SARS-CoV2 virus intercellular transmission pathways

The aim of the project is to study the routes of rapid movement of coronaviruses between cells in an infected body. Viruses can infect sensitive cells from the outside through binding with receptors present on the cell surface and gradual penetration into its interior. Over there viral particles are opened, the genetic material (DNA or RNA) that is there is released reproduced in the process of replication, production of viral proteins and assembly of newly formed particles viruses, which then escape and infect other cells. However, some viruses can also penetrate neighboring cells without being released into the environment extracellular. For this purpose, they use the places of direct contact between their neighbors cells or various types of intercellular connections. Thanks to such a direct path transmission, viruses can spread more quickly and avoid contact with antibodies and cells immune system. For some enveloped viruses (e.g. HIV, measles and human leukemia virus) it has been shown that this type of latent cell-tocell spread can account for over 60% of infections. It is believed that targeted braking is direct the intercellular transmission of viruses creates new, significant therapeutic possibilities, especially in neurological diseases, and for some animal herpesviruses mutants Viral viruses lacking the genes necessary for rapid transmission have been used as vaccines for years. It has not yet been shown that coronaviruses can also be transmitted between cells via "simplified" high-speed transmission. However, given the huge efficiency of the infection SARS-CoV2 and the variety of organs - the lungs, but also the intestines, kidneys, heart - that can be affected infection, it can be assumed that this virus also uses this way to travel in the body. In previous counters, we examined in detail the mechanism of the "cell-to-cell" transmission process for herpesviruses and hepatitis C virus (HCV). We have shown that such a process more often it occurs in primary cells than in typical cultured cells, and we found that herpesviruses can travel between distant cells through long cells projections made of actin, the so-called nanotubes ("tunneling nanotubes"), specialized bridges that has been identified in recent years. Moreover, we have shown that the presence of the virus is stimulating the formation of such intercellular connections and that one of the kinases is involved in this process viral. In the planned doctoral project, we want to conduct similar research for SARS-CoV2 virus to search for hidden routes of virus transport between loved ones and distant cells. For this purpose, the process of coronavirus penetration into cells will be studied different types and rates of plaque formation (reflecting viral transmission efficiency). Using the techniques of confocal microscopy and immunofluorescence, we will search connections between infected and uninfected cells, checking that the virus can spread through such connections, in particular through long and thin nanotubes, or prefers direct cell contacts (similar to the "virological synapses" described for HIV). Another issue that we will be looking for an answer to is whether and how SARS-CoV-2 can spread between cells of different types and origins, e.g. between peripheral blood cells and epithelial cells. This way the virus could get through even to cells lacking the appropriate receptors (such as ACE2). For this type In the experiments, co-cultures based on the simultaneous cultivation of such cells will be used such as epithelial cells and lymphocytes or cells of neuronal origin. They will also remain for research 3D cultures (organoids) were used, in particular for the analysis of less known infections intestinal. By expressing individual SARS-CoV-2 genes, we will search for factors viral influencing changes in the cytoskeleton of the cell, leading to the formation of projections cells needed to transport the virus. Search will be an important stage in the work inhibitors of intercellular viral transmission that could have potential applications therapeutic.