Human SARS-CoV-2 has evolved to increase U content and reduce genome size.

Infections caused by SARS-CoV-2 have brought great harm to human health. After transmission for over two years, SARS-CoV-2 has diverged greatly and formed dozens of different lineages. Understanding the trend of its genome evolution could help foresee difficulties in controlling transmission of the virus. In this study, we conducted an extensive monthly survey and in-depth analysis on variations of nucleotide, amino acid and codon numbers in 311,260 virus samples collected till January 2022. The results demonstrate that the evolution of SARS-CoV-2 is toward increasing U-content and reducing genome-size. C, G and A to U mutations have all contributed to this U-content increase. Mutations of C, G and A at codon position 1, 2 or 3 have no significant difference in most SARS-CoV-2 lineages. Current viruses are more cryptic and more efficient in replication, and are thus less virulent yet more infectious. Delta and Omicron variants have high mutability over other lineages, bringing new threat to human health. This trend of genome evolution may provide a clue for tracing the origin of SARS-CoV-2, because ancestral viruses should have lower U-content and probably bigger genome-size.

A Path toward SARS-CoV-2 Attenuation: Metabolic Pressure on CTP Synthesis Rules the Virus Evolution.

In the context of the COVID-19 pandemic, we describe here the singular metabolic background that constrains enveloped RNA viruses to evolve toward likely attenuation in the long term, possibly after a step of increased pathogenicity. Cytidine triphosphate (CTP) is at the crossroad of the processes allowing SARS-CoV-2 to multiply, because CTP is in demand for four essential metabolic steps. It is a building block of the virus genome, it is required for synthesis of the cytosine-based liponucleotide precursors of the viral envelope, it is a critical building block of the host transfer RNAs synthesis and it is required for synthesis of dolichol-phosphate, a precursor of viral protein glycosylation. The CCA 3'-end of all the transfer RNAs required to translate the RNA genome and further transcripts into the proteins used to build active virus copies is not coded in the human genome. It must be synthesized de novo from CTP and ATP. Furthermore, intermediary metabolism is built on compulsory steps of synthesis and salvage of cytosine-based metabolites via uridine triphosphate that keep limiting CTP availability. As a consequence, accidental replication errors tend to replace cytosine by uracil in the genome, unless recombination events allow the sequence to return to its ancestral sequences. We document some of the consequences of this situation in the function of viral proteins. This unique metabolic setup allowed us to highlight and provide a raison d'être to viperin, an enzyme of innate antiviral immunity, which synthesizes 3'-deoxy-3',4'-didehydro-CTP as an extremely efficient antiviral nucleotide.

Point mutation bias in SARS-CoV-2 variants results in increased ability to stimulate inflammatory responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces severe pneumonia and is the cause of a worldwide pandemic. Coronaviruses, including SARS-CoV-2, have RNA proofreading enzymes in their genomes, resulting in fewer gene mutations than other RNA viruses. Nevertheless, variants of SARS-CoV-2 exist and may induce different symptoms; however, the factors and the impacts of these mutations are not well understood. We found that there is a bias to the mutations occurring in SARS-CoV-2 variants, with disproportionate mutation to uracil (U). These point mutations to U are mainly derived from cytosine (C), which is consistent with the substrate specificity of host RNA editing enzymes, APOBECs. We also found the point mutations which are consistent with other RNA editing enzymes, ADARs. For the C-to-U mutations, the context of the upstream uracil and downstream guanine from mutated position was found to be most prevalent. Further, the degree of increase of U in SARS-CoV-2 variants correlates with enhanced production of cytokines, such as TNF-α and IL-6, in cell lines when compared with stimulation by the ssRNA sequence of the isolated virus in Wuhan. Therefore, RNA editing is a factor for mutation bias in SARS-CoV-2 variants, which affects host inflammatory cytokines production.

Mutation Patterns of Human SARS-CoV-2 and Bat RaTG13 Coronavirus Genomes Are Strongly Biased Towards C>U Transitions, Indicating Rapid Evolution in Their Hosts.

The pandemic caused by the spread of SARS-CoV-2 has led to considerable interest in its evolutionary origin and genome structure. Here, we analyzed mutation patterns in 34 human SARS-CoV-2 isolates and a closely related RaTG13 isolated from Rhinolophus affinis (a horseshoe bat). We also evaluated the CpG dinucleotide contents in SARS-CoV-2 and other human and animal coronavirus genomes. Out of 1136 single nucleotide variations (~4% divergence) between human SARS-CoV-2 and bat RaTG13, 682 (60%) can be attributed to C>U and U>C substitutions, far exceeding other types of substitutions. An accumulation of C>U mutations was also observed in SARS-CoV2 variants that arose within the human population. Globally, the C>U substitutions increased the frequency of codons for hydrophobic amino acids in SARS-CoV-2 peptides, while U>C substitutions decreased it. In contrast to most other coronaviruses, both SARS-CoV-2 and RaTG13 exhibited CpG depletion in their genomes. The data suggest that C-to-U conversion mediated by C deamination played a significant role in the evolution of the SARS-CoV-2 coronavirus. We hypothesize that the high frequency C>U transitions reflect virus adaptation processes in their hosts, and that SARS-CoV-2 could have been evolving for a relatively long period in humans following the transfer from animals before spreading worldwide.