pUL21a binding to the APC promotes degradation of APC subunits and APC dissociation.

<p>(A) pUL21a is required for HCMV to reduce APC4 and APC5 accumulation during infection. MRC-5 cells were infected in the presence or absence of PAA with AD<i>gfp</i> or AD<i>sub</i>UL21a. Cell lysates were analyzed by immunoblotting at 24 hpi. pUL21a, IE1-72, and actin were used as infection and loading controls, respectively. (B) HCMV infection does not alter APC4 or APC5 transcript levels. MRC-5 cells were infected with AD<i>gfp</i> or AD<i>sub</i>UL21a, total RNA was collected at indicated times, and APC4 and APC5 transcripts were measured by reverse transcription-coupled quantitative PCR (RT-qPCR) and normalized to that of GAPDH. (C) APC binding activity of pUL21a is required for proteasome-dependent degradation of APC4 and APC5 in HCMV infection. MRC-5 cells were infected with AD<i>gfp</i>, AD<i>sub</i>UL21a, AD<i>pm</i>UL21a_PH-AA, or AD<i>pm</i>UL21a_PR-AA. MG132 was added at 6 hpi, and cell lysates were analyzed at 20 hpi by immunoblotting. (D) APC binding ability of pUL21a is required for APC dissociation in HCMV infection. MRC-5 cells were infected with AD<i>pm</i>UL21a_PH-AA or AD<i>pm</i>UL21a_PR-AA, and treated with MG132 as described in panel C. Cells were collected at 20 hpi, cell lysates were immunoprecipitated with APC3 antibody, and both lysates and eluted proteins were analyzed by immunoblotting.