Marek's disease virus UL16 tegument protein is essential for secondary envelopment and infectious virion formation

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus whose productive infection and cell-to-cell spread rely on the formation of infectious mature virions, a process mediated by tegument proteins. UL16 is a conserved herpesviral tegument protein, in other herpesviruses, its deletion causes severe replication defects. However, its role in MDV remains unknown. In this study, a UL16-null MDV mutant was generated using BAC-based reverse genetics to investigate UL16's function. Deletion of UL16 completely abrogated the recovery of infectious virus from BAC DNA in chicken embryonic fibroblasts, whereas genetic restoration or transient complementation of UL16 fully restored viral replication. The rescued viruses exhibited plaque morphology and growth kinetics indistinguishable from those of the parental virus. Transcriptional profiling showed that UL16 deletion did not significantly alter the expression of representative immediate-early, early, or late viral genes. In contrast, transmission electron microscopy revealed that although nuclear capsid assembly proceeded normally, cytoplasmic virion maturation was severely impaired in the absence of UL16, leading to a failure in secondary envelopment and the absence of fully enveloped virions. In summary, these results demonstrate that UL16 is indispensable for MDV replication in vitro and plays a critical role in the late stages of virion maturation, offering new insights into the molecular mechanisms governing MDV assembly.