Jeffrey Mella and Brian Poole, MMBIO
My mentored research project was a terrific experience. I have been mentored with Dr. Brian Poole for the last three years. As a result of the project that was made possible by the ORCA grant, I was the first author on an abstract submitted and accepted by the International Immunology Summit. This last October I attended the International Immunology Summit in Las Vegas, Nevada. Dr. Poole and I presented our research and I responded to questions from the audience. I am currently an author on a manuscript that in progress that has been submitted for review. I have enjoyed my work on this project and I am grateful for the generous donations that made it possible.
My project involves a gene called EBI2. The name is an acronym for Epstein Barr Virus Induced G Protein Coupled Receptor 2. As the name implies, it is the most highly up regulated gene during EBV infection. It codes for a cellular receptor in the GPCR family. The receptor has a natural ligand called 7α,25-OHC, a polar lipid found both in lymphoid and non-lymphoid tissues. The interaction between the EBI2 receptor and the 7α,25- OHC ligand, plays a large role in the migration and differentiation of B cells inside lymphoid germinal centers. We wanted to determine whether or not the up regulation seen in EBV infection could be significantly altering B cell migration and could thus be important in viral latency.
Epstein-Barr Virus is thought to be present in 95 percent of the adult population. 40-50 percent of those infected during their teenage or early adult years develop infectious mononucleosis. This disease is characterized by swollen lymph glands, sore throat, high fever, and chronic fatigue. These symptoms usually resolve within 1-2 months. The virus, however, remains latent in select infected B-cells within the lymph nodes for life. EBV has been coo related with the pathogenesis of Burkitt’s lymphoma and nasopharyngeal carcinoma introduced by latent EBV. The ability of lymphocytes to properly home to targeted areas is crucial in viral infection, latency, and affects EBVinduced lymphomas. By studying EBI2, we gain critical understanding into how EBV regulates this important process, and can then apply that knowledge to combating EBV infection and tumorigenesis.
We first grew up stock of EBV that had recombinant DNA allowing them to express Green-Fluorescent Protein. We then isolated primary B cells from the peripheral blood of EBV-negative volunteers. The infected cells were then coinfected with EBI2 shRNA or control lentiviruses. Our cells exhibited distinct fluorescence profiles for each condition: uninfected-no fluorescence, EBV-infected-green fluorescence, EBI2 knockdown-red fluorescence, and EBV-infected/EBI2 knockdown-both green and red fluorescence. Unfortunately, after we had successfully infected our cells with our knockdown vectors, we discovered that the fluorescence we were using was not the proper wavelength to fit the spectrum of detection for the flow cytometer at BYU. We thus had to pause the migration assay until we order a different fluorescent vector.
With the migration assay on pause we continued our work with EBI2 to characterize the up regulation seen with EBV infection. Since EBI2 expression is highly up regulated post infection, it was of interest to determine the pattern of EBI2 expression during the course of EBV infection. To calculate this expression pattern, isolated naïve B cells were infected with EBV and EBI2 expression was measured at intervals over a 24 hour period of time. An expression time line was constructed (Fig. 4) starting with pre-infection and ending at 24 hours post infection. Between 3 and 6 hours post infection, there was a significant decrease in EBI2 expression (p < 0.005). There is also a significant increase in expression at 24 hours post infection observed when compared to the expression of EBI2 prior to infection as was previously observed (Fig 2B p < 0.05). During our study we have established that EBI2 is controlled during EBV infection of B cells. Different lytic cycle programs result in different levels of EBI2 expression. Higher levels of EBI2 mRNA expression result in higher levels of EBI2 protein. We have further established that an EBV gene induces EBI2. We are excited to continue our research on the migration assays and to corroborate that data with the timeline we have for B cell migration to synthesize a hypothesis about exactly how EBI2 is controlling EBV infection and latency.