Study: RNA polymerase inaccuracy underlies SARS-CoV-2variants and vaccine heterogeneity. Image Credit: gagalaguna/Shutterstock

The frequency and nature of RNA errors in SARS-CoV-2 and its vaccine

A recent article published on Research Square* The preprint server demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and vaccine variability stem from ribonucleic acid polymerase (RNAP) inaccuracy.

Study: RNA polymerase inaccuracy underlies SARS-CoV-2 variants and vaccine heterogeneity. Image Credit: gagalaguna/Shutterstock


Since the onset of the CoV disease 2019 (COVID-19) pandemic, the world has seen the emergence of concerning new variants of SARS-CoV-2 (VOC) and viral lineages that may evade vaccine protection. COVID-19 messenger RNA (mRNA) vaccines centered on the SARS-CoV-2 spike (S) protein have been commonly used to prevent COVID-19 and induce a protective immune response against COVs after multiple doses.

COVID-19 mRNA vaccines for their synthesis and SARS-CoV-2 for their replication require RNAPs. Nevertheless, these enzymes are inherently prone to breakdowns than their deoxyribonucleic acid (DNA) counterparts and could introduce SARS-CoV-2 mutants into RNA3.

To date, no empirical research has directly assessed the frequency of SARS-CoV-2 RNA-dependent RNAP (RdRp) defects during replication, a critical parameter for modeling viral evolution. Likewise, the frequency and nature of RNA variants produced during vaccine production are unclear. The distribution and extent of errors generated by RNAPs participating in each phase are crucial to understanding the evolution and vaccination of SARS-CoV-2 efficiency. Current approaches are not sensitive and specific enough to detect de novo RNA mutants in low input samples such as virus isolates.

About the study

In the present work, using a targeted and accurate consensus RNA sequencing (tARC-seq) approach, scientists establish the nature and frequency of RNA defects in SARS-CoV-2 and its vaccination. tARC-seq incorporates the core features of ARC-seq and the hybrid capture technique for target enhancement to enable in-depth probing of low input SARS-CoV-2 sample variants. The researchers propose a targeted sequencing approach to find RNA mutants in low abundance samples and infrequent transcripts.

The team initially validated tARC-seq by Escherichia coli (E.coli). They then examined SARS-CoV-2 RNA extracted from infected Vero cells using tARC-seq. To determine whether RNA variants were randomly distributed throughout the SARS-CoV-2 genome, frequencies were determined by position.

Since SARS-CoV-2 evolved into several distinct lineages, each with its own set of mutations and COVs, the researchers analyzed whether the frequency of RNA variants differed from viral lineage to virus lineage. other. They applied tARC-seq to SARS-CoV-2 Alpha and Delta variants.

Additionally, the team looked at the frequency and spectrum of RNA variants in the Pfizer vaccination, as vaccine mRNA was abundant and amenable to sequencing using consensus bulk RNA sequencing, i.e. ARC-seq. A sequence of T7 in vitro transcription reactions (IVT) were performed simultaneously at different temperatures on two distinct models: 1) the native S gene of the SARS-CoV-2 WT strain and 2) the codon-optimized S structure of the COVID-19 Pfizer vaccine.


Overall, the authors found that the SARS-CoV-2 RdRp creates an error per 10,000 nucleotides, more than previous estimates by sequencing three SARS-CoV-2 isolates. Although this frequency was higher than other predictions, it was equivalent to earlier findings for poliovirus, which uses an RdRp for replication but has no proofreading function. The team also found that the RNA mutants were not randomly scattered throughout the genome, although they were linked to specific genomic characteristics and genes, such as protein S.

Error frequency estimates were based on the finding of a 3′ to 5′ proofreading exoribonuclease (ExoN, nonstructural protein 14 (nsp14)) distinct from SARS-CoV-2 RdRp. The same proofreading process was tied to model switching, which the researchers found error-prone.

Large complex deletions, insertions and mutations were detected using tARC-seq, which could be simulated using an unprogrammed RdRp pattern flip. Many substantial genetic alterations identified in the evolution of several SARS-CoV-2 lineages worldwide, including the Omicron variant, can be explained by the pattern-switching function of RdRp. Subsequent sequencing of the Pfizer-BioNTech COVID-19 vaccine showed an RNA variant frequency of approximately one in 5,000, implying that the majority of vaccine transcripts generated in vitro by phage T7 RNAP contain a variant.

Taken together, these findings highlight the exceptional genetic variety of SARS-CoV-2 populations and the diverse trait of an mRNA vaccine fueled by the ineffectiveness of RNAP.


In summary, the study results show that the RdRp of SARS-CoV-2 was due to promiscuity due to nucleotide misincorporation and defective template flipping, both of which were regulated by the same exonuclease. ExoN could be a crucial protein in the control of viral evolution. These results demonstrate the basic biology that drove viral variety and evolution on such a large scale in the SARS-CoV-2 pandemic.

It is not yet clear what role vaccination heterogeneity plays in the immunological response. Data from Pfizer BioNTech SARS-CoV-2 vaccine analysis using ARC-seq may explain why mRNA COVID-19 vaccines provide broader immunity against new strains after the boost.

tARC-seq variant spectra, when combined with functional surveys and pandemic datasets, can help models anticipate the evolution of SARS-CoV-2. Ultimately, the current findings add to a growing body of medical and public health studies that promote mRNA-based therapeutic technology. As mRNA-based therapies gain traction, these findings could contribute to future COVID-19 vaccine development and research design.

*Important Notice

Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

#frequency #nature #RNA #errors #SARSCoV2 #vaccine

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