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

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

A recent article posted on the Research Square* preprint server showed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and vaccine variability arises from inaccuracy of ribonucleic acid polymerase (RNAP).

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

Background

Since the onset of the 2019 CoV disease (COVID-19) pandemic, the world has seen the emergence of new SARS-CoV-2 variants of care (VOC) and viral lines that can evade vaccine protection. COVID-19 messenger RNA (mRNA) vaccines, centering on the SARS-CoV-2 spike (S) protein, have often been used to avert COVID-19 and induce a protective immune response against VOCs after multiple doses.

COVID-19 mRNA vaccines for their synthesis and SARS-CoV-2 for replication require RNAP. Nevertheless, these enzymes are intrinsically error prone than their deoxyribonucleic acid (DNA) equivalents and could introduce SARS-CoV-2 mutants into the RNA3.

To date, no empirical study has directly evaluated 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 magnitude of errors generated by the RNAPs participating in each stage are critical to understanding the evolution and vaccination of SARS-CoV-2 effectiveness† Current approaches are not sensitive and specific enough to detect the new RNA mutants in low input samples such as virus isolates.

About the study

In the current work, using a targeted, accurate approach to RNA consensus sequencing (tARC-seq), the scientists determine the nature and frequency of RNA errors in both SARS-CoV-2 and its vaccination. tARC-seq integrates the core features of ARC-seq and the target enhancement hybrid capture technique to enable deep variants of low-input SARS-CoV-2 samples. The researchers provide a targeted sequencing approach for finding RNA mutants in samples with low abundance and rare transcripts.

The team initially validated tARC-seq in 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.

Because SARS-CoV-2 has evolved into 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 SARS-CoV-2 Alpha and Delta variants.

Furthermore, the team examined the frequency and spectrum of RNA variants in the Pfizer vaccination, as vaccine mRNA was abundant and amenable to sequencing using bulk RNA consensus sequencing, i.e. ARC-seq. A series of T7 in vitro transcription (IVT) reactions were performed simultaneously at different temperatures on two different templates: 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 .

Results

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

The error rate estimates were previously based on the discovery of a proofreading 3′-to-5′ exoribonuclease (ExoN, nonstructural protein 14 (nsp14)) separate from the SARS-CoV-2 RdRp. The same proofreading process is linked to switching templates, which the researchers found to be error-prone.

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

Overall, these findings highlight the exceptional genetic variety of the SARS-CoV-2 populations and the diverse property of an mRNA vaccine fueled by RNAP inefficiency.

conclusions

In summary, the study findings show that the RdRp of SARS-CoV-2 was promiscuous due to nucleotide misincorporation and template flipping, both of which were regulated by the same exonuclease. ExoN could be a crucial protein in tuning viral evolution. These findings demonstrate the fundamental biology that has driven viral variety and evolution on such a large scale in the SARS-CoV-2 pandemic.

It is still uncertain what role vaccination heterogeneity plays in the immune response. The data from the Pfizer BioNTech SARS-CoV-2 vaccine analysis using ARC-seq could explain why mRNA vaccines against COVID-19 provide broader immunity against new strains after boosting.

tARC-seq variant spectra, combined with functional research and pandemic datasets, can help models anticipate how SARS-CoV-2 will evolve. Ultimately, the current findings contribute to a growing corpus of medicine and public health studies advancing mRNA-based therapeutic technology. As mRNA therapies gain momentum, these findings may aid future COVID-19 vaccine development and research design.

*Important announcement

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

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