Structure of a coronavirus. Credit: Wikimedia
A brand new research describes how the functioning of RNA changes in cells infected by SARS-CoV-2, the COVID-19 virus. The findings provide clues as to how different variants can escape the immune system, and serve as a basis for the development of novel treatments.
For the first time, scientists have shown that infection by SARS-CoV-2, the virus that causes COVID-19, changes the functioning of host cell RNA. The researchers, from the Federal University of São Paulo (UNIFESP) in Brazil, arrived at this conclusion by analyzing 13 datasets obtained during four studies of viral, human, and animal cell RNA.
The most recent study, reported in an article published in the journal Frontiers in Cellular and Infection Microbiology, examined the epitranscriptome of Vero cells (derived from monkeys) and human Calu-3 cells by direct RNA sequencing. An epitranscriptome is the collection of biochemical modifications of cell RNA, such as methylation.
“Our first important finding in this study was that infection by SARS-CoV-2 increases the level of m6a [N6-methyladenosine], a sort of methylation, in host cells in contrast with non-infected cells,” Marcelo Briones, final creator of the article, informed Agência FAPESP. Briones is a professor at UNIFESP’s Medical School (EPM) and a researcher affiliated with its Center for Medical Bioinformatics.
Methylation is a biochemical modification involving the addition of a methyl group to a substrate. It happens in cells through the motion of enzymes able to transferring a part of one molecule to a different. This adjustments the habits of proteins, enzymes, hormones, and genes. The researchers demonstrated adjustments to contaminated cell RNA quantitatively by analyzing all of the RNAs current within the cells and qualitatively by finding on a map the variety of methylations per area within the nucleotides.
The research was a continuation of an earlier genomic analysis revealed in 2021, the place the researchers analyzed the methylation sample in SARS-CoV-2.
“Methylation has two functions in viruses. It regulates protein expression, and it defends the virus against the action of interferon, a potent antiviral substance produced by the host organism,” Briones mentioned.
In each research, the researchers analyzed m6a as a result of it’s the commonest kind of RNA nucleotide modification and is concerned in a number of important processes, resembling intracellular location and protein translation. RNA nucleotides include nitrogenous bases (adenine, guanine, uracil, or cytosine) operating alongside a single strand. The workforce additionally found that completely different strains of the virus displayed variations within the sequences of nitrogenous bases of their nucleotides. “Some strains may be much more methylated than others. If so, they can proliferate better inside host cells,” Briones mentioned.
They additionally discovered that nucleotide sequences often known as m6a DRACH motifs had been barely completely different in SARS-CoV-2 and in cells. In this acronym, which is incessantly utilized in epigenetics, the letter D stands for adenine, guanine, or uracil; R for adenine or guanine; A for the methylated residue; C for cytosine; and H for adenine, cytosine or uracil.
The virus makes use of cell enzymes for its personal methylation, producing evolutionary strain for adaptation of viral DRACH sequences in order that they develop into extra just like cell sequences. The viral strains that adapt finest are capable of escape interferon extra efficiently.
After finishing their investigation of how SARS-CoV-2 modifies m6A in host cells, the scientists’ subsequent step will likely be to research the saved knowledge seeking a correlation between viral RNA methylation ranges and the variety of viruses launched from every contaminated cell, often known as viral burst dimension.
“The more methylated the viruses, the more they grow in the cell cytoplasm and the larger the burst size,” Briones defined. Under regular circumstances, with out stimuli, a viral particle replicates a thousand occasions. “The findings pave the way to novel treatments for COVID-19 and repurposing of known drugs.” They additionally supply components for a deeper understanding of how viral strains escape the immune system.
Methodology
The Nanopore direct RNA sequencing technique (Oxford Nanopore Technologies) used within the research has a number of benefits, in response to the researchers. One of those is that it dispenses with the modifications required by the standard technique (reverse transcription polymerase chain response, or RT-PCR) to learn the RNA strand.
To look at a virus utilizing RT–PCR, scientists should first convert its RNA to DNA (reverse transcription). The result is cDNA, where the ‘c’ stands for complementary. This is because only DNA (which is double-stranded) can be copied. The cDNA is then amplified by being copied hundreds of thousands of times, creating billions of clones so that enough of the target sections of viral DNA are available for analysis, instead of a minuscule amount.
For Briones, researchers may be confused by distortions resulting from the production of viral sequences from cDNA. “Some scientists think nucleotides are switched owing to the presence of epigenetically modified bases. This needs to be investigated in a systematic manner,” he said.
The increase in cell methylation was mapped by two m6A detection programs. One of these (m6anet) used a machine learning technique called multiple instance learning (MIL). The other (EpiNano) validated the results using a technique called support vector machine (SVM).
Reference: “The epitranscriptome of Vero cells infected with SARS-CoV-2 assessed by direct RNA sequencing reveals m6A pattern changes and DRACH motif biases in viral and cellular RNAs” by João H. C. Campos, Gustavo V. Alves, Juliana T. Maricato, Carla T. Braconi, Fernando M. Antoneli, Luiz Mario R. Janini and Marcelo R. S. Briones, 16 August 2022, Frontiers in Cellular and Infection Microbiology.
DOI: 10.3389/fcimb.2022.906578
The study was part of a Thematic Project (“Investigation of induced host elements in response to immunization with ChAdOx1 nCOV-19 vaccine in a Phase III clinical trial”), for which the principal investigator is Luiz Mário Janini, penultimate author of the article.
The research team also included Juliana Maricato, Carla Braconi, Fernando Antoneli, João H. C. Campos, first author of the article supported by a postdoctoral fellowship from FAPESP, and Gustavo V. Alves, second author and an undergraduate in health information technology.
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