Group-specific structural features of the 5'-proximal sequences of coronavirus genomic RNAs

Abstract

Global predictions of the secondary structure of coronavirus (CoV) 5' untranslated regions and adjacent coding sequences revealed the presence of conserved structural elements. Stem loops (SL) 1, 2, 4, and 5 were predicted in all CoVs, while the core leader transcription-regulating sequence (L-TRS) forms SL3 in only some CoVs. SL5 in group I and II CoVs, with the exception of group IIa CoVs, is characterized by the presence of a large sequence insertion capable of forming hairpins with the conserved 5'-UUYCGU-3' loop sequence. Structure probing confirmed the existence of these hairpins in the group I Human coronavirus-229E and the group II Severe acute respiratory syndrome coronavirus (SARS-CoV). In general, the pattern of the 5' cis-acting elements is highly related to the lineage of CoVs, including features of the conserved hairpins in SL5. The function of these conserved hairpins as a putative packaging signal is discussed.

Fig. 1

Clustering and general features of the 5′ 420 nucleotides of CoVs. The tree is based on a multiple sequence alignment using ClustalW2 at the European Bioinformatics Institute webserver. The phylogenetic group, the start of core TRS-L, the region of upstream ORF (uORF), the start of ORF1ab, and GenBank accession number of each CoV are listed.

Fig. 2

The structural–phylogenetic analysis of the 5′-proximal sequences in group I CoVs. The predicted secondary structures of the 5′-proximal sequence of (A) group Ia TGEV-purdue, (B) group Ib HCoV-229E-inf-1, (C) group Ic PEDV-CV777, and (D) group Id BtCoV-1A coronaviruses are shown. Nucleotide variations located in the conserved elements in the other representative CoVs of each subgroup are indicated. The start codon of the ORF1ab is boxed, the core sequence of the transcription-regulating leader (TRS-L CS) is bracketed, and the length of the sequence insertion in SL5 is indicated.

Fig. 2

The structural–phylogenetic analysis of the 5′-proximal sequences in group I CoVs. The predicted secondary structures of the 5′-proximal sequence of (A) group Ia TGEV-purdue, (B) group Ib HCoV-229E-inf-1, (C) group Ic PEDV-CV777, and (D) group Id BtCoV-1A coronaviruses are shown. Nucleotide variations located in the conserved elements in the other representative CoVs of each subgroup are indicated. The start codon of the ORF1ab is boxed, the core sequence of the transcription-regulating leader (TRS-L CS) is bracketed, and the length of the sequence insertion in SL5 is indicated.

Fig. 3

The structural–phylogenetic analysis of the 5′-proximal sequences in group II CoVs. The predicted secondary structures of the 5′-proximal sequence of (A) group IIa BCoV, (B) group IIb SARS-CoV-Tor2, (C) group IIc BtCoV-HKU5-1, and (D) group IId BtCoV-HKU9-1 are shown. For details see Fig. 2.

Fig. 3

The structural–phylogenetic analysis of the 5′-proximal sequences in group II CoVs. The predicted secondary structures of the 5′-proximal sequence of (A) group IIa BCoV, (B) group IIb SARS-CoV-Tor2, (C) group IIc BtCoV-HKU5-1, and (D) group IId BtCoV-HKU9-1 are shown. For details see Fig. 2.

Fig. 4

The structural–phylogenetic analysis of the 5′-proximal sequences in group III CoVs. The predicted secondary structures of the 5′-proximal sequence of (A) group IIIa IBV-Beaudette, (B) group IIIb CoV-SW1, (C) group IIIc BuCoV-HKU11/796, and (D) group IIId HKU13/3514 are shown. For further details see Fig. 2.

Fig. 5

The substructural hairpins of SL5 in group I and II CoVs. The secondary structure of the SL5 substructural hairpins, SL5a–c, in (A) group Ia TGEV-purdue, (B) group Ib HCoV-229E-inf-1, (C) group Ic PEDV-CV777, (D) group Id BtCoV-1A, (E) group IIa BCoV, (F) group IIb SARS-CoV-Tor2, (G) group IIc BtCoV-HKU5-1, and (H) group IId BtCoV-HKU9-1 are shown. The start codon of the BtCoV-HKU5-1 ORF1ab is located in SL5b as indicated. SL5.1 which is located upstream of SL5 in BtCoV-HKU9-1 also contains the conserved UUUCGU motif.

Fig. 5

The substructural hairpins of SL5 in group I and II CoVs. The secondary structure of the SL5 substructural hairpins, SL5a–c, in (A) group Ia TGEV-purdue, (B) group Ib HCoV-229E-inf-1, (C) group Ic PEDV-CV777, (D) group Id BtCoV-1A, (E) group IIa BCoV, (F) group IIb SARS-CoV-Tor2, (G) group IIc BtCoV-HKU5-1, and (H) group IId BtCoV-HKU9-1 are shown. The start codon of the BtCoV-HKU5-1 ORF1ab is located in SL5b as indicated. SL5.1 which is located upstream of SL5 in BtCoV-HKU9-1 also contains the conserved UUUCGU motif.

Fig. 6

Structure probing of the inserted sequences in SL5 of group Ib HCoV-229E and group IIb SARS-CoV-Tor2. The secondary structures of the SL5 substructural hairpins of (A) the HCoV-229E and (B) the SARS-CoV are analyzed by enzymatic and chemical structure probing. Annotation of the denaturing electrophoresis: Un, untreated; D, DMS treated; R, RNase A treated; T₁, RNase T₁ treated; V₁, RNase V₁ treated; S₁, S₁ nuclease treated; G, U, C and A, the RNA sequencing ladder.

Fig. 7

Multiple alignment of the CoV nsp15 sequence corresponding to the group IIa packaging signal. The amino acid sequences of the group IIa CoV nsp15 are aligned with the sequences of other CoV groups, showing the underlined sequence insertion of the packaging signal corresponding region in group IIa CoVs.