Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories (preprint)

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic: Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.

Conflicting and Ambiguous Names of Overlapping ORFs in SARS-CoV- 2: A Homology-Based Resolution (preprint)

At least six small alternate-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 ICTV. 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 due to prior usage of alternative names.

Mathematical analysis of Cordoba calcifediol trial suggests strong role for Vitamin D in reducing ICU admissions of hospitalized COVID-19 patients (preprint)

A randomized controlled trial of calcifediol (25-hydroxyvitamin D3) as a treatment for hospitalized COVID-19 patients in Cordoba, Spain, found that the treatment was associated with reduced ICU admissions with very large effect size and high statistical significance, but the study has had limited impact because it had only 76 patients and imperfect blinding, and did not measure vitamin D levels pre- and post-treatment or adjust for several comorbidities. Here we reanalyze the results of the study using rigorous and well established statistical techniques, and find that the randomization, large effect size, and high statistical significance address many of these concerns. In particular, we show that decreased ICU admissions were not due to uneven distribution of comorbidities or other prognostic indicators, to imperfect blinding, or to chance, but were instead associated with the calcifediol intervention. We conclude that the Cordoba study provides sufficient evidence to warrant immediate, well-designed pivotal clinical trials of calcifediol in a broader cohort of inpatients and outpatients with COVID-19, and to consider broad adoption of calcifediol treatment for vitamin-D-deficient hospitalized COVID-19 patients.

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

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.

Sarbecovirus comparative genomics elucidates gene content of SARS-CoV-2 and functional impact of COVID-19 pandemic mutations (preprint)

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.