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. 2000 Feb 18;97(6):2864–2869. doi: 10.1073/pnas.050504297

Figure 1.

Figure 1

α1-PDX is a potent inhibitor of HCMV. (A) Schematic of HCMV gB. The 907-amino acid pro-gB is cleaved during transit through the TGN at the C-terminal side of the consensus furin site (black bar) to yield the disulfide-linked gp110/55 heterodimer. The inserted FLAG tag (hatched) and transmembrane domain (stippled) are also shown. (B) Inhibition of the furin-dependent processing of HCMV gB by α1-PDX. U-373 MG cells on replicate plates were coinfected with adenovirus recombinants expressing the tet transactivator [ta; multiplicity of infection (moi) = 5] and HCMV pro-gB (gB; total moi = 10) or triple-infected with the ta and gB recombinants together with adenovirus recombinants expressing either α1-antitrypsin (α1-NAT) or α1-PDX for 24 h (total moi = 15). Alternatively, U-373 MG cells were pretreated with 8 μM α1-PDX for 24 h and then coinfected with the ta and gB recombinants for an additional 24 h in the presence of 8 μM α1-PDX. As a control, furin-deficient LoVo cells were infected with either adenovirus ta alone (moi = 5) or together with adenovirus gB (total moi = 10) for 24 h. Cells were harvested in RIPA minus SDS and analyzed by Western blotting (SDS/8% PAGE) by using anti-gB mAb 27-156. The gp55 band could also be detected with the anti-FLAG mAb M1 (requires free N terminus), indicating correct cleavage of pro-gB (data not shown). (C) Dose response of inhibition of HCMV by α1-PDX and foscarnet. U373 cells on replicate plates were infected with the HCMV Towne (moi = 0.1) in the absence or presence of increasing concentrations of α1-PDX (triangles) or the pyrophosphate analog foscarnet (squares). At 5 days after infection, cells were harvested, and infectious cell-associated HCMV was quantified by plaque assay on human foreskin fibroblast cells (HCMV remains cell-associated in U373 MG cells; J.A.N., unpublished results). Data represent averages of three experiments. Error bars depict standard deviation. (Inset) U-373 MG cells on replicate plates were infected with HCMV Towne (moi = 0.2). At 24 h after infection, the medium was replaced with fresh medium containing either 0 or 8 μM α1-PDX. At 72 h after infection, cells were metabolically labeled with [35S]Met/Cys for 3 h, chased for 2 h in the absence of label, and harvested. gB proteins were immunoprecipitated with mAb 7-17. The minor protein band migrating below the 116-kDa marker likely represents coimmunoprecipitated gp110.