Toward Understanding Molecular Bases for Biological Diversification of Human Coronaviruses: Present Status and Future Perspectives

Abstract

Human coronaviruses (HCoVs) are of zoonotic origins, and seven distinct HCoVs are currently known to infect humans. While the four seasonal HCoVs appear to be mildly pathogenic and circulate among human populations, the other three designated SARS-CoV, MERS-CoV, and SARS-CoV-2 can cause severe diseases in some cases. The newly identified SARS-CoV-2, a causative virus of COVID-19 that can be deadly, is now spreading worldwide much more efficiently than the other two pathogenic viruses. Despite evident differences in these properties, all HCoVs commonly have an exceptionally large genomic RNA with a rather peculiar gene organization and have the potential to readily alter their biological properties. CoVs are characterized by their biological diversifications, high recombination, and efficient adaptive evolution. We are particularly concerned about the high replication and transmission nature of SARS-CoV-2, which may lead to the emergence of more transmissible and/or pathogenic viruses than ever before. Furthermore, novel variant viruses may appear at any time from the CoV pools actively circulating or persistently being maintained in the animal reservoirs, and from the CoVs in infected human individuals. In this review, we describe knowns of the CoVs and then mention their unknowns to clarify the major issues to be addressed. Genome organizations and sequences of numerous CoVs have been determined, and the viruses are presently classified into separate phylogenetic groups. Functional roles in the viral replication cycle in vitro of non-structural and structural proteins are also quite well understood or suggested. In contrast, those in the in vitro and in vivo replication for various accessory proteins encoded by the variable 3' one-third portion of the CoV genome mostly remain to be determined. Importantly, the genomic sequences/structures closely linked to the high CoV recombination are poorly investigated and elucidated. Also, determinants for adaptation and pathogenicity have not been systematically investigated. We summarize here these research situations. Among conceivable projects, we are especially interested in the underlying molecular mechanism by which the observed CoV diversification is generated. Finally, as virologists, we discuss how we handle the present difficulties and propose possible research directions in the medium or long term.

Keywords: COVID-19; HCoV; MERS-CoV; SARS-CoV; SARS-CoV-2; adaptive evolution; biological diversification; recombination.

FIGURE 1

Genome organization of human coronaviruses. (A) Schematic representation of the variable 3′ genomic region. The genome organization of viruses in the four viral groups (Su et al., 2016; Forni et al., 2017) including SARS-CoV-2 (Tse et al., 2020; Wang N. et al., 2020) are shown. Those for various accessory proteins are omitted. The HE orf sequences of HCoV-HKU1 and HCoV-OC43 (Table 3 footnote) are also omitted. The beta-CoV lineage D group that contains some bat viruses only is not presented in this panel. (B) Comparison of SARS-CoV-2, SARS-CoV, and MERS-CoV genomes. All orf sequences in the full-length viral genome are shown (Totura and Baric, 2012; Su et al., 2016; Forni et al., 2017; Cui et al., 2019; Biswas et al., 2020; Hou et al., 2020; Thao et al., 2020; Wang N. et al., 2020; Wu A. et al., 2020; Xie et al., 2020). It has been reported that SARS-CoV-2 contains orf 9b (Cagliani et al., 2020), suggesting a more similar genome structure between SARS-CoV and SARS-CoV-2 than shown in panel B.

FIGURE 2

Replication cycle of coronaviruses. The replication process of coronaviruses is schematically shown from the virus attachment to target cells up to the virus release from infected cells (de Haan and Rottier, 2005; Fehr and Perlman, 2015; de Wilde et al., 2018; Oberfeld et al., 2020; Tse et al., 2020). An entry receptor and a co-factor for efficient viral entry (ACE2 and TMPRSS2, respectively, in this case) are indicated. For details of the replication steps, see the text. A schema of the coronavirus virion is also shown in a box at the top-left. Abbreviations (Fehr and Perlman, 2015; de Wilde et al., 2018): ACE2, angiotensin-converting enzyme 2; TMPRSS2, transmembrane protease, serine 2; CM, cell membrane; gRNA, genomic RNA; pp, polyprotein; PLpro, papain-like protease; Mpro, main protease; sgRNA, subgenomic RNA; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; ER, endoplasmic reticulum.

FIGURE 3

Reverse genetics systems for studies on CoVs. Outlines of the three major methods to produce CoVs by RNA transfection are shown. For details, see Thao et al. (2020) for panel (A), Thiel et al. (2001) for panel (B), and Hou et al. (2020); Xie et al. (2020) for panel (C). The BAC system (Table 4) is essentially quite similar with that in panel (A), but a complete full-length CoV-DNA must be constructed in vitro as BAC before transformation into bacteria and the following preparation of plasmid DNA (Almazán et al., 2000). Red arrow heads in this figure indicate the T7 promoter sequence. r, recombinant.