of a particular nucleotide sequence by a nucleocapsid protein. Such ''packaging signals'' have been identified at the 3 0 end of the viral genomes of mouse hepatitis virus (MHV) (Fosmire et al, 1992) and bovine coronavirus (BCV) (Cologna and Hogue, 2000) and at the 5 0 end of the TGEV genome (Escors et al, 2003), but not unambiguously for the IBV genome. In elegant structural studies performed in other viral families with RNA genomes, such as HIV (De Guzman et al, 1998) and the MS2 bacteriophage (Valegard et al, 1997), the packaging signals were seen to form a stem-loop structure that is recognized by the nucleocapsid protein.…”