5. Thema: Detection of viral proteins

Supervisor: Assel Nurbekova, Annica Preikschat
AG Tenzer, Institute for Immunologie, University Medical Center Mainz

Background of the project:

Viruses are tiny pathogens that can cause serious illnesses. One example are herpesviruses. Typically, every human is infected with at least one kind of herpesvirus throughout their lifetime. In contrast to other viruses, herpesviruses can never be fully eradicated by the immune system and therefore stay inside us for our whole life. One member of the herpesvirus (Herpesviridae) family is the cytomegalovirus (CMV). Our immune system can defend efficiently against CMV, so that we do not even realize a primary infection of CMV. However, CMV can evade immune system to some extent. Therefore, it is never fully eradicated from the body, resulting in balanced state called viral latency. This balance is disrupted when the immune system is suppressed by other causes, as it is the case for AIDS patients or patients that receive an organ transplantation, causing an active replication of a virus. In this project we investigate how CMV can avoid the immune system and, on the other hand, the mechanisms used by the immune system to keep the virus in check. To approach this, we determine changes of the host-cell proteome in the course of infection. Liquid chromatography- coupled to mass spectrometry (LC-MS) is a modern state-of the art instrument that identifies thousands of proteins in a single sample and is a powerful tool to characterize various protein complexes that exist in a cell. In this project we use such method to identify viral proteins in infected cells and understand how they interact with host cells over time.

Project description:

The Institutes of Virology and Immunology at the University Medical Center work together in a joint project to identify viral proteins and their host targets. Our aim is to better understand the interactions between virus and a host on a molecular level. In this project, we are infecting mouse fibroblast cells (NIH3T3) with a murine CMV at different time points to identify viral proteins and compare protein changes in infected host cells to non-infected cells. We will perform SDS-PAGE to visualize the proteins of whole cell lysates followed by immunoblotting to monitor/detect specific mCMV proteins in infected cells. Further, we will analyse changes in mCMV infected cells using a data set derived from mass spectrometric protein analysis.

Methods:

• Cell lysis and isolation of proteins
• Determination of the total protein content of a sample
• SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis)
• Coomassie Blue staining
• Western Blot
• Protein digestion for Mass Spectrometer
• Statistical evaluation of a proteome dataset derived from mass spectrometric analysis