Human beta interferon scaffold attachment region inhibits de novo methylation and confers long-term, copy number-dependent expression to a retroviralvector

Moloney murine leukemia virus-based retroviral vector expression is gradual ly lost during prolonged in vitro culture of CEMSS T cells. However, when t he human beta interferon scaffold attachment region (IFN-SAR) was inserted into the vector immediately upstream of the 3' long terminal repeat (LTR), expression was maintained for the length of the study (4 months). Clonal an alysis of the retrovirus vector-infected CEMSS cells showed that SAR-contai ning retroviral vector expression levels were positively correlated with th e proviral copy numbers (P < 0.0001), while there was no correlation betwee n the proviral copy numbers and expression levels in control vector-infecte d clones. Thirty-three percent of the CEMSS cell clones infected with the c ontrol vector showed evidence of partial or complete methylation in the 5' LTR region. In sharp contrast, we detected no methylation in the clones inf ected with the SAR-containing vector. To demonstrate a direct inhibitory ef fect of methylation on retroviral vector expression, we have transfected 29 3 cells with in vitro-methylated proviral DNA. In transiently transfected c ells, expression of methylated LTR was reduced but not completely inhibited , irrespective of the presence of the IFN-SAR sequence, In stably transfect ed cells, however, methylation completely abolished expression of the contr ol vector but not of the SAR-containing vector. Furthermore, the expression of the SAR-containing vector was stable over time, indicating the ability of the SAR sequence to alleviate methylation-mediated transcriptional repre ssion of a vector. This study extends our understanding of the mechanisms o f retroviral vector inactivation by methylation and provides insight into a functional role for the SAR elements.