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

A recent article published in the Research Square * prepress server showed that coronavirus 2 (SARS-CoV-2) mutants in severe acute respiratory syndrome and vaccine variability are derived from the inaccuracy of acid polymerase. ribonucleic acid (RNAP).

Study: RNA polymerase inaccuracy is the basis for SARS-CoV-2 variants and vaccine heterogeneity. Image credit: gagalaguna / Shutterstock


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

COVID-19 mRNA vaccines for synthesis and SARS-CoV-2 for replication require RNAP. However, these enzymes are intrinsically prone to fail their deoxyribonucleic acid (DNA) equivalents 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. Similarly, the frequency and nature of RNA variants produced during vaccine production are unclear. The distribution and extent of RNAP-generated errors involved in each phase are crucial to understanding the evolution of SARS-CoV-2 and the effectiveness of vaccination. Current approaches are not adequately sensitive and specific for detecting de novo RNA mutants in low-input samples, such as virus isolates.

About the study

In the present work, using a targeted and accurate RNA consensus sequencing approach (tARC-seq), scientists establish the nature and frequency of RNA failures in both SARS-CoV-2 and its vaccination. . tARC-seq integrates the basic features of ARC-seq and the hybrid capture technique for goal enhancement to allow for in-depth probing of low-input SARS-CoV-2 sample variants. Researchers offer a targeted sequencing approach to find RNA mutants in low-abundance samples and infrequent transcripts.

The team initially validated tARC-seq in Escherichia coli (E. coli). They then examined SARS-CoV-2 RNA extracted from Vero cells infected with 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 has developed in multiple separate lineages, each with its own set of mutations and VOCs, the researchers analyzed whether the frequency of RNA variants differed between viral lineages. They applied tARC-seq to the Alpha and Delta variants of SARS-CoV-2.

In addition, the team examined the frequency and spectrum of RNA variants in Pfizer vaccination, as vaccine mRNA was abundant and susceptible to sequencing by bulk RNA consensus sequencing. , that is, ARC-seq. An in vitro T7 transcription reaction (IVT) sequence was performed simultaneously at several temperatures in two different templates: 1) the native S gene of the SARS-CoV-2 WT strain and 2) the S structure optimized for codons of the Pfizer vaccine against COVID-19.


Overall, the authors found that SARS-CoV-2 RdRp creates one 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 previous findings in poliovirus, which uses an RdRp for replication but has no proofreading function. The team also found that RNA mutants did not scatter randomly throughout the genome, although they were related to specific genomic features and genes, such as the S protein.

Error frequency estimates were previously based on the discovery of a 3′-to-5 ‘exoribonuclease (ExoN, nonstructural protein 14 (nsp14)) test patch separate from SARS-CoV-2 RdRp. The same correction process has been linked to the change in staff, which the researchers found prone to errors.

Large deletions, insertions, and intricate mutations were detected using tARC-seq, which could be simulated by an unscheduled RdRp template change. Many of the substantial genetic alterations identified in the evolution of multiple SARS-CoV-2 lineages worldwide, including the Omicron variant, can be explained by the RdRp template change function. Subsequent sequencing of the Pfizer-BioNTech COVID-19 vaccine showed an RNA variant frequency of approximately one in 5,000, implying that most vaccine transcripts generated in vitro by RNA from beech T7 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 RNAP inefficiency.


In summary, the study’s findings show that the SARS-CoV-2 RdRp was promiscuous due to poor nucleotide incorporation and defective template change, both regulated by the same exonuclease. ExoN could be a crucial protein in adjusting viral evolution. These findings demonstrate the fundamental biology that drove viral variety and evolution on such a large scale in the SARS-CoV-2 pandemic.

It is not yet known what role the heterogeneity of vaccination plays in the immune response. Pfizer BioNTech SARS-CoV-2 vaccine analysis data using ARC-seq could explain why COVID-19 mRNA vaccines provide broader immunity against new strains after augmentation.

Spectra of tARC-seq variants, when combined with functional research and pandemic data sets, can help models predict how SARS-CoV-2 will evolve. Ultimately, the current findings add to a growing corpus of medical and public health studies that promote mRNA-based therapeutic technology. As mRNA therapies gain traction, these findings may help the future development of the COVID-19 vaccine and the design of research.

* Important news

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

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