FIG 5.
Requirement of the TM domain for ISGylation inhibition by pUL50. (A) 293T cells in six-well plates were cotransfected with plasmids (0.25 μg) expressing Myc-ISG15GG, HA-UBE1L, Flag-UbcH8, and HA-Herc5 and a plasmid (1 μg) expressing pUL50-HA (wild type [Wt] or the ΔTM or 1-358 mutant), as indicated. Forty-eight hours after transfection, immunoblotting was performed with anti-Myc or anti-HA antibody. Levels of β-actin are shown as a loading control. The positions of the wild-type pUL50 protein and its 26-kDa isoform (UL50-p26) are indicated. (B) Structures of the UL50 constructs used for panel A. The α-helix, globular domain, and transmembrane (TM) regions and the UL53 binding region are indicated. The relatively conserved regions (CR1 and CR2) in other herpesvirus homologs are also indicated. The numbers indicate amino acid positions. (C) 293T cells in six-well plates were cotransfected with plasmids encoding pUL50-HA (wild type or mutant) (1 μg) and Myc-UBE1L (0.5 μg), as indicated. Forty-eight hours after transfection, cell lysates were prepared and immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-Myc antibody. The expression level of each protein in total cell lysate was also determined by immunoblotting with anti-UBE1L or anti-HA antibody. The position of endogenous (endo.) UBE1L is indicated. Levels of β-actin are shown as a loading control. (D) HF cells in chamber slides were transfected with UL50 plasmids. Forty-eight hours after transfection, cells were fixed with 4% paraformaldehyde and stained with anti-HA antibody. Hoechst dye was used to stain cell nuclei. Representative confocal laser scanning microscopic images are shown.