New Research Reveals That COVID Virus Alters RNA in Infected Cells

Construction of a coronavirus. Credit score: 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 College (EPM) and a researcher affiliated with its Middle for Medical Bioinformatics. 

Methylation is a biochemical modification involving the addition of a methyl group to a substrate. It happens in cells by way of the motion of enzymes able to transferring a part of one molecule to a different. This modifications the conduct of proteins, enzymes, hormones, and genes. The researchers demonstrated modifications 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 features in viruses. It regulates protein expression, and it defends the virus towards the motion of interferon, a potent antiviral substance produced by the host organism,” Briones stated.

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 vital processes, comparable to intracellular location and protein translation. RNA nucleotides comprise nitrogenous bases (adenine, guanine, uracil, or cytosine) operating alongside a single strand. The workforce additionally found that totally different strains of the virus displayed variations within the sequences of nitrogenous bases of their nucleotides. “Some strains could also be rather more methylated than others. In that case, they will proliferate higher inside host cells,” Briones stated.

In addition they discovered that nucleotide sequences referred to as m6a DRACH motifs have been barely totally different in SARS-CoV-2 and in cells. On this acronym, which is ceaselessly 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 much like cell sequences. The viral strains that adapt greatest 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 might be to investigate the saved knowledge in quest of a correlation between viral RNA methylation ranges and the variety of viruses launched from every contaminated cell, referred to as viral burst measurement.

“The extra methylated the viruses, the extra they develop within the cell cytoplasm and the bigger the burst measurement,” Briones defined. Underneath regular circumstances, with out stimuli, a viral particle replicates a thousand instances. “The findings pave the way in which to novel therapies for COVID-19 and repurposing of identified medicine.” In addition they supply parts for a deeper understanding of how viral strains escape the immune system.

Methodology

The Nanopore direct RNA sequencing methodology (Oxford Nanopore Applied sciences) used within the research has a number of benefits, based on the researchers. One among these is that it dispenses with the modifications required by the traditional methodology (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.