Two New Studies Shows That SARS-CoV-2 Is Evolving Rapidly Due To Human Host Factors, Leading To More Genetic Variants

Two recently published study findings are indicating that the SARS-CoV-2 virus is evolving in the human host due to a multitude of factors and hence will give rise to more genetic variants.


 
SARS-CoV-2 Undergoes ADAR-Mediated A-To-I RNA Editing In Human Cells
 
In the first study, scientists explored severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) ribonucleic acid (RNA) editing in host cells.
 
The SARS-CoV-2 virus is a positive-sense, single-stranded RNA virus responsible for the COVID-19 pandemic. It remains unclear whether and to what extent the virus in human host cells undergoes RNA editing, a major RNA modification mechanism.
 
The SARS-CoV-2 Variants study team performed a robust bioinformatic analysis of metatranscriptomic data from multiple bronchoalveolar lavage fluid samples of COVID-19 patients, revealing an appreciable number of A-to-I RNA editing candidate sites in SARS-CoV-2.
 
The study findings confirmed the enrichment of A-to-I RNA editing signals at these candidate sites through evaluating four characteristics specific to RNA editing: the inferred RNA editing sites exhibit (i) stronger ADAR1 binding affinity predicted by a deep-learning model built from ADAR1 CLIP-seq data, (ii) decreased editing levels in ADAR1-inhibited human lung cells, (iii) local clustering patterns, and (iv) higher RNA secondary structure propensity.
 
The study findings have critical implications in understanding the evolution of SARS-CoV-2 as well as in COVID-19 research, such as phylogenetic analysis and vaccine development.
 
The study findings were published in the peer reviewed journal: PLOS Genetics.
https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010130
 
The COVID-19 pandemic caused by SARS-CoV-2 has resulted in almost half a billion cases globally and over 28 million deaths if excess death rates were also included.
 
In the last 27 months, the research community has extensively studied and characterized SARS-CoV-2 with thousands of viral genomes sequenced from COVID-19 patients.
 
The identified detailed characterizations have been vital to understanding the evolutionary and pathogenic features of SARS-CoV-2 and designing effective therapeutics.
 
There is however limited data on nucleotide variation and viral genome plasticity, particularly the RNA modifications caused by host cells.
 
Typically, in humans, adenosine to inosine (A-to-I) change catalyzed by adenosine deaminase acting on RNA 1 (ADAR1) is the most common RNA modification, and the translation machinery recognizes the resultant inosine as guanosine (G).
 
However, the extent to which the SARS-CoV-2 genome is subject to RNA editing inside host cells remains unknown. It is crucial to evaluate whether RNA editing occurs in the SARS-CoV-2 genome because the virus uses the minus (-) strand of its RNA as the template for replication. Any nucleotide change, if induced, could cause genetic variations that subsequent generations could inherit. Besides, the genome of SARS-CoV-2 is vastly protein -coding, unlike human genomes.
 
Hence any nucleotide change in the RNA would, with high probability, reflect changes in the amino acid sequences of protein.
 
The study team identified a nucleotide variant pool from meta-transcriptomic sequence reads with a bioinformatics pipeline and tested if actual RNA editing sites were present in the collection.
 
Meta-transcriptomic sequence reads from the bronchoalveolar lavage fluid samples were systematically analyzed. A bioinformatics platform was developed to detect single nucleotide variants (SNVs) that could remove low-quality reads, identify viral reads with Fastv, trim end nucleotides, generate high-quality alignment, and detect SNVs with significant significance variable allele frequency (VAF).
 
Interestingly of the 19 samples assessed, one had no detectable SNVs; in the rest of the samples, a consistent pattern of A > G and thymidine (T) > cytidine (C) substitutions. The SNVs, A > G and T > C, correspond to the A-to-I transition in positive (+) and (-) strands, respectively, of SARS-CoV-2 RNA. The authors observed about 144 recurrent SNVs with VAF ranging from 0.5 % to 70 % and obtained a set of A-to-I candidate sequences.
 
