sgDI-tector: defective interfering viral genome bioinformatics for detection of coronavirus subgenomic RNAs.

Coronavirus RNA-dependent RNA polymerases produce subgenomic RNAs (sgRNAs) that encode viral structural and accessory proteins. User-friendly bioinformatic tools to detect and quantify sgRNA production are urgently needed to study the growing number of next-generation sequencing (NGS) data of SARS-CoV-2. We introduced sgDI-tector to identify and quantify sgRNA in SARS-CoV-2 NGS data. sgDI-tector allowed detection of sgRNA without initial knowledge of the transcription-regulatory sequences. We produced NGS data and successfully detected the nested set of sgRNAs with the ranking M > ORF3a > N>ORF6 > ORF7a > ORF8 > S > E>ORF7b. We also compared the level of sgRNA production with other types of viral RNA products such as defective interfering viral genomes.

The Heterogeneous Landscape and Early Evolution of Pathogen-Associated CpG Dinucleotides in SARS-CoV-2

SARS-CoV-2 infection can lead to acute respiratory syndrome in patients, which can be due in part to dysregulated immune signalling. We analyze here the occurrences of CpG dinucleotides, which are putative pathogen-associated molecular patterns, along the viral sequence. Carrying out a comparative analysis with other ssRNA viruses and within the Coronaviridae family, we find the CpG content of SARS-CoV-2, while low compared to other betacoronaviruses, widely fluctuates along its primary sequence. While the CpG relative abundance and its associated CpG force parameter are low for the spike protein (S) and comparable to circulating seasonal coronaviruses such as HKU1, they are much greater and comparable to SARS and MERS for the 3'-end of the viral genome. In particular, the nucleocapsid protein (N), whose transcripts are relatively abundant in the cytoplasm of infected cells and present in the 3'UTRs of all subgenomic RNA, has high CpG content. We speculate this dual nature of CpG content can confer to SARS-CoV-2 high ability to both enter the host and trigger pattern recognition receptors (PRRs) in different contexts. We then investigate the evolution of synonymous mutations since the outbreak of the COVID-19 pandemic. Using a new application of selective forces on dinucleotides to estimate context driven mutational processes, we find that synonymous mutations seem driven both by the viral codon bias and by the high value of the CpG force in the N protein, leading to a loss in CpG content. Sequence motifs preceding these CpG-loss-associated loci match recently identified binding patterns of the Zinc Finger anti-viral Protein (ZAP) protein. Funding: This work was partially supported by the ANR19 Decrypted CE30-0021-01 grants. B.G. was supported by National Institutes of Health grants 7R01AI081848-04, 1R01CA240924-01, a Stand Up to Cancer - Lustgarten Foundation Convergence Dream Team Grant, and The Pershing Square Sohn Prize - Mark Foundation Fellow supported by funding from The Mark Foundation for Cancer Research.

The Heterogeneous Landscape and Early Evolution of Pathogen-Associated CpG Dinucleotides in SARS-CoV-2.

COVID-19 can lead to acute respiratory syndrome, which can be due to dysregulated immune signaling. We analyze the distribution of CpG dinucleotides, a pathogen-associated molecular pattern, in the SARS-CoV-2 genome. We characterize CpG content by a CpG force that accounts for statistical constraints acting on the genome at the nucleotidic and amino acid levels. The CpG force, as the CpG content, is overall low compared with other pathogenic betacoronaviruses; however, it widely fluctuates along the genome, with a particularly low value, comparable with the circulating seasonal HKU1, in the spike coding region and a greater value, comparable with SARS and MERS, in the highly expressed nucleocapside coding region (N ORF), whose transcripts are relatively abundant in the cytoplasm of infected cells and present in the 3'UTRs of all subgenomic RNA. This dual nature of CpG content could confer to SARS-CoV-2 the ability to avoid triggering pattern recognition receptors upon entry, while eliciting a stronger response during replication. We then investigate the evolution of synonymous mutations since the outbreak of the COVID-19 pandemic, finding a signature of CpG loss in regions with a greater CpG force. Sequence motifs preceding the CpG-loss-associated loci in the N ORF match recently identified binding patterns of the zinc finger antiviral protein. Using a model of the viral gene evolution under human host pressure, we find that synonymous mutations seem driven in the SARS-CoV-2 genome, and particularly in the N ORF, by the viral codon bias, the transition-transversion bias, and the pressure to lower CpG content.

The Heterogeneous Landscape and Early Evolution of Pathogen-Associated CpG Dinucleotides in SARS-CoV-2.

SARS-CoV-2 infection can lead to acute respiratory syndrome in patients, which can be due in part to dysregulated immune signalling. We analyze here the occurrences of CpG dinucleotides, which are putative pathogen-associated molecular patterns, along the viral sequence. Carrying out a comparative analysis with other ssRNA viruses and within the Coronaviridae family, we find the CpG content of SARS-CoV-2, while low compared to other betacoronaviruses, widely fluctuates along its primary sequence. While the CpG relative abundance and its associated CpG force parameter are low for the spike protein (S) and comparable to circulating seasonal coronaviruses such as HKU1, they are much greater and comparable to SARS and MERS for the 3'-end of the viral genome. In particular, the nucleocapsid protein (N), whose transcripts are relatively abundant in the cytoplasm of infected cells and present in the 3'UTRs of all subgenomic RNA, has high CpG content. We speculate this dual  nature of CpG content can confer to SARS-CoV-2 high ability to both enter the host and trigger pattern recognition receptors (PRRs) in different contexts. We then investigate the evolution of synonymous mutations since the outbreak of the COVID-19 pandemic. Using a new application of selective forces on dinucleotides to estimate context driven mutational processes, we find that synonymous mutations seem driven both by the viral codon bias and by the high value of the CpG force in the N protein, leading to a loss in CpG content. Sequence motifs preceding these CpG-loss-associated loci match recently identified binding patterns of the Zinc Finger anti-viral Protein (ZAP) protein. Funding: This work was partially supported by the ANR19 Decrypted CE30-0021-01 grants. B.G. was supported by National Institutes of Health grants 7R01AI081848-04, 1R01CA240924-01, a Stand Up to Cancer - Lustgarten Foundation Convergence Dream Team Grant, and The Pershing Square Sohn Prize - Mark Foundation Fellow supported by funding from The Mark Foundation for Cancer Research.