Innate Immunity

The purpose of this project was to determine whether the Coxsackievirus B (CVB) uses toll-like receptors (TLR) to enter cells. In the signal transduction pathway of Toll-like Receptors, phosphorylation of the inhibitor protein IkB (by either of the IkB kinases (IKK)1 or IKK2) leads to the degradation of IkB. IkB is always associated with NF-kB, which is a transcription signaling molecule. When IkB is degraded, NF-kB is translocated into the nucleus, and ultimately causes cytokines, especially interleukin-8 (IL-8) to be synthesized and released. This project involved the creation of a DNA reporter plasmid that in the presence of free cellular NF-kB expresses the reporter protein dsRed-Express-1. This NF-kB reporter plasmid was transiently transfected into several different Human Embryonic Kidney (HEK 293) cell lines which were each stably transfected with different TLRs. Known ligands for these TLRs were used to test the specificity of the expression of the fluorescent signal. Once the system was shown to work well with positive control ligands, Coxsackievirus was used as a ligand, and it was shown that Coxsackievirus does indeed activate NF-kB, but not by the classic pathway, no IL8 synthesis was detected. So CVB does not appear to interact with any of the TLRs used in this specific HEK cell line, but it does not fully rule out an interaction between TLRs and CVB in other cells