The article is part of the Science section, research – our chance.
The Covid-19 pandemic has caused a panic of unprecedented dimensions worldwide and has paralyzed the normal functioning of each of us for several years.
In addition to intense efforts to develop sensitive diagnostic methods for virus detection in the population, the issue of the biological interaction between the virus and the cell was also examined.
For many, the notion of investigating this interaction can act as science-fiction, because to track these processes in real time on living cells was until recently something like looking for a needle in a haystack … with a light-off light.
What does the virus actually consist of?
Imagine the virus as a sophisticated puzzle. Each piece is an important part without which the viral particle is not working properly.
The functionality of the virus depends on the correct composition of the individual components into the fully functional viral particle. In brief, SARS-CO-2 virus consists of four structural proteins (S, E, M and N), 16 non-structural proteins (NsP-1 to NsP-16) and several auxiliary proteins.
Only by combining all these components will create a full -fledged virus that can recognize the appropriate cellular receptor, transfer its genetic information to the cell, use its proteosynthetic apparatus and multiply effectively.
One virus, 2 roads
To understand the way the virus enters the cell, we will describe two basic mechanisms that SARS-CO-2 uses to transfer its genetic information.
In order for the virus to consider the cell to be a suitable goal, it must first recognize the ACE2 receptor (angiotensin converting enzyme 2) on its surface. An interesting process begins here.
Without unnecessary biochemistry details, we can summarize this as follows: If the cell does not have an active transmembrane seic prostéaz 2 (TMPRSS2, an enzyme that cleaves proteins) or if this protease is insufficiently active, the virus penetrates directly into the cell process that is known as endocytosis. Endocytosis is a process in which the cell receives substances from the environment by surrounding them with part of its membrane and draws inside into the cytoplasm (the internal environment of the cell). Following the entrance to the cell, a RNA viral is released, which triggers the virus replication process in the intracellular space.
In the latter case, entering the cell (in the presence of TMPRSS2 on the surface) the process is slightly different. In this process, the ability to split viral with protein through TMPRSS2 protease is applied. As a result, this will cause the virus membrane to merge with the cell membrane and the release of the viral RNA into the host cell cytoplasm.
Monitoring of a virus in a living cell
The above -mentioned interaction of the virus with the cell, as well as many others, are unique and help us better understand the biology of the virus itself. But are there technologies that allow us to follow these processes in real time? The answer is clearly yes. One option is the formation of fluorescently marked infectious viruses.
But is working with infectious pathogens always ideal? There is no clear answer to this question. Working with infectious materials requires special spaces and strict protocols. Normally, laboratories certification at BSL-3 level is required, which is not available in every scientific facility, and not at all in Slovakia.
Therefore, it is more effective to use systems that make it possible to monitor the interactions of the virus-cell without the need to build special laboratories, which is both economically and technically demanding. The solution to these problems is the use of virus -like viral particles (VLP), which allow safe and reliable study of these interactions.
Is it a virus or is it not a virus?
VLP particles are structures that mimic viruses but do not contain viral genetic material. This is the main reason why they cannot replicate as real viruses and are therefore non -infectious.
What can be studied using these structures? In addition to the solution of the interaction of the virus-cell, these particles can also be applied in other areas of medicine, including the development of vaccines, drug delivery directly to cells or gene therapy.
Lighting Coronavirus: How to brighten SARS-COC-2 under a microscope
As already mentioned, the virus is a comprehensive puzzle in which each piece has its own role. SARS-Cov-2 form, among other things, four basic proteins-S, E, M and N-whose presence is crucial for the assembly of SARS-cove-2 VLP.
Real art and creativity are to connect the fluorescent brand to these basic proteins. This simple modification allows us to visualize VLP particles in real time using fluorescent microscopy.
But how to create these modified proteins? Theoretically, it is demanding and practically (unfortunately) even more. In principle, it is necessary to use methods of molecular biology, which means preparing a circular DNA (so -called plasmide), which carries information about the desired protein of the virus. This “carrier” of genetic information contains in addition to the gene itself but also an additional sequence for the fluorescent brand. This will allow the viral protein to shine in the fluorescent microscope, thus directly monitoring it.
