Human telomerase reverse transcriptase-immortalized MRC-5 and HCA2 human fibroblasts are fully permissive for human cytomegalovirus

MRC-5 cells are a well-characterized human diploid fibroblast cell line app roved for vaccine production and favoured for the routine propagation of hu man cytomegalovirus (HCMV). Ectopic expression of telomerase in fibroblasts is capable of overcoming replicative senescence induced by telomere shorte ning. Following delivery of the hTERT gene to MRC-5 cells using a retroviru s vector three clones were generated that (i) expressed functional telomera se activity, (ii) exhibited telomere extension and (iii) were sustained for > 100 population doublings, Immortalized MRC-5-hTERT and also HCA2-hTERT h uman fibroblasts were both fully permissive for HCMV as determined by plaqu e assay, studies of virus growth kinetics and measurement of virus yields. Furthermore, telomerase-immortalized HCA2 cells proved capable of Supportin g the stable maintenance of an EBV-based episomal vector with efficient tra nsgene expression when driven by the HCMV immediate early promoter. An indi cator cell line suitable for the efficient detection of HCMV infection was also generated using an episome containing a reporter gene (lacZ) under the control of the HCMV beta -2.7 early promoter. Telomerase immortalization o f human fibroblasts will thus facilitate the growth and detection of HCMV a nd also the generation of helper cell lines for the propagation of HCMV del etion mutants. Immortalization of fibroblasts by telomerase does not affect cell morphology or growth characteristics. The MRC-5-hTERT clones may ther efore be suitable for additional applications in virology, cell biology, va ccine production and biotechnology.