Nucleocytoplasmic shuttling of the herpesvirus tegument protein UL47_TEG5 is required for its functional role in horizontal transmission in chickens
Mareks disease virus (MDV) UL47 tegument protein 5 (UL47_TEG5) is a major tegument protein essential for viral replication and horizontal transmission in chickens. The protein undergoes dynamic nucleocytoplasmic shuttling, likely regulated by host and viral factors, including post-translational modifications (PTMs). PTMs, in particular phosphorylation, on homologous herpesviral UL47_TEG5 proteins regulate nucleocytoplasmic shuttling through nuclear localization (NLS) and export (NES) motifs. Mass spectrometry-based proteomics of feather follicle epithelial skin from chickens infected with MDV recently showed that MDV UL47_TEG5 is phosphorylated. In silico analysis identified putative NLS and NES motifs near or within these phosphorylation sites. To test whether these motifs act as predicted, we systematically deleted the predicted NLS and NES motifs and assessed subcellular localization in transient transfection assays. Individual NLS deletions had minimal effect on nuclear localization, whereas NES deletions caused cytoplasmic accumulation and abolished nucleocytoplasmic shuttling. Next, we generated recombinant MDV lacking UL47_TEG5 (v{Delta}47) or the NES2 motif (aa 664-678) of UL47_TEG5 (v47{Delta}NES2) using two-step Red recombination. In experimentally infected chickens, replication and MD incidence were not different from those of wild-type MDV. However, in contact chickens, only wild-type MDV transmitted between birds, whereas both v{Delta}47 and v47{Delta}NES2 failed to transmit horizontally. These results demonstrate that nucleocytoplasmic shuttling of UL47_TEG5 is essential for horizontal transmission. In addition, our studies suggest that UL47_TEG5 expression, or lack thereof, in cells may be related to protein stability. Our findings link the nucleocytoplasmic shuttling of UL47_TEG5 to its essential function during natural infection and suggest this tegument protein may be a promising target for next-generation MDV vaccines.