Viral metagenomics, protein structure, and reverse genetics: Key strategies for investigating coronaviruses

Abstract

Viral metagenomics, modeling of protein structure, and manipulation of viral genetics are key approaches that have laid the foundations of our understanding of coronavirus biology. In this review, we discuss the major advances each method has provided and discuss how future studies should leverage these strategies synergistically to answer novel questions.

Fig. 1

The expanding phylogeny of coronaviruses. The Spike protein sequences of 83 coronaviruses were aligned and phylogenetically compared. The four coronavirus genera are grouped in shades of orange (Alphacoronavirus), blue (Betacoronavirus), red (Gammacoronavirus), and green (Deltacoronavirus). Classic subgroup designations (1a-b, 2a-d, 3, and 4) are also shown. Sequences designated as 1b* group with 1b viruses in proteins other than Spike. Individual viral species and strains are colored based on the original publication dates of their sequences: black (pre-2002), red (2002–2005), blue (2006–2011), green (2012–2014), and purple (2015–2017). Sequences were aligned using the MUSCLE package in Geneious R9. The tree was constructed using the neighbor-joining method based on the multiple sequence alignment, also in Geneious R9. Numbers in parentheses following virus species names indicate the number of sequences represented at that tree position. The radial phylogram was visualized and rendered for publication using CLC Sequence Viewer 7 and Adobe Illustrator CC 2017.

Fig. 2

Coronavirus trimer, a structure providing new insights. A) Overall structure of the SARS-CoV Spike ectodomain trimer as previously described (Yuan et al., 2017) was subdivided into the N-terminal domain (NTD, orange), receptor-binding domain (RBD, red), C-terminal to S1 cleavage (CTS1, magenta), and entire S2 domain (gray). B & C) Two previously predicted confirmation states of the SARS RBD regions with the B) “lying” conformation and C) “standing” conformation. D) Spike protein sequences of the indicated viruses were aligned according to the bounds of the NTD, RBD, CTS1, S1, and S2. Sequence identities were extracted from the alignments, and a heatmap of sequence identity using SARS-CoV-Urbani as the reference sequence was constructed using EvolView (www.evolgenius.info/evolview). The heatmap was further rendered and edited in Adobe Illustrator CC 2017.

Fig. 3

Dual approaches to leverage reverse genetics. Utilizing coronavirus molecular clones, two strategies have been employed to explore the emergence and pathogenic potential of sequences derived from zoonotic populations. A) Replacing the wild-type spike proteins, this strategy explores the capacity of the spike proteins within the context of a viral backbone known to be capable of replication. These studies provide insights into the potential of spike proteins to mediate infection of human cells and cause in vivo disease and aid in examinations of the broad efficacy of therapeutics directed against CoV spike proteins. B) Utilizing portions or whole spike proteins of replication-competent CoVs, this approach examines the capacity of the viral backbone in mediating infection and pathogenesis. These studies provide insights into whether the backbone has the capacity to infect and cause disease if paired with receptor binding/entry. This approach can also evaluate the efficacy of therapeutics targeting portions of the CoV genome other than spike. Both approaches have been used to examine bat viruses currently circulating in animal populations around the world.