ORF9p (homologous to herpes simplex virus 1 [HSV-1] VP22) is a varicella-zoster virus (VZV) tegument
protein essential for viral replication. Even though its precise functions are far from being fully described, a role in the secondary envelopment of the virus has long been suggested. We performed a yeast two-hybrid screen to identify cellular
proteins interacting with ORF9p that might be important for this function. We found 31 ORF9p interaction partners, among which was AP1M1, the μ subunit of the
adaptor protein complex 1 (AP-1).
AP-1 is a heterotetramer involved in intracellular vesicle-mediated transport and regulates the shuttling of cargo
proteins between endosomes and the trans-Golgi network via clathrin-coated vesicles. We confirmed that
AP-1 interacts with ORF9p in infected cells and mapped potential interaction motifs within ORF9p. We generated VZV mutants in which each of these motifs was individually impaired and identified
leucine 231 in ORF9p to be critical for the interaction with
AP-1. Disrupting ORF9p binding to
AP-1 by mutating
leucine 231 to
alanine in ORF9p strongly impaired viral growth, most likely by preventing efficient secondary envelopment of the virus.
Leucine 231 is part of a dileucine motif conserved among alphaherpesviruses, and we showed that VP22 of
Marek's disease virus and HSV-2 also interacts with
AP-1. This indicates that the function of this interaction in secondary envelopment might be conserved as well.IMPORTANCE Herpesviruses are responsible for
infections that, especially in immunocompromised patients, can lead to severe complications, including neurological symptoms and
strokes. The constant emergence of viral strains resistant to classical
antivirals (mainly
acyclovir and its derivatives) pleads for the identification of new targets for future
antiviral treatments. Cellular adaptor
protein (AP) complexes have been implicated in the correct addressing of herpesvirus
glycoproteins in infected cells, and the discovery that a major constituent of the varicella-zoster virus tegument interacts with
AP-1 reveals a previously unsuspected role of this tegument
protein. Unraveling the complex mechanisms leading to virion production will certainly be an important step in the discovery of future therapeutic targets.