Identification of a surface-accessible tegument protein bearing a neutralizing epitope in varicella-zoster virus

Varicella-Zoster Virus (VZV) causes primary varicella and can reactivate later in life to induce herpes zoster with severe complications, posing a persistent threat to human health. Previous studies of VZV immunogens have focused primarily on the major envelope glycoproteins (e.g., gE, gH, and gB), whereas other potential immunogenic components remain unexplored. To broaden the repertoire of anti-VZV targets, we established a biotin-avidin membrane-protein capture strategy to screen for surface-accessible viral antigens that elicit neutralizing antibodies. Using this approach, we identified the conserved Alphaherpesvirinae-specific tegument protein pORF9 as a previously unrecognized surface-accessible antigen bearing a neutralizing epitope, which is unexpected because tegument proteins are generally considered internal virion components. Immunofluorescence, flow cytometry, and cell fractionation analysis showed that pORF9 undergoes late-stage plasma membrane association and surface accessibility, with about 30% of infected cells becoming surface positive for pORF9 by 72 hpi; this phenotype was not observed for the non-essential homolog VP22 from herpes simplex virus type 1. We mapped the neutralizing epitope to amino acid residues 111-125, which are highly conserved among 136 VZV strains spanning five phylogenetic clades, and confirmed key antibody-binding residues by alanine scanning. Moreover, chimeric hepatitis B core virus-like particles displaying this epitope induced pORF9-specific neutralizing antibodies in mice. Collectively, these findings refine current views of VZV tegument protein localization and immunological function, establish pORF9 as a dual B- and T-cell target, and support further exploration of pORF9 as a potential target for anti-VZV interventions.