Recent Progress in Torovirus Molecular Biology

Abstract

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. ToVs are associated with enteric diseases in animals and humans. In contrast to CoVs, which are recognised as pathogens of veterinary and medical importance, little attention has been paid to ToVs because their infections are usually asymptomatic or not severe; for a long time, only one equine ToV could be propagated in cultured cells. However, bovine ToVs, which predominantly cause diarrhoea in calves, have been detected worldwide, leading to economic losses. Porcine ToVs have also spread globally; although they have not caused serious economic losses, coinfections with other pathogens can exacerbate their symptoms. In addition, frequent inter- or intra-recombination among ToVs can increase pathogenesis or unpredicted host adaptation. These findings have highlighted the importance of ToVs as pathogens and the need for basic ToV research. Here, we review recent progress in the study of ToV molecular biology including reverse genetics, focusing on the similarities and differences between ToVs and CoVs.

Keywords: coronavirus; enteric diseases; non-structural proteins; replication; reverse genetics; structural proteins; torovirus; transcription.

Figure 1

(a) Schematic diagrams of torovirus (ToV) and coronavirus (CoV) virions: spike (S), envelope (E), membrane (M), haemagglutinin-esterase (HE), and nucleocapsid (N) proteins. (b) Topology of the four structural envelope proteins. All proteins are depicted as monomers, but the S and HE proteins form homotrimers and homodimers, respectively. Oligosaccharides on the S and HE proteins are shown. Although a number are omitted, the S and HE proteins contain 19 to 28 and 7 to 12 N-glycosylation sites, respectively. (c) Electron micrograph of BToV (left) and SARS-CoV (right). Bar: 100 nm.

Figure 2

Genome organisation and gene expression of (a) BToV and (b) MHV (lineage A beta-CoV). The subgenomic (sg) mRNA species are numbered from large to small, with the genome designated RNA1. BToV and MHV polyprotein processing of pp1a and pp1ab and the conserved domains of nonstructural proteins (nsps) are shown. U1 and U2 proteins may be translated from an unconventional CUG initiation codon. The internal (I) protein is encoded within the N gene in some beta-CoVs. The 5′-leader sequence present in the genome and sg mRNA 2 of ToV and the 5′-leader sequence of CoV in all RNA species are indicated by a small red box, and terminator/promoter signals of ToV in sg mRNA3 to 5 by a small green box. Red and green arrows indicate the locations of transcription-regulating sequences (TRSs). Arrowheads indicate (deduced) cleavage sites by papain-like (orange) or 3C-like protease/main protease (red). Vertical dashed line shows a ribosomal frameshifting site. 3CL^pro, 3C-like protease (main protease); EndoU, endoribonuclease; ExoN, exoribonuclease; HEL, helicase; Mac, macrodomains including ADP-ribose-1″phosphatase; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; N-MT, guanosine N7-methyltransferase; O-MT, ribose 2′-O-methyltransferase; PDE, 2’, 5’-phosphodiesterase; PL^pro, papain-like protease; RdRp, RNA-dependent RNA polymerase; TM, transmembrane domain; ZBD, zinc-binding domain.

Figure 3

Homologous and heterologous recombination of ToVs. (a) Genome organisation of BToV by interspecies homologous recombination between BRV-like BToV (red) and PToV (green). Recombination of HE from an as-yet-unknown ToV is shown in orange. European and Asian isolates were from Italy, Hungary, the Netherlands, and Japan and China, respectively. Years of isolation are shown. BToV HE gene can be divided into two lineages, the BRV and B150 lineages. NA, not analysed. (b) Two lineages of PToV based on the HE gene and genome organisation of Japanese PToV (Iba/2018) with a mosaic sequence. The HE gene of the Markelo lineage is shown in blue. PToV (Iba/2018) may be a result of intraspecies homologous recombination among three related PToVs (named below). (c) Heterologous recombination between ToV and picornavirus. The ToV-like PL^pro gene is inserted into the EV-G genome at the 2C/3A junction or is completely replaced by a viral structural gene up to the VP1/2A junction.

Figure 4

Discontinuous and continuous transcription of CoV and ToV. (a) Model of TRS-driven discontinuous transcription of CoV. (-) RNA synthesis (light grey) initiates at the 3’-end of the genome and is terminated or attenuated when the replication and transcription complex (light blue circle) encounters B-TRS (in orange). The discontinuous step is driven by base-pairing between the anti-B-TRS and L-TRS within the hairpin, resulting in template switch from nascent (-) RNA to the leader. Next, (-) RNA synthesis re-initiates and a leader sequence (red) is added. This complete (-) sg RNA in turn serves as a template for (+) sg mRNAs. (b) Model of structure-driven discontinuous (left) and continuous (right) transcription of ToV. In sg mRNA2, nascent (-) RNA synthesis is terminated by the hairpin (HP) structure and base pairing between homologous regions (blue), followed by L-TRS (green) and HP, promotes the template switch. After switching, the 5’ genome-derived sequence including a short leader (red) and L-TRS and additional nucleotides are added to nascent (-) RNA, resulting in complete (-) sg RNA synthesis. In sg mRNA 3-5, semiconserved sequences in the genome, CACN_3-4CUUUAGA (yellow and green) including B-TRS (green) act as a termination signal, and complete (-) sg RNA synthesis is terminated at this region and is then detached from genome. (+) sg mRNA contains ACN_3-4CUUUAGA at the 5’-end (without the C residue of the 5’-genome). A small portion of mRNA 5 contains a leader sequence that is subjected to discontinuous transcription.

Figure 5

(a) Schematic diagrams of the S proteins of ToV and CoV. BToV contains two furin sites, an S1/S2 site and an additional site near the N-terminal end, whereas MHV (CoV) has two well-defined protease cleavage sites, S1/S2 and S2′ (arrowheads). The S protein consists of two subunits, the S1 receptor-binding subunit and the S2 fusion subunit. NTD: N-terminal domain of S1, CTD: C-terminal domain of S1, S1/S2, and S2′ cleavage sites, furin-site: Additional furin cleavage site, FP: putative fusion peptide, HR1: heptad repeat 1, HR2: heptad repeat 2, TM: transmembrane domain, CT: cytoplasmic tail. (b) C-terminal ends of three ToV and six CoV S proteins. Amino acid sequence alignment was performed using the ClustalW program. Shaded box indicates the deduced TM domain. Cysteine residues in the cysteine-rich domain of CoVs are indicated in red. Orange and red boxes indicate potential ER retrieval signals (KxHxx- or KKxx-motif) and tyrosine-dependent localisation signals/internalisation signals (Yxxθ motif, where θ can be F, I, L, M, or V), respectively.

Figure 6

(a) Schematic diagrams of the N protein of ToV, with two nuclear/nucleolar localisation signals (NLS/NLoS) and nuclear export signals (NES). (b) Subcellular localisation of BToV or MHV (CoV) N proteins in infected cells. The cells were fixed and permeabilised at the indicated hours post-infection (hpi). N proteins and nucleolar marker fibrillarin were detected using mouse anti-N antiserum (green) and anti-fibrillarin rabbit mAb (red), respectively. The nucleus was stained with Hoechst solution (blue).