The role of ORF9p in VZV-induced cell-cell fusion

VZV infection is characterized by the formation of syncytia resulting principally from the fusion of neighboring infected cells, more rarely from the fusion of infected cells with uninfected ones. One naturally occuring hyperfusogenic strain (VZV-MSP), discovered in the late nineties exhibits a single-point mutation on gE. Exaggerated cell-cell fusion is also observed with laboratory-engineered strains in which gH cytoplasmic strain is deleted or containing mutations within gB cytoplasmic domain. These VZV viral strains poorly replicates in skin models. On the other hand, to our knowledge, spontaneous non-fusogenic VZV strains are not described in the literature and the generation of gH mutant with cell fusion defect can lead to the appearance of compensatory mutations. Cell-cell fusion regulation seems then to be crucial to maintain viral fitness.
We previously identified on ORF9p an acidic dileucine motif (E227GLNLI232) important for its interaction with the cellular adaptor protein complex-1 (AP-1), an heterotetramer regulating the shuttling of cargo proteins between endosomes and the TGN. Disrupting ORF9p binding to AP-1 by mutating leucine 231 to alanine strongly impacts viral growth but also completely abolishes cell-cell fusion. Strikingly, after about 15-20 passages, the phenotype spontaneously reverts to an apparent WT cell-cell fusion efficiency. This prompted us to sequence the whole VZV genome of nine spontaneous revertants. Five had a point mutation introducing a stop codon within gH cytoplasmic tail, removing the last seven amino acids, the region already known to regulate cell fusion. Two had a two distinct point mutation on gE, one in the extracellular domain (D436H) and one on the cytoplasmic tail (D620Y). The last two revertants had both the gHΔcyt and the gE-D620Y mutations. We now are trying to understand the role of ORF9p in the maintenance of the gB/gH/gL complex and the role of the gE/gI complex in the regulation of cell-cell fusion.