Generation of a Novel SARS-CoV-2 Sub-genomic RNA Due to the R203K/G204R Variant in Nucleocapsid: Homologous Recombination has Potential to Change SARS-CoV-2 at Both Protein and RNA Level

Abstract

Background: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host’s anti-viral immune response, in turn affecting the frequency of variants over time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. Methods: Deep-sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Results: Sequence analysis suggests that the 3 adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep-sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. Conclusions: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans, resulting in both coding changes and novel sub-genomic RNA transcripts, suggests this as a mechanism for diversification and adaptation within its new host.

Human coronaviruses disassemble processing bodies

“…One difference that we observed by immunoblotting was the presence of a lower molecular weight ~37 kDa band recognized by our anti-N antibody for SARS-CoV-2. We did not observe the 37kDa N product after OC43 infection, transduction with be produced [68,117,118]. Translation of the N* ORF is predicted to start at an internal in-frame methionine residue (Met210) within the N protein [117,118].…”

“…Steady state levels of the lower molecular weight N product increased over the course of SARS-CoV-2 infection, consistent with the timing of PB disassembly. Other groups have noted that the SARS-CoV-2 variant of concern (VOC), Alpha, produces an additional subgenomic mRNA from which a truncated version of N, termed N*, can be produced [68,117,118]. Translation of the N* ORF is predicted to start at an internal in-frame methionine residue (Met210) within the N protein [117, 118].…”

“…Other groups have noted that the SARS-CoV-2 variant of concern (VOC), Alpha, produces an additional subgenomic mRNA from which a truncated version of N, termed N*, can be produced [68,117,118]. Translation of the N* ORF is predicted to start at an internal in-frame methionine residue (Met210) within the N protein [117, 118]. Alignment of the SARS-CoV-2 N protein sequence against other N proteins revealed that of the human CoVs, only SARS-CoV-1 and SARS-CoV-2 N retained a methionine at position 210 (229E: NC_002645.1, HKU1: NC_006577.2, MERS: NC_019843.3, NL63: NC_005831.2, OC43: NC_006213.1, SARS-1:…”

Emergence of SARS-CoV-2 Omicron (B.1.1.529) variant, salient features, high global health concerns and strategies to counter it amid ongoing COVID-19 pandemic

“…RNA-dependent RNA polymerase (Nsp12) and nonstructural protein 14 (Nsp14) are the proteins indispensable for viral replication, and it is still questionable whether mutations in these regions might confer higher mutation rates of Omicron ( Gao et al, 2021 ). Omicron also possess mutations in the nucleocapsid protein (R203K and G204R), which are though not unique to the Omicron but are linked with enhanced subgenomic RNA expression ( Leary et al, 2021 ) and viral replication ( Mourier et al, 2021 Quarleri et al, 2021 ). The original SARS-CoV-2 has an R0 of 2.5, while Delta variant had an R0 below 7.…”

Culture and identification of a “Deltamicron” SARS‐CoV‐2 in a three cases cluster in southern France

“… 10 , 11 , 12 , 13 Furthermore, genetic recombinations were reported or suspected, based on the concurrent detection in consensus genomes of signature mutations of different mutants or variants. 10 , 12 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 A study detected up to 1175 (0.2%) putative recombinant genomes among 537 360 genomes and estimated that up to 5% of SARS‐CoV‐2 having circulated in the USA and UK could be recombinants. 16 …”