SARS-CoV-2 gene content and COVID-19 mutation impact by comparing 44 Sarbecovirus genomes

Abstract

Despite its overwhelming clinical importance for understanding and mitigating the COVID-19 pandemic, the protein-coding gene content of the SARS-CoV-2 genome remains unresolved, with the function and even protein-coding status of many hypothetical proteins unknown and often conflicting among different annotations, thus hindering efforts for systematic dissection of its biology and the impact of recent mutations. Comparative genomics is a powerful approach for distinguishing protein-coding versus non-coding functional elements, based on their characteristic patterns of change, which we previously used to annotate protein-coding genes in human, fly, and other species. Here, we use comparative genomics to provide a high-confidence set of SARS-CoV-2 protein-coding genes, to characterize their protein-level and nucleotide-level evolutionary constraint, and to interpret the functional implications for SARS-CoV-2 mutations acquired during the current pandemic. We select 44 complete Sarbecovirus genomes at evolutionary distances well-suited for protein-coding and non-coding element identification, create whole-genome alignments spanning all named and putative genes, and quantify their protein-coding evolutionary signatures using PhyloCSF and their overlapping constraint using FRESCo. We find strong protein-coding signatures for all named genes and for hypothetical ORFs 3a, 6, 7a, 7b, and 8, indicating protein-coding roles, and provide strong evidence of protein-coding status for a recently-proposed alternate-frame novel ORF within 3a. By contrast, ORF10 shows no protein-coding signatures but shows unusually-high nucleotide-level constraint, indicating it has important but non-coding functions, and ORF14 and SARS-CoV-1 ORF3b, which overlap other genes, lack evolutionary signatures expected for dual-coding regions, indicating they do not produce functional proteins. ORF9b has ambiguous protein-coding signatures, preventing us from resolving its protein-coding status. ORF8 shows extremely fast nucleotide-level evolution, lacks a known function, and was deactivated in SARS-CoV-1, but shows clear signatures indicating protein-coding function worthy of further investigation given its rapid evolution and potential role in replication. SARS-CoV-2 mutations are preferentially excluded from evolutionarily-constrained amino acid residues and synonymously-constrained nucleotides, indicating purifying constraint acting at both coding and non-coding levels. In contrast, we find a conserved region in the nucleocapsid that is enriched for recent mutations, which could indicate a selective signal, and find that several spike-protein mutations previously identified as candidates for increased transmission and several mutations in isolates found to generate higher viral load in-vitro disrupt otherwise-perfectly-conserved amino-acids, consistent with adaptations for human-to-human transmission.

The coding capacity of SARS-CoV-2

“…Analysis of the conservation of these outof-frame iORFs in SARS-CoV and in related viruses (Sarbecoviruses) revealed 3a.iORF1 is highly conserved in Sarbecoviruses (Table S6). This ORF was also identified by three independent comparative genomic studies that demonstrate this ORF has a significant purifying selection signature, implying it is a functional polypeptide [28][29][30] . In combination with our expression measurements, these findings indicate this internal ORF is a novel and likely functional transmembrane protein, conserved throughout sarbecoviruses and as was suggested by Jungreis et al 30 should be named ORF3c.…”

“…The internal S-ORF is situated just downstream of the ORF-S AUG, suggesting ribosomes might initiate translation via leaky scanning. This region in the S-protein shows extremely-rapid evolution 30 but in the SARS-CoV-2 isolates that have been sequenced its coding capacity is not impaired. Future work will have to delineate if this ORF, which is highly expressed ( Figure 4B and Figure S20), represents a functional protein.…”

Single-nucleotide conservation state annotation of the SARS-CoV-2 genome

“…The positive and negative bases were matched by their nucleotide composition such that the proportions of each nucleotide in positive and negative bases were the same. As a comparison, we also used existing sequence constraint annotations learned from the sequence alignment identical to or containing the same strains as those provided to ConsHMM as input, which include PhastCons scores 4 , PhyloP scores 5 , and a five-way annotation based on mutation type and codon constraint in Sarbecoviruses 16 (Methods). For PhastCons and PhyloP scores, we additionally generated discrete versions of the annotation by binning the scores and evaluated these bins in an analogous way to state annotations (Methods).…”

“…Specifically, for annotation of genes, genes were ranked based on their enrichment for positive training bases, which was later used to rank test bases. The same procedure was applied to an annotation of possible intergenic, synonymous, missense, and nonsense mutations and separately to a five-way annotation based on mutation type and codon constraint in Sarbecoviruses 16 . In each of these categorical predictors, if a test base was assigned to multiple categories, the category with the stronger enrichment for positive training bases was assigned to the test base.…”

The stem loop 2 motif is a site of vulnerability for SARS-CoV-2

“…For instance, high SHAPE reactivities were found at the loop region (G71-A75), indicating strong possibility of a single-stranded nature, while low SHAPE reactivities were found on nucleotides predicted to be base-paired, such as nucleotides from 54 to 58, and nucleotides from 90 to 94. We further performed SHAPE probing on an extended version of the 3' UTR RNA (extended 3ʹ UTR) that additionally includes ORF10 and the region immediately upstream from it (ORF10 may not be protein coding; Taiaroa et al, 2020;Jungreis et al, 2020) (Fig. 1A and S1 and Table 1).…”