Replication of wild-type and mutant human cytomegalovirus in life-extended human diploid fibroblasts

Abstract

A cDNA encoding the catalytic subunit of human telomerase was used to generate life-extended derivatives of primary human diploid fibroblasts. The life-extended cells supported efficient human cytomegalovirus (HCMV) replication. A subclone of the life-extended cells was generated containing the HCMV UL82 gene and used to isolate and propagate a virus that exhibited a profound growth defect after infection at a low input multiplicity.

FIG. 1

Telomerase activity in stable HFF clones. Stable HFF clones obtained by transfection with pGRN145 expressing the catalytic subunit of telomerase (Tel) were analyzed for telomerase activity by the TRAP assay using a TRAPEZE kit (Intergen) according to the manufacturer's instructions. Two thousand cells were analyzed in all samples. TRAP products were separated by gel electrophoresis (11.25% polyacrylamide) and detected by autoradiography. As a negative control, cell lysates were incubated at 85°C for 10 min (ΔH) to inactivate telomerase. TSR8, positive control for the PCR; IC, internal control for all reactions.

FIG. 2

Replicative life span of telomerase-positive and -negative clones. Cells were counted and passaged every 5 to 7 days, and new population doublings were calculated. Cultures were considered senescent (*) if they had undergone less than one population doubling in 2 weeks.

FIG. 3

Comparison of immediate-early and late gene expression in HFF and telomerase-positive cells. HFF or telomerase 18 cells were infected with HCMV at an MOI of 5. Cell lysates were prepared at the indicated times postinfection (P.I.), and 50 μg of protein from each sample was analyzed by Western blotting using antibodies against either IE1 (Chemicon 810) or pp28 (9, 11).