VP22 is a tegument protein specific of alpha-herpesviruses encoded by the UL49 gene. Unlike most species from this subfamily, VP22 is essential for Marek’s disease virus (MDV) and Varicella-Zoster virus (VZV) replication. Several properties have been associated to MDV VP22, including virus cell-to-cell spread, DNA binding and cell cycle arrest. This 249aa-long protein contains three regions: a disordered N-terminus, a central conserved domain and a disordered C-terminus. To decipher the structure-function of VP22, we generated three series of recombinant MDV genomes harboring (i) orthologous UL49, from Varicelloviruses (VZV and PRV) and infectious laryngotracheitis virus (ILTV), in place of UL49 MDV, (ii) chimeric genes between UL49 from MDV and ILTV, by substituting one of the three regions of MDV by ILTV (r22IMM, r22MIM, r22MMI), or (iii) mutated MDV UL49 encoding truncated C-term VP22. All mutant viruses were assessed for spread in primary avian fibroblasts. The WT and mutated VP22 were also tested for their ability to arrest the cell cycle in S-phase, their sub-cellular location and their histones association after transient transfection in an avian cell line. We found that VP22 proteins and domains are not functionally homologous between alphaherpesviruses in term of MDV spread and cell cycle modulation, and this independently that the % in AA identity in the central domain. We showed that these two VP22 functions are supported by the N-terminus associated to the central domain and independent of the C-terminus from AA190. Interestingly, the region between AA173 and AA190 is essential for cell cycle arrest and MDV spread. All VP22 which cis-complemented MDV for spread were able to efficiently arrest the cell cycle and showed a nuclear localization and histones association. The relationship between cell cycle arrest and MDV spread will be discussed.