HIRI and JMU researchers visualize the fight between the virus and the host cell

Cells are equipped with effective defense mechanisms to fight intruders. The battle plan and construction is written in the genes that must be activated during an enemy attack. Scientists from the Helmholtz Institute for Research on RNA Infections (HIRI) in Würzburg, a site of the Braunschweig Helmholtz Center for Infection Research (HZI), and the Julius-Maximilians-Universität Würzburg have put to the point a new method called scSLAM-seq, with which study the activity of thousands of genes in individual cells and follow them precisely for a few hours. For the first time, researchers have been able to explain why some cells are successfully infected with a virus while others are not. In addition, they have gained fundamentally new knowledge about gene regulation. Their results are published in the latest issue of the journal Nature.

If viruses invade our body – such as during a flu or a gastrointestinal infection – the processes in the affected cells change: in the worst case, the virus takes over and the cell is reprogrammed. It then produces viral components and the intruder multiplies explosively. However, in another cell, the virus can lose and is successfully removed by the activated protection mechanisms. But how come one cell is saturated and the other controls the virus? How fast do individual cells respond to a virus attack and what protective genes are activated? "Until now, little was known about these issues at the cell level," says Dr. Antoine-Emmanuel Saliba of the Helmholtz Institute for Research on RNA-related Infections ( HIRI). "With our current study, we have made great progress in answering these questions."

In close collaboration with Professor Florian Erhard from the Institute of Virology and Immunobiology of the Julius-Maximilians University of Würzburg, the HIRI research team around Saliba and Professor Lars Dölken , working in both institutes, have studied the link between gene activity and identity. and reflects the physiological state of a cell – impaired after infection with cytomegalovirus within individual infected mouse cells. Cytomegaloviruses are widespread, over 80% of people are infected. It can usually be healthy, but it can be dangerous for newborns and transplant patients, with serious neurological consequences. With a frequently used experimental procedure, called single-cell RNA sequencing (scRNAseq), it is possible to determine which genes in a cell are currently active. Short-term changes in gene activity, such as those that occur during viral infections, can only be detected to a very limited extent. In addition, each cell can be examined only once. Thus, we still do not know how cells react to external influences, for example to a viral infection. To study the molecular processes in infected cells, researchers have developed a new method called scSLAM-seq, which allows them to visualize for the first time the genes activated in individual cells within hours. When a gene is activated, its code is translated into RNA (English: ribonucleic acid). RNA consists of four different building blocks, the organic bases adenine, cytosine, guanine and uracil. These are chained as on a chain, in a characteristic combination of the original gene. The RNA chain then serves as a template for the production of a protein that fulfills a specific function in the cell.

scSLAM-seq sorts RNA in old and new

In order to be able to distinguish which RNA was already present before the viral infection and which had been recently added, the researchers used a marker trick: they simultaneously added to the infecting virus a chemically slightly modified form of the uracil RNA component in the nutrient medium compared to the natural variant. add to the cells. The cells then incorporated labeled uracil in their newly prepared RNA. After two hours, the experiment was stopped. A chemical reaction transformed the labeled uracil into another constitutive block of RNA, cytosine. "The RNA sequence is exactly where uracil should be, followed by cytosine," says Dölken. "The underlying idea is that RNA produced after viral infection now carries a label that allows us to identify it as new in the subsequent sequencing of RNA." " With the help of a complex bioinformatic technique, the researchers studied the RNA of each individual. Cell, they assigned more than 4000 known genes per cell and separated them into new and old RNAs. "The data we can collect with the scSLAM seq procedure is spectacular," says Erhard. "We can actually determine for each individual cell the percentage of its RNA that will be reproduced within two hours of a disorder, in this case a viral infection." For the first time, dose-response analyzes at a cell level are possible. "Overall, the research team studied the RNA of 100 unique cells. "It was already enough to have a completely new vision of the activation of cellular genes," says Saliba. "With scSLAM-seq, we can now analyze for the first time accurately the behavior of an individual cell in a short period of time in the event of a disturbance such as a virus or bacterial infection, which genes are increased or diminished in the sequence, and thus understand what plan of battle it is in the fight against the intruder ".

Each cell is different

In addition, scientists have been able to show that the reading of genes is not done continuously, but in bursts (English: bursts): thus, the viral infection causes hundreds of genes of their sleep and causes them to read a few hours after the entry of the virus into the cell. In particular, these activated genes help our cells fight infection. "In our analyzes, many cell genes exhibited pronounced burst behavior.Either in a cell, almost all the RNA of a gene was new or old," says Erhard. "And we have been able to show that burst behavior depends on the structure of certain regions of the gene sequence – what are called promoters – that control the reading of genes." This is not upregulated or downward, but occurs in every cell after an on / off principle. Saliba: "This also explains why the RNA profiles of cells often differ so clearly and that some cells can attack viruses immediately, for example, and others at a time." Each cell runs at its own pace initially identical RNA profiles, after a few days, completely different RNAs inside their cell. Dölken: "Thanks to our research, we were able to acquire new fundamental knowledge about the timing of cell gene activation, which completely changed my understanding of activation."

Protection against autoimmune diseases

The principle of activation / deactivation of cellular genes probably has a very important function for our body. If all the genes used to fight viruses were permanently produced by all the cells of the body, this could lead to false reactions and autoimmune diseases. "Lighting at the right time, according to the slogan" Walk the water! "Our immune system can create a protective environment without the risk of causing false reactions," but it only works if it is hot, "says Dölken, so in a small part of the body's cells, some mechanisms are fully operational. These "sentinel" cells are then able to recognize, for example, an invading virus and fight it effectively, and they inform the other cells, which also start the entire defense arsenal and activate the corresponding genes to control the infection. and ward off danger.

Thanks to the patent-pending bioinformatics analysis procedure required to evaluate the extremely complex experimental data, researchers in Würzburg were able to obtain fundamental information on cellular processes. "For the first time, we can really see how one cell reacts," says Dölken. In future surveys, it can be used to answer a wide variety of questions at the cell level. "ScSLAM-seq is ideal for knock-out studies to determine which genes play a key role in controlling pathogens or triggering a disease," says Saliba. "It's methodically simple, the data quality is high and ideal for dose-response studies, even over time." The research group is therefore confident: it will be exciting!

The Helmholtz Center for Infection Research:
At the Helmholtz Center for Infection Research (HZI), scientists are studying the mechanisms of infection and their defense. What makes bacteria or viruses pathogenic: Understanding this is the key to developing new drugs and vaccines. HZI is a member of the German Infection Research Center (DZIF).

Helmholtz Institute for Research on RNA Infections:
The Helmholtz Institute for Research on RNA Infections (HIRI) was founded in May 2017 as a joint institution of the Braunschweig Helmholtz Infection Research Center (HZI) and the Julius-Maximilians- Universität Würzburg (JMU). Based at the Würzburg University Hospital campus, HIRI will be the first institute of its kind in the world dedicated to the role of ribonucleic acids (RNAs) in infection processes. On the basis of these results, new therapeutic approaches will be developed in an integrative research approach and made clinically applicable by the development of pharmaceutical applications.

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Original publication:
Florian Erhard, Marisa AP Baptista, Tobias Krammer, Thomas Hennig, Marius Lange, Panagiota Arampatzi, Christopher Jürges, Fabian J. Theis, Antoine-Emmanuel Saliba and Lars Dölken: scSLAM-seq reveals the fundamental characteristics of the dynamics of transcription in single cells. Nature 2019, doi: 10.1038 / s41586-019-1369-y

idw 2019/07