Nucleocapsid protein-dependent assembly of the RNA packaging signal of Middle East respiratory syndrome coronavirus

Abstract

BackgroundMiddle East respiratory syndrome coronavirus (MERS-CoV) consists of a positive-sense, single-stranded RNA genome and four structural proteins: the spike, envelope, membrane, and nucleocapsid protein. The assembly of the viral genome into virus particles involves viral structural proteins and is believed to be mediated through recognition of specific sequences and RNA structures of the viral genome.Methods and ResultsA culture system for the production of MERS coronavirus-like particles (MERS VLPs) was determined and established by electron microscopy and the detection of coexpressed viral structural proteins. Using the VLP system, a 258-nucleotide RNA fragment, which spans nucleotides 19,712 to 19,969 of the MERS-CoV genome (designated PS258(19712–19969)ME), was identified to function as a packaging signal. Assembly of the RNA packaging signal into MERS VLPs is dependent on the viral nucleocapsid protein. In addition, a 45-nucleotide stable stem-loop substructure of the PS258(19712–19969)ME interacted with both the N-terminal domain and the C-terminal domain of the viral nucleocapsid protein. Furthermore, a functional SARS-CoV RNA packaging signal failed to assemble into the MERS VLPs, which indicated virus-specific assembly of the RNA genome.ConclusionsA MERS-oV RNA packaging signal was identified by the detection of GFP expression following an incubation of MERS VLPs carrying the heterologous mRNA GFP-PS258(19712–19969)ME with virus permissive Huh7 cells. The MERS VLP system could help us in understanding virus infection and morphogenesis.

An in vivo cell-based assay for investigating the specific interaction between the SARS-CoV N-protein and its viral RNA packaging sequence

“…These findings support previously published results in which a two-domain fragment containing both the NTD and CTD demonstrated the greatest nucleotide-binding affinity to the oligonucleotides in vitro [15]. Furthermore, studies of Middle East respiratory syndrome (MERS), another coronavirus, also showed that both the NTD and CTD of the MERS-CoV N-protein harbored binding activity to its RNA packaging signal PS258 (nt 19712 to 19969) [26].…”

Coronavirus genomic RNA packaging

“…The encoded polypeptide corresponds to a surface loop of MHV nsp15 (Xu et al, 2006) that does not exist in the nsp15 structures of SARS-CoV (Joseph et al, 2007) or MERS-CoV (Zhang et al, 2018). Thus, there is no basis for the presumption that the same region of nsp15 contains the PS for either SARS-CoV (Hsieh et al, 2005) or MERS-CoV (Hsin et al, 2018). The ancillary positioning of the PS-encoded protein loop suggests that this PS was a late acquisition during the evolution of the coronavirus family.…”

Identification of a Novel Linear B-Cell Epitope on the Nucleocapsid Protein of Porcine Deltacoronavirus

“…Moreover, NS7a is contained into NS7 ORF [16]. The N protein is a phosphoprotein and binds to RNA genome, which supplies a structural basis to the helical nucleocapsid [17,18]. The common characteristics for all CoVs N proteins are high expression levels early in the infection and high anti-N antibody levels.…”

“…The common characteristics for all CoVs N proteins are high expression levels early in the infection and high anti-N antibody levels. N protein also owns multiple functions in pathogenesis, viral replication, and immune system interference [17]. These characteristics make the N protein an ideal target for development of serological methods based on purified protein [19] or antigenic epitopes [20].…”