Structural and functional conservation of cis-acting RNA elements in coronavirus 5'-terminal genome regions

Abstract

Structure predictions suggest a partial conservation of RNA structure elements in coronavirus terminal genome regions. Here, we determined the structures of stem-loops (SL) 1 and 2 of two alphacoronaviruses, human coronavirus (HCoV) 229E and NL63, by RNA structure probing and studied the functional relevance of these putative cis-acting elements. HCoV-229E SL1 and SL2 mutants generated by reverse genetics were used to study the effects on viral replication of single-nucleotide substitutions predicted to destabilize the SL1 and SL2 structures. The data provide conclusive evidence for the critical role of SL1 and SL2 in HCoV-229E replication and, in some cases, revealed parallels with previously characterized betacoronavirus SL1 and SL2 elements. Also, we were able to rescue viable HCoV-229E mutants carrying replacements of SL2 with equivalent betacoronavirus structural elements. The data obtained in this study reveal a remarkable degree of structural and functional conservation of 5'-terminal RNA structural elements across coronavirus genus boundaries.

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

“…We sequenced total RNA samples obtained from Huh7 cells infected with serially passaged recombinant human coronaviruses: wild-type (WT) HCoV-229E, HCoV-229E_SL2-SARS-CoV, and HCoV-229E_SL2-BCoV, respectively. In the latter two viruses, a conserved stem-loop structure (SL2) residing in the HCoV-229E 5 ′ UTR was replaced with the equivalent SL2 element from SARS-CoV and BCoV, respectively (Madhugiri et al 2018). Total RNA samples obtained for the latter two (chimeric) viruses were pooled before sequence analysis.…”

“…The two total RNA samples used in this study for DRS (ONT MinION) and Illumina sequencing were prepared at 24 h post infection from Huh7 cells infected at an MOI of three with recombinant HCoV-229E WT, HCoV-229E_SL2-SARS-CoV, and HCoV-229E_SL2-BCoV, respectively (Madhugiri et al 2018). Before sequence analysis, the two RNA samples obtained from HCoV-229E_SL2-SARS-CoV-infected and HCoV-229E_SL2-BCoV-infected cells were pooled (SL2 sample) (see Supplemental Fig.…”

“…Generation of recombinant viruses and total RNA isolation were performed as described previously (Madhugiri et al 2018). Briefly, full-length cDNA copies of the genomes of HCoV-229E (GenBank accession number NC_002645), HCoV-229E_SL2-SARS-CoV, and HCoV-229E_SL2-BCoV, respectively, were engineered into recombinant vaccinia viruses using previously described methods (Thiel et al 2001;Hertzig et al 2004;Thiel and Siddell 2005).…”

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

“…The two total RNA samples used in this study for DRS (ONT MinION) and Illumina sequencing were prepared at 24 h post infection from Huh-7 cells infected at an MOI of 3 with recombinant HCoV-229E WT, HCoV-229E_SL2-SARS-CoV and HCoV-229E_SL2-BCoV, respectively 41 . Prior to sequence analysis, the two RNA samples obtained from HCoV-229E_SL2-SARS-CoVand HCoV-229E_SL2-BCoV-infected cells were pooled (SL2 sample, see SFig.…”

“…We sequenced total RNA samples obtained from Huh-7 cells infected with serially passaged recombinant human coronaviruses: wild-type (WT) HCoV-229E, HCoV-229E_SL2-SARS-CoV, and HCoV-229E_SL2-BCoV, respectively. In the latter two viruses, a conserved stem-loop structure (SL2) residing in the HCoV-229E 5'-UTR was replaced with the equivalent SL2 element from SARS-CoV and BCoV, respectively 41 . Total RNA samples obtained for the latter two (chimeric) viruses were pooled prior to sequence analysis.…”