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.
Highlights
Middle 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
A MERS-oV RNA packaging signal was identified by the detection of green fluorescent protein (GFP) expression following an incubation of MERS virus-like particles (VLPs) carrying the heterologous mRNA GFP-PS258(19712–19969)ME with virus permissive Huh7 cells
The results indicate that the RNA fragment spanning nt 19,712 to 19,969 of the viral genome is sufficient to function as a packaging signal and assemble into the MERS VLPs
Summary
Middle 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. Our previous studies on severe acute respiratory syndrome coronavirus (SARS-CoV), which is a lineage B betacoronavirus, identified a 580-nt RNA fragment with a stable stem-loop structure as a functional signal to drive packaging of coexpressed RNA of a green fluorescent protein (GFP) into virus-like particles (VLPs) [19]. This SARS-CoV packaging signal spanned the viral genome from nt 19,715 to 20,294 and was localized to a subdomain of the nsp gene different from the packaging signal of lineage A betacoronaviruses. Studies on alphacoronavirus transmissible gastroenteritis virus (TGEV) localized the RNA packaging signal to the 5′-end of the viral genome [20, 21], and sequences in the 5’-UTR and/or 3’-UTR may be required for the RNA packaging of gammacoronavirus infectious bronchitis virus (IBV) [22, 23]
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