Characterization of the Interaction Between Protein Kinase R (PKR) and Epstein- Barr Virus Inhibitor RNA (EBER1)

A variety of virus-based infectious diseases cause a host of problems for human health. Humans have developed a variety of defenses against viral infection, but unfortunately viruses have evolved clever and sophisticated ways to elude host cell defenses. The protein kinase R (PKR) plays a variety of important functions in a cell, but most importantly PKR is able to sense viral byproducts (dsRNA) and inhibit viral translation, leading to the death of virus-infected cells. However, viruses have found ways of preventing PKR from performing its function through a variety of methods. We looked at how the Epstein-Barr virus, which is one of the viruses that can inhibit PKR, produces a double-stranded RNA molecule (EBER1) that interacts with the PKR to inhibit it. We first had to clone a suitable plasmid containing the RNA molecule of interest. After transcribing the RNA and purifying it, we were able to run both Nuclear Magnetic Resonance tests (NMR) and gel shifts to examine the properties of the RNA. NMR experiments were able to confirm the basic sequence and structural composition of the RNA. The gel shifts showed that PKR, when introduced to the EBER-CS RNA, formed a specific protein/RNA complex, likely containing two protein and one RNA molecules. We also showed that both RNA binding domains of PKR were required to form a complex with the EBER-CS RNA. Lastly, the full length PKR and the dsRBD PKR act the same in their interaction with the EBER-CS RNA, indicating that dsRBD PKR is a useful construct to investigate. It is vital that, as a race, we are able to stay a few steps ahead of the viruses and prevent dangerous outbreaks; these novel results have important implications for continued studies in this field.