Identification of host proteins which interact with vaccinia virus encoded E3L
thesisposted on 15.12.2014, 10:33 authored by Sarah J. Rogan
The E3L protein is one of a number of proteins encoded by Vaccinia Virus (VV) which antagonise the IFN cellular response. E3L is a dsRNA binding protein which sequesters dsRNA that would ordinarily activate PKR. Therefore, within a VV infected system, protein translation is maintained. Both PKR and E3L are members of a family of proteins of diverse function which interact with dsRNA via a dsRNA binding motif (dsRBM). The amino acid sequences of these dsRBMs are highly conserved and probably form a conserved structural motif.;One of the aims of my PhD project was to determine the structure of a dsRBM by NMR spectroscopy. Studies involved the over-expression of the dsRBM region of PKR, Met1-170 and three constructs of E3L, Metl-190, Met38-190 and Met94-190, using an E. coli based expression system. The proteins were expressed and purified to homogeneity, however, structural analysis of these constructs was not possible as the maximum concentration of the proteins was insufficient for NMR analysis. The self association of E3L Met94-190 was also investigated. The protein was found to form dimers at a concentration up to 3.4 mg/ml as determined by analytical ultra centrifugation and gel filtration, however, crosslinking analysis indicated the protein was able to form multimers.;A yeast-two-hybrid screen was undertaken to identify cellular proteins that interact with E3L. Three proteins were identified, PIC-1, a nuclear protein, H3.3 a histone protein and L23a, a ribosomal protein. All three of the proteins were found to interact with the dsRBM or E3L. Attempts to demonstrate biochemical interactions between E3L and PIC-1 and H3.3 were unsuccessful.