IE1 assembles a Hes1 ubiquitination complex and leads to ubiquitination of Hes1 in vitro.

(A) Interaction between IE1 and Ubc5a in HCMV infected NPCs. NPCs were infected with HCMV at an MOI of 3, and harvested at 12hpi. Cell lysates were subjected to IE1- or Ubc5a-directed IP followed by IB against IE1 and Ubc5a. Normal IgG was applied as the nonspecific antibody control. (B) Effect of IE1 on assembling the Hes1 and Ubc5a ubiquitination complex. The purified His-Hes1 (1μg) was mixed with Ubc5a (1μg) in the presence or absence of purified His-IE1 (1μg). The mixtures were then subjected to Hes1- or Ubc5a-directed IP, followed by IB for the indicated component proteins. Normal IgG was applied as the nonspecific antibody control. The input components in the reaction were examined. (C-D) Effect of IE1 AA451-475 on ubiquitination of Hes1 in vitro. Purified His-tagged proteins (His-Hes1, His-IE1, His-Δ451–475) were mixed with ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme Ubc5a (E2) and ubiquitin as described in Materials and Methods, and then incubated at the indicated temperature for 2 h to perform the in vitro ubiquitination reaction. The mixtures were subjected to Hes1-directed IP and subsequent IB for ubiquitin (Ub) (C). Alternatively, the products from in vitro ubiquitination reactions were directly subjected to IB with anti-Hes1 (D). According to protein size, mono-ubiquitinated Hes1 is indicated by an asterisk (*). The input components in the reaction were examined, and the indicated proteins are shown.