There is evidence that SARS-CoV-2 alters the RNA of infected cells

Scientists at the Federal University of São Paulo (UNIFESP) in Brazil have demonstrated for the first time that infection with the SARS-CoV-2 virus that causes COVID-19 alters the function of
host cell RNA.
They came to this conclusion
by analyzing 13 datasets obtained from four studies of viral, human and animal cell RNA.

An article published in Frontiers in Cellular and Infection Microbiology reports on a recent study that examined the transcriptome
of Vero cells (derived from monkeys) and human Calu-3 cells by direct RNA sequencing.
The transcriptome is a collection of biochemical modifications of cellular RNA, such as methylation
.

"Our first important finding in this study is that infection with SARS-CoV-2 increases levels of m6A (N6-methyladenine), a type of methylation, in host cells compared to non-infected cells," Marcelo Briones, the paper's last author, told Agência FAPESP
.
Briones is a professor at the United Nations Agency for International Development School of Medicine (EPM) and a researcher at
the Faculty of Medicine and Bioinformatics Centre.

Methylation is a biochemical modification that involves the addition of a methyl group to a
substrate.
It occurs in cells through the action of enzymes that are able to transfer parts of one molecule to another
.
This alters the behavior of
proteins, enzymes, hormones, and genes.
The researchers quantitatively demonstrated changes in the RNA of infected cells by analyzing all the RNA present in the cells, and qualitatively showed the changes
by locating the number of methylations in each region of the nucleotide on a map.

The study is a continuation of an earlier genomic analysis published in 2021 in which researchers analyzed methylation patterns
in SARS-CoV-2.

"Methylation has two functions
in viruses.
It regulates protein expression and protects viruses from interferon, a potent antiviral substance
produced by the host body.
”

In both studies, the researchers analyzed m6A because it is the most common type of RNA nucleotide modification, involved in several important processes such as intracellular localization and protein translation
.
RNA nucleotides contain nitrogenous bases (adenine, guanine, uracil, or cytosine)
arranged along a single strand.
The team also found that different strains of the virus exhibited differently
in the sequence of nitrogenous bases in their nucleotides.
"Some strains may have a much
higher degree of methylation than others.
If so, they can proliferate
better within the host cell.
”

They also found that the nucleotide sequence, known as the m6A DRACH motif, differed
slightly in SARS-CoV-2 and cells.
In this acronym commonly used in epigenetics, the letter D stands for adenine, guanine, or uracil; R stands for adenine or guanine; A is a methylation residue; C is cytosine; H stands for adenine, cytosine, or uracil
.
Viruses use cellular enzymes for their own methylation, creating evolutionary pressure for adaptation of viral DRACH sequences, bringing them closer to cell sequences
.
The best-adapted strains are more successful in escaping interferon
.

After completing the study of how SARS-CoV-2 modifies m6A in host cells, the scientists' next step will be to analyze the stored data to look for correlations between viral RNA methylation levels and the amount of virus released by each infected cell, i.
e.
, the size of the viral outbreak
.

"The higher the degree of methylation of viruses, the more they grow in the cytoplasm and the larger the outbreak
," Briones explains.
Under normal circumstances, without stimulation, virions can replicate a thousand times
.
These findings pave the way
for new treatments for COVID and the repurposing of existing drugs.
They also provide elements
for a deeper understanding of how virus strains escape the immune system.
”

method

According to the researchers, the nanopore direct RNA sequencing method used in the study has several advantages
.
One of them is that it does not require the modifications required by traditional methods (reverse transcription polymerase chain reaction, or RT-PCR) to read RNA strands
.

To detect a virus with RT-PCR, scientists must first convert its RNA into DNA (reverse transcription).

The result is cDNA, where "c" stands for complementarity
.
This is because only DNA (double strands) can be copied
.
This cDNA is then copied hundreds of thousands of times, producing billions of clones, leaving enough target fragments of viral DNA to analyze, rather than just a tiny fraction.

For Briones, researchers may be bewildered
by the distortion that produces viral sequences from cDNA.
"Some scientists believe that the conversion of nucleotides is due to the presence of
epigenetically modified bases.
This needs to be investigated
in a systematic manner.
”

Two m6A assay procedures reflect an increase
in cell methylation.
One of them, m6anet, uses a machine learning technique
called multi-instance learning (MIL).
The other group (EpiNano) verified the results
using a technique called support vector machines (SVMs).

The study is part of the thematic project (Study of Host Factor Response Induced in ChAdOx1 nCOV-19 Vaccine Immunophase III Clinical Trial) led by Luiz Mário Janini, penultimate author
of this article.