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. 1999 Oct 26;96(22):12536–12541. doi: 10.1073/pnas.96.22.12536

Figure 1.

Figure 1

Identification of a UL37 gene product as an inhibitor of apoptosis. (a) CMV-infected human fibroblasts acquire resistance to Fas- and TNF-α-mediated apoptosis. Cells were infected with CMV(Towne-RIT) (3 plaque-forming units per cell), or left uninfected (N). At the indicated times after infection, cells were exposed for an additional 12 h to medium alone (no treatment), TNF-α + CHX, or anti-Fas antibody + CHX. Viable cells remaining after these treatments were quantified by counting representative fields under a phase-contrast microscope. (b) CMV genomic sequences contained in plasmids with anti-apoptotic activity. (Upper) The structure of the CMV(AD169) genome (5) and UL37 region and associated ORFs are shown schematically. (Lower) CMV DNA inserts in plasmids isolated from three independent library pools (designated 176, 209, and 135). Nucleotide numbering is as described in ref. 4. Arrows indicate the orientation of inserts relative to the promoter in the expression vector. (c) pUL37x1 suppresses Fas-mediated apoptosis in transfected HeLa cells. Cells were transiently transfected with expression plasmids (1 μg) coding for the following proteins: pUL37x1myc, pUL37x1Δ2–23myc, E1B19K, baculovirus p35, CMV IE1491aa, CMV IE2579aa, or a 1:1 mixture of the plasmids containing IE1 and IE2, or they were transfected with an empty vector (pcDNA3). 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. (d) (Upper) Structures of pUL37x1, gpUL37M, and gpUL37 produced by alternative splicing. The amino acid numbers used in gpUL37M correspond to those in gpUL37. (Lower) Protection against Fas-mediated apoptosis by pUL37x1myc, gpUL37M, and gpUL37. HeLa cells were transfected with 1 μg of plasmid DNA expressing the indicated proteins and assayed as in c. (e) RNA blot analysis of UL37 exon 3 region transcripts expressed by CMV(AD169) and CMV(Towne-RIT) at 4 and 8 h after infection (Left) or UL37 exon 1 region transcripts expressed by CMV(Towne-RIT) at 4, 8, 24, 48, and 72 h after infection (Right).