SARS-CoV-2 Subgenomic RNAs: Characterization, Utility, and Perspectives

Abstract

SARS-CoV-2, the etiologic agent at the root of the ongoing COVID-19 pandemic, harbors a large RNA genome from which a tiered ensemble of subgenomic RNAs (sgRNAs) is generated. Comprehensive definition and investigation of these RNA products are important for understanding SARS-CoV-2 pathogenesis. This review summarizes the recent progress on SARS-CoV-2 sgRNA identification, characterization, and application as a viral replication marker. The significance of these findings and potential future research areas of interest are discussed.

Keywords: COVID-19; SARS-CoV-2; sgRNA; subgenomic RNA.

Figure 1

Schematic representation of SARS-CoV-2 genome organization and the canonical subgenomic mRNAs. The genome features two large genes, ORF1a and ORF1b, which encode a total of 16 non-structural proteins (nsp1-nsp16) (primary translation); structural genes encoding structural proteins include spike (S), envelope (E), membrane (M), and nucleocapsid (N), respectively; and genes encoding several small accessory proteins (3a, 6, 7a, 7b, 8 and 10). Depicted in the lower right are 10 canonical subgenomic mRNAs. Figure is adapted from [12].

Figure 2

Schematic depiction of SARS-CoV-2 replication and discontinuous transcription. In addition to serving as a template for producing (−) genomic RNA (which enables genome replication), the full length (+) genomic RNA also serves as a template to produce (−) subgenomic RNAs (sgRNAs), which are subsequently used to synthesize (+) subgenomic mRNAs encoding structural and accessory proteins. (−) sgRNA synthesis involves a template switch from a body transcription regulatory sequence (TRS-B) (located upstream of most open reading frames in the 3′ one-third of the viral genome) to the leader TRS (TRS-L, located at about 70 nucleotides from the 5′ end of the genome). This discontinuous transcription process, leading to leader-body fusion, can occur at any TRS-B, and eventually results in the synthesis of a characteristic nested set of (+) subgenomic mRNAs. SARS-CoV-2 subgenomic mRNAs are structurally polycistronic, but are assumed to be functionally monocistronic [3,13], in that only the first open reading frame in each sgRNA, which is absent in the next smaller sgRNA, is translated. Depicted in the lower left and lower middle is the conserved TRS motif (ACGAAC) in the leader and body sequences. In addition, in SARS-CoV-2, extensive base pairing with 7–12 consecutive base pairs beyond the conserved motif between TRS-L and anti-TRS-B has been observed [6]. Figure is adapted from [14].

Figure 3

SARS-CoV-2 sgRNA recombination sites. Depicted are three types of fusion/junction sites. (Green, turquoise, or purple bracket lines represent the 5′ and 3′ locations of junctions.) (A) TRS-L- and TRS-B-dependent discontinuous transcription, which gives rise to canonical sgRNAs. Note that each canonical junction represents a group of subgenomes that have similar, yet distinct fusion junction sites upstream of a common first annotated gene downstream of the junction [6]. (B) TRS-L-dependent noncanonical fusions between TRS-L and unanticipated 3′ sites in the middle of ORFs or UTR (i.e., noncanonical 3′ sites) in the body. (C) TRS-L-independent fusion between sequences that share no similarity to the leader, resulting in long-distance fusions and smaller deletions mainly in the structural and accessory genes when the fusion occurs between proximal sites. Hundreds of noncanonical sgRNAs have been identified [5,6,12,20], and in (B,C), only several representative fusion patterns are illustrated. In addition, both in-frame and out-of-frame fusion products can be generated in (B,C), with out-of-frame noncanonical sgRNAs significantly outnumbering in-frame noncanonical sgRNAs (by ~60%) [12].