Evolution of enhanced innate immune evasion by SARS-CoV-2.

The emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6-all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection4. The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants.

Genomic RNA Elements Drive Phase Separation of the SARS-CoV-2 Nucleocapsid.

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33°C and 37°C) and reduced at room temperature (22°C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

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

Despite its clinical importance, the SARS-CoV-2 gene set remains unresolved, hindering dissection of COVID-19 biology. We use comparative genomics to provide a high-confidence protein-coding gene set, characterize evolutionary constraint, and prioritize functional mutations. We select 44 Sarbecovirus genomes at ideally-suited evolutionary distances, and quantify protein-coding evolutionary signatures and overlapping constraint. We find strong protein-coding signatures for ORFs 3a, 6, 7a, 7b, 8, 9b, and a novel alternate-frame gene, ORF3c, whereas ORFs 2b, 3d/3d-2, 3b, 9c, and 10 lack protein-coding signatures or convincing experimental evidence of protein-coding function. Furthermore, we show no other conserved protein-coding genes remain to be discovered. Mutation analysis suggests ORF8 contributes to within-individual fitness but not person-to-person transmission. Cross-strain and within-strain evolutionary pressures agree, except for fewer-than-expected within-strain mutations in nsp3 and S1, and more-than-expected in nucleocapsid, which shows a cluster of mutations in a predicted B-cell epitope, suggesting immune-avoidance selection. Evolutionary histories of residues disrupted by spike-protein substitutions D614G, N501Y, E484K, and K417N/T provide clues about their biology, and we catalog likely-functional co-inherited mutations. Previously reported RNA-modification sites show no enrichment for conservation. Here we report a high-confidence gene set and evolutionary-history annotations providing valuable resources and insights on SARS-CoV-2 biology, mutations, and evolution.

Conflicting and ambiguous names of overlapping ORFs in the SARS-CoV-2 genome: A homology-based resolution.

At least six small alternative-frame open reading frames (ORFs) overlapping well-characterized SARS-CoV-2 genes have been hypothesized to encode accessory proteins. Researchers have used different names for the same ORF or the same name for different ORFs, resulting in erroneous homological and functional inferences. We propose standard names for these ORFs and their shorter isoforms, developed in consultation with the Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. We recommend calling the 39 codon Spike-overlapping ORF ORF2b; the 41, 57, and 22 codon ORF3a-overlapping ORFs ORF3c, ORF3d, and ORF3b; the 33 codon ORF3d isoform ORF3d-2; and the 97 and 73 codon Nucleocapsid-overlapping ORFs ORF9b and ORF9c. Finally, we document conflicting usage of the name ORF3b in 32 studies, and consequent erroneous inferences, stressing the importance of reserving identical names for homologs. We recommend that authors referring to these ORFs provide lengths and coordinates to minimize ambiguity caused by prior usage of alternative names.

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

Despite its overwhelming clinical importance, the SARS-CoV-2 gene set remains unresolved, hindering dissection of COVID-19 biology. Here, we use comparative genomics to provide a high-confidence protein-coding gene set, characterize protein-level and nucleotide-level evolutionary constraint, and prioritize functional mutations from the ongoing COVID-19 pandemic. We select 44 complete Sarbecovirus genomes at evolutionary distances ideally-suited for protein-coding and non-coding element identification, create whole-genome alignments, and quantify protein-coding evolutionary signatures and overlapping constraint. We find strong protein-coding signatures for all named genes and for 3a, 6, 7a, 7b, 8, 9b, and also ORF3c, a novel alternate-frame gene. By contrast, ORF10, and overlapping-ORFs 9c, 3b, and 3d lack protein-coding signatures or convincing experimental evidence and are not protein-coding. Furthermore, we show no other protein-coding genes remain to be discovered. Cross-strain and within-strain evolutionary pressures largely agree at the gene, amino-acid, and nucleotide levels, with some notable exceptions, including fewer-than-expected mutations in nsp3 and Spike subunit S1, and more-than-expected mutations in Nucleocapsid. The latter also shows a cluster of amino-acid-changing variants in otherwise-conserved residues in a predicted B-cell epitope, which may indicate positive selection for immune avoidance. Several Spike-protein mutations, including D614G, which has been associated with increased transmission, disrupt otherwise-perfectly-conserved amino acids, and could be novel adaptations to human hosts. The resulting high-confidence gene set and evolutionary-history annotations provide valuable resources and insights on COVID-19 biology, mutations, and evolution.