FIG 3.

Efficient interactions require all three terminase proteins. (A) Schematic representations of the expression plasmids used in this study. The major immediate early promoter of HCMV (CMV), sequences for Strep-tag II, FLAG, and HA epitope tags (SF/HA) and polyadenylation signal sequence (pA) are indicated. (B) RPE-1 cells were transfected with the expression plasmids depicted in panel A, either alone or in the indicated combinations, or were mock transfected. On day 2 posttransfection, whole-cell lysates were prepared and analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), against pUL56, pUL89, or GAPDH (which served as a loading control). The asterisks denote unspecific reactivity with a cellular protein. (C) The epitope-tagged pUL51 was pulled down using Strep-Tactin beads, and pUL56 or pUL89 bound to it was assessed by immunoblotting. (D) Following immunoprecipitation (IP) of pUL56, coprecipitated pUL51 or pUL89 was detected by immunoblotting. IgG HC, immunoglobulin heavy chain. (E) The samples displayed in panel D, lanes 6 and 7, were diluted to adjust immunoprecipitated pUL56 to comparable levels, and interacting pUL51 and pUL89 was analyzed by immunoblotting.