Covid virus changes RNA from infected cells, study says

For the first time, scientists from Unifesp (Federal University of São Paulo) were able to show that infection with the SARS-CoV-2 virus, which causes Covid-19, changes the functioning pattern of cell RNAs. To do this, they examined 13 datasets obtained over four studies that analyzed viral RNA as well as that from animal and human cells.

The most recent of them, published in the journal Frontiers in Cellular and Infection Microbiology, examined the epitranscriptome of Vero cells (derived from monkeys) and the human Calu-3 lineage using a direct RNA sequencing technique. The epitranscriptome corresponds to the set of biochemical modifications of RNA (for example the addition of a methyl group to the molecule, a phenomenon known as methylation) within a cell.

“Our first important finding in this work is that SARS-CoV-2 infection increases the overall level of m6A-type methylation in the cell’s RNA pool. [N6-metiladenosina]compared to uninfected cells”, says Marcelo Briones, researcher at the Center for Medical Bioinformatics at Escola Paulista de Medicina (EPM-Unifesp) and coordinator of the investigation.

Methylation is a biochemical modification that occurs in the cell by the action of enzymes capable of transferring part of one molecule to another. This changes the behavior of proteins, enzymes, hormones and genes. The researchers demonstrated the changes in the RNA of infected cells in a quantitative way by analyzing the entire set of RNAs in them; and, in a qualitative way, by individually indicating on a map the number of methylations per base region of the nucleotides that make up the RNA of these cells.

The study is a continuation of work published in 2021, which analyzed the epigenome of the virus and showed the pattern of methylation in its RNA.

“In viruses, methylation has two functions: to regulate the expression of proteins and to defend the pathogen from the action of interferon, a potent antiviral substance manufactured by the host organism”, says Briones.

In both papers, the researchers analyzed the most common type of RNA nucleotide modification, m6A, which is involved in several crucial processes of RNAs, such as intracellular localization and the ability to produce proteins. Nucleotides are composed of four different nitrogenous bases (adenine, guanine, cytosine and uracil) distributed along the RNA strands found in each cell. The team also observed that different strains of the virus have variations in the sequence of nitrogenous bases that make up their nucleotides. “In this way, some strains can be better methylated than others and, thus, proliferate better within cells”, says Briones.

It was also observed that a nucleotide sequence known as “DRACH”, receptor for m6A methylation, is slightly different in the RNAs of SARS-CoV-2 compared to the RNAs of cells. In this acronym, often used in studies of the type, the letter D indicates the nitrogenous bases adenine, guanine or uracil; the letter R indicates adenine or guanine; the letter A is the methylated residue; the letter C corresponds to cytosine; and the letter H indicates adenine, cytosine or uracil.

As the virus uses the cell’s enzymes for its own methylation, this puts evolutionary pressure on viruses to adapt their “DRACH” sequences to be more cell-like. The viral strains that best make this adaptation will also be more efficient at evading interferon.

After completing the analysis of the action of SARS-CoV-2 on the pathogen-host binomial in relation to the m6A modification, the next step for the scientists will be to analyze the stored data to trace a correlation between the level of viral RNA methylation and the burst size of the virus. virus, that is, the viral multiplication quotient.

“The more methylated the virus is, the more it will grow in the cell cytoplasm and the greater its burst size”, explains Briones. In a normal situation, without stimuli, a viral particle replicates itself in a thousand others. “The findings open the perspective for new treatments for Covid-19 and the repositioning of known drugs”, says the researcher. In addition, it brings elements to a better understanding of the ability of sublines to escape the immune system.

Methodology

Unifesp scientists used a direct RNA sequencing method called Nanopore (Oxford Nanopore Technologies). One of the advantages of this choice, according to the researchers, is to avoid the modifications made to read the RNA strand by the conventional method, RT-PCR (reverse transcriptase polymerase).

To undergo an RT-PCR test, the RNA molecule is copied and converted into a complementary DNA, the cDNA. In this process, the molecule that previously had a single strand of nucleotides now has two strands. Then the cDNA molecules are amplified, generating billions of clones. In Briones’ view, as many labs are making the coronavirus sequences from cDNA, this can generate some biases and confuse researchers. “Some think that nucleotide exchanges actually occur because there was an epigenetically modified base there. This needs to be investigated in a systematic way”, says the researcher.

The global increase in methylation in cells was mapped by an m6A detection program known as m6anet, which uses multiple instance learning (MIL) machine learning technology. Then, the findings were validated by a second program, EpiNano, which uses the support vector machine (SVM) technique.

The study was conducted within the scope of the thematic project “Investigation of elements induced by the vaccine response in individuals undergoing clinical trials with the ChAdOx1 nCOV-19 vaccine”, coordinated by Professor Luiz Mário Janini. The researchers Juliana Maricato, Carla Braconi and Fernando Antoneli also participated. The first author, João HC Campos, is a Fapesp postdoctoral fellow. The study also had the participation, as the second author, of Gustavo V. Alves, graduating in health informatics technology.