A conserved alphaherpesvirus UL20 homolog is required for cytoplasmic virion maturation and secondary envelopment of Marek's disease virus.

Abstract

Marek's disease virus (MDV), an oncogenic alphaherpesvirus, induces severe immunosuppression and T-cell lymphomas in chickens, posing a major threat to poultry production. Viral morphogenesis in alphaherpesviruses depends on coordinated membrane remodeling processes mediated by conserved viral membrane proteins. Although MDV encodes a homolog of UL20 protein, its functional role remains unknown. In this study, we employed an integrated approach combining bioinformatic analysis, bacterial artificial chromosome (BAC)-based mutagenesis, and ultrastructural characterization to elucidate the role of MDV UL20 in viral replication and morphogenesis. Sequence analysis revealed that MDV UL20 is a conserved four-pass transmembrane protein with a membrane topology similar to that of UL20 homologs in other alphaherpesviruses. To assess its functional relevance, a UL20 deletion mutant (Md5BACΔUL20) was generated, and its biological properties were evaluated in chicken embryonic fibroblasts. Deletion of UL20 completely abolished the production of infectious virus, resulting in the absence of plaque formation and impaired cell-to-cell spread. In contrast, genetic reconstitution of UL20 fully restored viral replication to wild-type levels. Transmission electron microscopy demonstrated that UL20 deletion did not affect nuclear capsid assembly but markedly reduced the number of cytoplasmic capsids, secondary envelopment intermediates, and extracellular virion accumulation. UL20 re-expression assays confirmed that these defects were specifically attributable to the loss of UL20. Collectively, these findings demonstrate that MDV UL20 is dispensable for nuclear capsid formation but essential for cytoplasmic secondary envelopment and infectious virion production, providing new insights into the membrane-associated mechanisms underlying MDV morphogenesis.