Sequence alignment of ORF translations of pUL36 encoded by several laboratory CMV strains. The predicted amino acid sequences for the UL36-coding region obtained from the cosmid pCM1017 containing a segment of the genome of AD169varDE and those from gUL36_AD169varDE/pcR3.1 and from UL36_AD169varDEmyc/pcDNA3 were identical, and their translation is shown here as AD169varDE. The sequences for the UL36-coding region obtained from a cosmid containing the genomic DNA for TownevarRIT and from gUL36_TownevarRIT/pcDNA3 were identical. The sequence obtained from UL36_AD169varATCCmyc/pcDNA3 was identical to that published previously (5). The amino acid sequence for the translation from gUL36_TownevarATCC/pcDNA3 was identical to that of pUL36_TownevarATCC published previously (14). The sequences for the pUL36 translations obtained from TownevarATCC (denoted as Towne) and Toledo were taken from ref. 14.

pUL36 interrupts Fas-mediated apoptosis at the step of caspase-8 activation. (A) Effects of pUL36 expression on biochemical events associated with Fas-mediated apoptosis. BJAB/pcDNA3 cells and BJAB/UL36 cells were treated for 24 h with anti-Fas or were left untreated. Cell lysates were prepared, separated by SDS/PAGE, and blotted onto nitrocellulose. Then procaspase-8, Bid, and PARP were detected by immunoblot analysis. (B) Association of pUL36 with pro-caspase-8. Lysates from BJAB/UL36 cells or from control BJAB/pcDNA3 cells were immunoprecipitated (IP) with the anti-myc antibody 9E10 covalently linked to Affi-Prep-10 beads. Lanes designated IP are proteins bound with anti-myc antibody. Pro-caspase-8 and FADD were detected by immunoblot analysis. Lanes designated L are samples of the transfected cell lysate taken before immunoprecipitation. (C) Pro-caspase-8 fails to coimmunoprecipitate with pUL36_AD169varATCC in transiently transfected 293T cells. Cells were transfected with pcDNA3 (lane 1), UL36_AD169varDEmyc/pcDNA3 (lane 2), or UL36_AD169varATCCmyc/pcDNA3 (lane 3). The cells were lysed 24 h later, and pUL36 immunoprecipitated with anti-myc antibody. Cell lysates and immunoprecipitates were probed with either anti-pro-caspase-8 antibody or anti-myc antibody. Pro-caspase-8 was easily detectable in an IP sample of pUL36_AD169varDE diluted to the concentration that gave a similar pUL36 signal to that of the pUL36_AD169varATCC sample, but no pro-caspase-8 signal (data not shown). (D) pUL36_AD169varDE, but not pUL36_AD169varATCC, coimmunoprecipitates with either HA-tagged pro-caspase-8 or the HA-tagged pro-domain fragment of pro-caspase-8 in transiently transfected 293T cells. Cells were transfected with the following pairs of plasmids: UL36_{AD169varDEmyc} plus either vector (lane 1), HA-caspase-8-pro-domain (lane 2), or HA-pro-caspase-8 (lane 3); UL36_{AD169varATCCmyc} plus either vector (lane 4); HA-caspase-8-pro-domain (lane 5); or HA-pro-caspase-8 (lane 6). The cells were lysed 24 h later, immunoprecipitated with anti-HA antibody, and coprecipitated pUL36myc was detected by immunoblot with anti-myc antibody.

Antiapoptotic activity of pUL36 in HeLa and BJAB cells. (A) HeLa cells (4 × 10⁴ cells per sample) were transfected transiently with an expression vector containing genomic UL36_AD169varDE, cDNA UL36_AD169varDEmyc, or one of the respective empty vectors. Twenty-four hours after transfection, the cells were exposed to anti-Fas + CHX for an additional 24 h, and surviving cells were scored under a microscope. The data are presented as means ± SEM (n = 4). (B) Expression of pUL36 in stably transfected HeLa and BJAB cells. Lysates from HeLa and BJAB cells stably transfected with either UL36_AD169varDEmyc/pcDNA3 or an empty vector (pcDNA3myc) were immunoprecipitated with the anti-myc antibody 9E10 covalently linked to Affi-Prep-10 beads. Bound pUL36 was detected by immunoblot analysis with anti-myc 9E10 antibody. (C) Resistance of BJAB/pcDNA3 cells and BJAB/UL36 cells to Fas-mediated apoptosis. Cells were exposed to anti-Fas antibody or to medium alone (untreated) for 24 h, and then the surviving fractions of cells were determined in a clonogenic assay as described (11). The data are presented as the means ± SEM (n = 2). (D) Resistance of HeLa/UL36 cells to Fas-mediated apoptosis. Cells were incubated for 24 h either in DMEM containing 10% (vol/vol) FBS or in serum-free DMEM. The following reagents were added to the medium: anti-Fas antibody + CHX, CHX, or none (untreated). Then the cells were photographed under a phase-contrast microscope. The cells in cultures exposed to CHX alone ceased to proliferate, whereas the control cells nearly doubled in number during the 24-h incubation.

Comparison of antiapoptotic activities of pUL36 proteins encoded by CMV_AD169varDE, CMV_AD169varATCC, CMV_TownevarATCC, and CMV_TownevarRIT. Cells (4 × 10⁴ cells per sample) were transfected transiently with an expression vector that was either empty or contained an ORF for one of these genes, and 24 h later the cells were exposed to anti-Fas + CHX for an additional 24 h. Surviving cells were scored under a microscope. The data are presented as means ± SEM (n = 4).