Nucleocytoplasmic shuttling of the herpesvirus tegument protein UL47_TEG5 is required for its functional role in horizontal transmission in chickens

Abstract

Marek’s 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 (vΔ47) or the NES2 motif (aa 664-678) of UL47_TEG5 (v47ΔNES2) 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 vΔ47 and v47ΔNES2 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. AUTHOR SUMMARY Marek’s disease virus (MDV) causes a highly contagious cancer in chickens and spreads efficiently from bird to bird. The viral protein UL47_TEG5 is essential for this horizontal transmission. We discovered that UL47_TEG5 constantly shuttles between the nucleus and cytoplasm, a process controlled by specific export signals (NES). Using molecular techniques, we deleted either NES motif, which trapped the protein in the cytoplasm and prevented shuttling. When we removed one key export signal (NES2) from the virus and infected chickens, the mutant virus replicated but completely failed to transmit to new birds. Our results show that UL47_TEG5’s ability to move between the nucleus and cytoplasm is critical for MDV transmission in chickens, likely through its function in virion assembly. These findings show that nucleocytoplasmic shuttling of UL47_TEG5 is mediated through a non-canonical mechanism and highlight UL47_TEG5 as a potential target for improved vaccines to better control this economically important disease.