The battle between virus and host cell observed live

Cells are equipped with effective defense mechanisms to combat intruders. Battle and building plans are written in the genes that must be activated when an enemy attack takes place. Scientists developed a new method to study the activity of thousands of genes in single cells.

Single-cell analysis allows a view into cellular processes

Cells are equipped with effective defense mechanisms to combat intruders. Battle and building plans are written in the genes that must be activated when an enemy attack takes place. Scientists developed a new method to study the activity of thousands of genes in single cells.

If viruses invade the body - for example in the case of influenza or a gastrointestinal infection - the processes in the affected cells change: In the worst case, the virus completely takes over the infected cell and its metabolism is reprogrammed. The cell then produces virus components and the virus multiplies explosively. In another cell, however, the virus may lose out and is successfully eliminated by the activated protective mechanisms. But how does it come to the phenomenon that one cell is overrun and the other brings the virus under control? How quickly do individual cells react to a virus attack and which protective genes become active?

To date, hardly anything has been known about these questions at the single-cell level. In a recent study, however, researchers were able to make considerable progress in answering these questions. They investigated how gene activity - which reflects the identity and physiological state of a cell - changed within individual infected mouse cells after infection with the cytomegalovirus (CMV).

Single-cell analysis in murine cell culture

Using a frequently-used experimental method, the so-called single-cell RNA sequencing (scRNAseq), it is possible to determine which genes of a cell are currently active. However, short-term changes in gene activity, such as those that occur in a viral infection, can only be detected to a very limited extent. In addition, each individual cell can only be examined once. Thus, it remains unclear how individual cells react to external influences, for example, a viral infection.

In order to investigate the molecular processes within individual infected cells, the researchers have now developed a new method called scSLAM-seq, which enables them for the first time to visualize which genes are activated how strongly in individual cells within a few hours. When a gene is activated, its code is translated into RNA.

scSLAM-seq sorts RNA

In order to be able to distinguish which RNA was already present before the virus infection and which was newly added, the researchers used a marker trick: At the same time as the infecting virus, they added a chemically slightly modified form of the RNA building block uracil to the culture medium of the cells, which was slightly different from the natural variant. The cells then incorporated the labeled uracil into their newly produced RNA. After two hours the experiment was finished. A chemical reaction was used to convert the labeled uracil into another RNA building block, cytosine.

The RNA sequence contains cytosine instead of where uracil should have been. The idea behind this is that the RNA produced after the virus infection now has a label with which the researchers can identify it as new during the subsequent RNA sequencing.

Using a complex bioinformatic procedure, the researchers examined the RNA of each individual cell, assigned more than 4000 known genes per cell and separated them into new and old RNAs. For the first time, dose-response analyses at the single-cell level are now possible. In total, the research team investigated the RNA of 100 individual cells. This was already sufficient to gain a completely new insight into cellular gene activation.

Using scSLAM-seq, the researchers were now able for the first time to precisely analyse how a single cell reacted within a short time window to a disorder such as a virus or bacterial infection, which genes were subsequently increased or decreased in readings, and thus understand which battle plan it had prepared in the fight against the intruder.

Every cell ticks differently

The scientists were also able to show that the reading of genes does not take place continuously, but in pulses: the virus infection awakens hundreds of genes from their hibernation and causes them to be read within hours of the virus penetrating the cell. In particular, genes are activated, helping the cells to fight the infection.  This also explains why cells often differ so considerably in their RNA profiles and why some cells are able to fight viruses immediately and others not at that point in time. Each cell ticks according to its own rhythm: cells with initially identical RNA profiles exhibit completely different RNAs in their cell interior after only a few days.

Protection against autoimmune diseases?

The On/Off principle of cellular gene activation probably has a very important function for our bodies. If all genes used in the fight against viruses were permanently produced by every body cell, this could lead to false reactions and autoimmune diseases. By switching it on at the right moment, our immune system can build a protective environment without the risk of harmful false reactions.

Only in a small part of the body cells are certain mechanisms fully functional. These "sentinel" cells are then able, for example, to detect an invading virus and combat it efficiently. And they inform the other cells, which then also start up the entire defense arsenal and activate the corresponding genes in order to control the infection and avert the danger.