Detailed analysis of sequences flanking the candidate sites revealed G depletion and enrichment preference at the editing sites’ -1 and +1 positions, respectively. RNA editing at about 55% of sites resulted in non-synonymous changes, mainly in the ORF1ab and spike protein.
 
Since it is already known that SARS-CoV-2 RNA replicates in the host cell cytoplasm, harboring the p150 ADAR1 isoform, the study team speculated that the inferred RNA editing sites would have a higher binding affinity to ADAR1. A hybrid neural network was built based on the ADAR1 crosslinking immunoprecipitation (CLIP) followed by high-throughput sequencing (CLIP-seq) peak set to predict binding affinity. It was noted that many RNA editing sites had a greater tendency to bind to ADAR1.
 
RNA-seq or RNA sequencing data were obtained from a cell line infected with SARS-CoV-2.
 
Ruxolitinib, an immunosuppressive agent, significantly reduced the expression levels of ADAR1 in the infected cells. The inhibition was more apparent for its p110 isoform, and RNA editing was lower at the candidate sites.
 
It was found that of the 84 candidate editing sites, 75% exhibited reduced editing levels, indicating the direct effect of ADAR1 of host cells on the A-to-I viral RNA editing. RNA editing sites were enriched in clusters, i.e., the distance between RNA editing sites was shorter. CROSS, a computational RNA structure prediction algorithm, revealed that RNA editing sites had a significantly high propensity to form secondary structures. The amino acid substitutions induced by the RNA editing events were studied in the context of binding affinity of T cell epitope to human leucocyte antigen (HLA) and improved binding affinity with the edited peptide relative to wild-type peptide was noted.
 
The study findings showed that ADAR1 mediates A-to-I RNA editing of SARS-CoV-2 RNA in host cells. Thus, RNA editing might represent another factor of genetic variation of the SARS-CoV-2 genome shaping its evolution and plasticity. In addition to the p150 ADAR1 isoform, the p110 isoform was also observed as mediating viral RNA editing.
 
Importantly the RNA editing induced by the host cell might accelerate the viral evolution. Still, its fate is dependent on the fitness effect and subject to selection pressures such as purifying selection if found deleterious and positive selection if found advantageous. One of the few limitations of the study includes the non-validation of the RNA editing sites experimentally.
 
Large-Scale Analysis Of SARS-Cov-2 Synonymous Mutations Reveals The Adaptation To The Human Codon Usage During The Virus Evolution
 
In the second study by Italian researchers, a large-scale analysis of SARS-CoV-2 synonymous mutations revealed the adaptation to the human codon usage during the virus evolution. The study findings showed that synonymous SARS-CoV-2 mutations related to the activity of different mutational processes may positively impact viral evolution by increasing its adaptation to the human codon usage.
 
To date, numerous national and transnational studies have been dedicated to the analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genome, most of which focused on missense and nonsense mutations.
 
However, approximately 30 per cent of the SARS-CoV-2 variants are synonymous, therefore changing the target codon without affecting the corresponding protein sequence.
 
The study team by performing a large-scale analysis of sequencing data generated from almost 400,000 SARS-CoV-2 samples, found that silent mutations increasing the similarity of viral codons to the human ones tend to fixate in the viral genome overtime. This indicates that SARS-CoV-2 codon usage is adapting to the human host, likely improving its effectiveness in using the human aminoacyl-tRNA set through the accumulation of deceitfully neutral silent mutations.
 
The study findings were published in the peer journal: Virus Evolution. https://academic.oup.com/ve/article/8/1/veac026/6553895
 
Global as well as time-series analyses of silent mutations indicate that Codon Usage or CU adaptation may play a relevant role in the evolution of SARS-CoV-2, suggesting that the evolution of the viral genome has been intense.
 
Further studies will be required to thoroughly dissect the overall impact and clinical relevance of these findings. Soon, it will be important to assess if specific viral genomes with a high CU adaptation also show an increased infectiousness and/or clinical aggressiveness, which, at the time being, is very difficult, owing to the limited availability of clinical data in publicly available SARS-CoV-2 sequencing studies. In this regard and given the profound impact of the COVID-19 pandemic, every effort should be made to share clinical information together with sequencing data, which is unfortunately rarely done.
 
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