Total RNA was separated on 10% TBE-Urea acrylamide gels (Bio-Rad) and electroblotted onto Hybond N + membranes (GE Healthcare). After UV cross-linking the membranes were pre-hybridized and hybridized with labeled probes at 48°C as per the DIG manual (Roche). Probe sequences are CGATGGTCGAAAAGGAACTCGA-TACGGCTATGCGGTTCT (RNA1), AGTTCACGA Proteasome-Dependent Disruption of the E3 Ubiquitin Ligase Anaphase-Promoting Complex by HCMV Protein pUL21a The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109–110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity. Regulation of protein degradation plays a key role in many cellular processes ranging from cell cycle progression, innate immunity, and antigen presentation to the turnover of misfolded or oxidized proteins. Most degradation is carried out by the ubiquitin-proteasome system (UPS). Ubiquitin is added to proteins by a cascade of ubiquitin conjugating enzymes, resulting in a polyubiquitinated protein which is subsequently degraded by the 26S proteasome. As a means to regulate protein function, it is no surprise that many viruses have co-opted the UPS for their own benefit. Viruses can promote proteasome degradation of antiviral host proteins either by encoding their own E3 ubiquitin ligase, targeting proteins to a cellular E3 ligase, or even inducing ubiquitin-independent degradation of targets. Examples of viral E3 ligases include the herpes simplex virus-1 protein ICP0 [1] and Kaposi's sarcoma-associated herpesvirus proteins K3 and K5 (for a review, see [2] ). Viral proteins that can hijack a cellular E3 ligase include human immunodeficiency virus-1 vpr and vif (for a review, see [3] ), paramyxovirus V [4] , and human papillomavirus E6 and E7 (for a review, see [5] ). Finally, the human cytomegalovirus (HCMV) protein pp71 uses a ubiquitin-independent mechanism to target the Rb and hDaxx proteins [6, 7] . In fact, pharmacological inhibition of the proteasome blocks multiple stages of the viral life cycle, suggesting that viruses rely on activities of the UPS for their replication [8] [9] [10] [11] [12] . On the other hand, viruses must also modulate cellular E3 ligase activity in order to replicate because ubiquitination regulates many important cellular processes central to virus infection. The SV40 large T antigen inhibits the SCF fbw7 ubiquitin ligase to increase cyclin E levels [13] , and influenza virus NS1 inhibits TRIM 25-mediated ubiquitination of RIG-I, thereby attenuating interferon production [14] .