DNA methylation of helper virus increases genetic instability of retroviral vector producer cells

Retroviral vector producer cells (VPC) have been considered genetically sta ble. A clonal cell population exhibiting a uniform vector integration patte rn is used for sustained vector production, Here, we observed that the vect or copy number is increased and varied in a population of established LTKOS N.2 VPC, Among five subclones of LTKOSN.2 WC, the vector copy number ranged from 1 to approximately 29 copies per cell, A vector superinfection experi ment and Northern blot analysis demonstrated that suppression of helper vir us gene expression decreased Env-receptor interference and allowed increase d superinfection. The titer production was tightly associated with helper v irus gene expression and varied between 0 and 2.2 x 10(5) CFU/ml in these s ubclones, In one analyzed subclone, the number of integrated vectors increa sed from one copy per cell to nine copies per cell during a 31-day period. Vector titer was reduced from 1.5 x 10(5) CFU to an undetectable level. To understand the mechanism involved, helper virus and vectors were examined f or DNA methylation status by methylation-sensitive restriction enzyme diges tion, We demonstrated that DNA methylation of helper virus 5' long terminal repeat occurred in approximately 2% of the VPC population per day and corr elated closely with inactivation of helper virus gene expression. In contra st, retroviral vectors did not exhibit significant methylation and maintain ed consistent transcription activity. Treatment with 5-azacytidine, a methy lation inhibitor, partially reversed the helper virus DNA methylation and r estored a portion of vector production. The preference for methylation of h elper virus sequences over vector sequences may have important implications for host-virus interaction, Designing a helper virus to overcome cellular DNA methylation may therefore improve vector production, The maintenance of increased viral envelope-receptor interference might also prevent replicat ion-competent retrovirus formation.