1D). This partial RING domain is insufficient to confer E3 ubiquitin ligase activity on viral Pellino since a recombinant form of the latter failed to catalyse the in vitro generation of polyubiquitin chains in the presence of E1 and E2 enzymes, whereas the mammalian member Pellino3S shows strong catalytic activity (Fig.
1E). Western immunoblotting using an anti-myc Tofacitinib chemical structure antibody shows that the lack of activity of viral Pellino relative to Pellino3 cannot be attributed to differences in protein quantity since both proteins show comparable levels of immunoreactivity. Interestingly, viral Pellino has a mobility corresponding to its predicted size of 25.4 kDa but it also shows a fainter immunoreactive band of slower electrophoretic mobility. The identity of this protein is unknown but its lack of reactivity with the anti-ubiquitin
antibody excludes selleck chemical the possibility of the protein being modified by ubiquitination. The above analysis suggests that viral Pellino resembles its mammalian counterparts in containing a core FHA domain but differs in lacking both a wing appendage to the FHA domain and a functional RING-like motif. The emerging roles of Pellino proteins in TLR signalling coupled to the discovery of a viral homolog prompted studies on the ability of viral Pellino to regulate TLR signal transduction. Viral Pellino is encoded by the genome of MsEPV and given that the natural host of MsEPV is insect cells, the highly AT-rich sequence of the viral Pellino gene reflects an adaptation to this environment. In order
to ensure expression of viral Pellino in both insect and human cells, a form of the gene was chemically synthesised with codon sequences optimised for recognition by human translation machinery. This involved replacing As or Ts in the third position of each codon with a G or C, without altering the amino acid sequence of the translated protein. Such an approach was previously shown to enhance expression of poxviral genes in human cells 24. We initially Thiamine-diphosphate kinase assessed the effects of viral Pellino on Toll signalling in macrophage-like Drosophila S2 cells. A myc-tagged version of the viral protein showed uniform cytoplasmic distribution after transfection in these cells (Fig. 2A). The effects of increasing levels of viral Pellino expression on signalling by the Toll ligand C-106 was then assessed (Fig. 2B). C-106 is the active C-terminal fragment of the Spätzle protein and induced activation of a firefly luciferase reporter under the control of the drosomycin promoter. Toll signalling can induce expression of this antimicrobial peptide through the Rel family transactivators Dorsal and Dif. Thus, the activation of the drosomycin promoter was an especially relevant readout for Toll signalling in the present studies in light of the demonstration that Drosophila Pellino plays a key role in driving expression of drosomycin 13.