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Review
. 2019 Nov:537:198-207.
doi: 10.1016/j.virol.2019.08.031. Epub 2019 Aug 30.

Coronavirus genomic RNA packaging

Paul S Masters  1
Affiliations
Review

Coronavirus genomic RNA packaging

Paul S Masters. Virology. 2019 Nov.

Abstract

RNA viruses carry out selective packaging of their genomes in a variety of ways, many involving a genomic packaging signal. The first coronavirus packaging signal was discovered nearly thirty years ago, but how it functions remains incompletely understood. This review addresses the current state of knowledge of coronavirus genome packaging, which has mainly been studied in two prototype species, mouse hepatitis virus and transmissible gastroenteritis virus. Despite the progress that has been made in the mapping and characterization of some packaging signals, there is conflicting evidence as to whether the viral nucleocapsid protein or the membrane protein plays the primary role in packaging signal recognition. The different models for the mechanism of genomic RNA packaging that have been prompted by these competing views are described. Also discussed is the recent exciting discovery that selective coronavirus genome packaging is critical for in vivo evasion of the host innate immune response.

Keywords: Coronavirus; Innate immunity; Membrane protein; Mouse hepatitis virus; Nucleocapsid protein; Packaging signal; RNA virus; Viral genome packaging.

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Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Coronavirus RNA species and virions. Top, a schematic of the coronavirus genome (gRNA), typified by that of mouse hepatitis virus (MHV). The 5′ end contains the rep1a and rep1b genes, which encode the viral replicase-transcriptase, while the 3′ end contains the structural protein genes S, E, M, and N. Also at the 3′ end of the genome are accessory genes (unlabeled), the locations and numbers of which vary for different viruses; those shown are for MHV. Under the genome, to the right, are the nested set of subgenomic mRNAs (sgRNAs), of which MHV has six. To the left is a schematic of the virion, depicting the essential structural proteins: spike protein (S); membrane protein (M); envelope protein (E); and nucleocapsid protein (N).
Fig. 2
Fig. 2
Betacoronavirus lineage A packaging signals. Top, a map of the lineage A betacoronavirus genome, showing the loci of PS and PS-like elements. (A) Structure of the MHV PS, modeled by Chen et al. (2007). Repeat units are highlighted in orange. (B) Phylogenetic conservation of the PS among lineage A betacoronaviruses. Circles denote basepairs in double-stranded sections that co-vary between MHV and one of the other viruses; additional base-pairing covariation is found among different strains of MHV and of HCoV-HKU1 (not shown). Triangles denote bases in single-stranded sections that differ from MHV; the shaded rectangle indicates the part of the structure that is deleted in EqCoV. (C) Amino-acid alignment of part of the nsp15 subunit of the replicase-transcriptase. Highlighted in red is the segment encoded by the PS of lineage A betacoronaviruses. This segment is absent from all other lineages and genera, here represented by SARS-CoV (betacoronavirus, lineage B), MERS-CoV (betacoronavirus, lineage C), TGEV (alphacoronavirus), IBV (gammacoronavirus), and PDCoV (deltacoronavirus). (D) Upstream PS-like structures, denoted PS(2), in RbCoV and EqCoV. In each, broken-line brackets show the region that is a duplication of PS sequence in nsp15; repeat units are highlighted in yellow. GenBank accession numbers for the sequences shown: MHV, AY700211; BCoV, U00735; HCoV-HKU1, AY597011; HCoV-OC43, AY903460; EqCoV, EF446615; RbCoV-HKU14, JN874559; SARS-CoV, AY278741; MERS-CoV, JX869059; TGEV, AJ271965; IBV, AJ311317; PDCoV, JQ065042.
Fig. 3
Fig. 3
Other coronavirus PSs. (A) The genomic segment to which the PS for the alphacoronavirus TGEV has been localized (Morales et al., 2013); nucleotides 1–598 include the 5′ UTR and almost all of the nsp1 gene. (B) The TGEV PS predicted by Chen and Olsthoorn (2010), corresponding to the shaded area in (A). (C) A potential PS, containing multiple repeats of a loop motif, found at the nsp13-nsp14 junction of a subset of deltacoronaviruses, including BuCoV. In (B) and (C), repeat units are highlighted in yellow. GenBank accession numbers for the sequences shown: TGEV, AJ271965; BuCoV-HKU11, FJ376620.
Fig. 4
Fig. 4
Function and recognition of the PS. (A) Role of the PS in the viral genome. Left, the primary sequence and RNA secondary structure of the wild-type (wt) MHV PS was disrupted with 20 coding-silent mutations to create a mutant designated silPS (Kuo and Masters, 2013). Altered nucleotides are those highlighted in blue in the wt PS structure and in green in the silPS structure. Right, a Northern blot of RNA from highly purified virions of wt MHV and the silPS mutant detected with a probe specific for the 3′ end of the genome, which is common to gRNA and all sgRNAs. (B) Linear schematics of the MHV N and M proteins. In the N protein, there are two RNA-binding structural domains (NTD and CTD) and the carboxy-terminal domain (N3). At the amino terminus, and connecting the three domains, are unstructured segments; the central spacer contains a serine- and arginine-rich region (SR). In the M protein, the amino-terminal ectodomain (ecto) is connected to the carboxy-terminal endodomain (endo) by three transmembrane segments (Tm). Red, loci of substitutions or point mutations of N protein, in either the CTD (Kuo et al., 2014) or in N3 (Kuo et al., 2016b), which separately abolish packaging selectivity in viruses that contain a wt PS. Green, loci of critical residues in N protein (Hurst et al., 2005; Verma et al., 2006) and M protein (Escors et al., 2001; Kuo and Masters, 2002; Verma et al., 2007) essential for N-M virion assembly interactions (arrow).
Fig. 5
Fig. 5
Models for the mechanism of coronavirus gRNA packaging. (A) The N protein CTD binds to the PS, concomitantly releasing domain N3; N3 then binds to the M protein endodomain. (B) The M protein binds to the PS, initiating encapsidation of the genome by N protein. (C) Complex formation by the N and M proteins precedes PS recognition. See text for details. For simplicity, in all three models subsequent cooperative N-RNA, N-N, N-M, and M-M interactions have been omitted.
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