These plasmids, which contain genes for all basic proteins (some even with a fluorescent brand), are placed in suitable cells used to produce viruses. These cells subsequently produce the required fluorescently marked VLP particles.
It shines, it recognizes the right receptor. Okay, but ….
Creating SARS-COC-2 VLP, which recognize the right receptor, enter the cell through endocytosis and illuminate in a fluorescent microscope, although it is crucial, it is also important to study the interaction between the virus and the cell to choose the optimal cell model.
Many human body cells contain the ACE2 receptor on their surface, which is essential for virus entry (or in our case VLP) into the cell. Cells of the heart, kidneys, intestines and especially the lungs have a large number of this receptor. But how to choose the right model?
The ideal model should be available, easy to maintain and immortal. Therefore, cancer cells are a suitable candidate as they meet all these requirements. Lines of colorectal cancer, pulmonary adenocarcinoma and hepatocellular carcinoma have been shown to be optimal cells to study SARS-CO-2 interaction.
We have the choice of the appropriate model. What next? Modern gene manipulation methods allow us to improve these model systems. One of the most effective tools that science uses for this purpose is lentiviruses.
As viruses to use in our favor
With the method of gene engineering, we can create compound (compound) lentiviruses, which are the most common viral vector for effective introduction of genes into mammal cells. The main advantage of this approach is that it gives us the ability to integrate a foreign gene directly into the genome of the host cell, thus gaining the opportunity to use this gene to study the interactions between the virus and the cell.
In our study, aimed at analyzing this interaction, we modified the hepatocellular carcinoma line. In such a way, we put a gene for the production of small fluorescent proteins, so -called. nanobody, which were marked with a red mark.
These nanobody are specifically binding to a modified N protein, which is also fluorescently labeled, but this time green, and is located in our VLP particles. The clever solution of this bond is that N protein contains, in addition to information about the protein itself and the florescent signal, additional information about the so -called. Alpha-Tag. Alpha-Tag is a short sequence of amino acid added to a protein that serves as a mark.
This alpha-Tag acts as a high-specific puzzle piece, which is only linked to another specific piece-in our case nanobody. The result is a combination of both proteins in the cell, and can be monitored thanks to different fluorescent colors through fluorescent microscopy. This approach allows us to analyze in detail the time-spatial dynamics of SARS-CO-2 VLP into the cell.
What awaits us in the coming years in the field of study of the interaction of the Virus-Bunka in the world and in Slovakia?
Until recently, the only possibility of visualization of viruses was electron microscopy. However, this approach has a fundamental disadvantage – the virus is killed, which makes it impossible to study viruses in living cells.
A breakthrough in this area has brought fluorescent technology in combination with the development of gene engineering, which allows you to directly observe the interaction between viruses and live host cells in real time.
From the perspective of a person who had the opportunity to participate in a similar project abroad, I can say that although we still have some reserves in Slovakia in Slovakia, we compensate for this shortcoming by highly qualified experts in demand worldwide. Maintaining and developing international cooperation thus allows Slovak science to participate in world research projects.
Funded by the Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA no. 1-0261-22.
RNDr. Marek Samec, PhD.
- Graduate of the Faculty of Science, Charles University in Bratislava in the field of molecular biology.
- During his thesis, he devoted himself to identifying new bacteriophages infecting opportunistic pathogenic strains of bacteria.
- Assistant assistant at the Institute of Medical Biology of the Jessenius Faculty of Medicine, Charles University in Martin.
- The co-researcher of the project aimed at analyzing the time-spatial dynamics of SARS-CO-2 VLP into the cell. Co-author of work focused on detecting SARS-COC-2 in wastewater methods of molecular biology.
- A graduate of a foreign internship in Montpellier in CNRS (Center National de la Recherche Scientifique), namely at the Institute Irim (Institut de Recherche en Infectiology de Montpellier). Within this internship, I have learned the latest practices and technologies in the field of virology and cell biology, which I am currently trying to apply in Slovakia.
