The Role of Viral Protein pUL21a in Human Cytomegalovirus Infection

The double-stranded DNA virus human cytomegalovirus: HCMV) is a ubiquitous human pathogen that causes severe disease in immunocompromised individuals and neonates. The biology of HCMV remains largely undefined and functions of many of the roughly 200 putative HCMV genes are still unknown. One of these genes, UL21a, was shown to be critical for efficient viral replication in two large-scale mutagenesis studies. UL21a encodes for a putative protein that is 123 amino acids and has no homology to any known proteins. The gene products of UL21a, its role during infection, and its function(s) remain undefined. Here, we characterized UL21a and demonstrated its role in HCMV infection. We identified a single UL21a transcript which was expressed with early gene kinetics. UL21a encoded a protein termed pUL21a which localized to the cytoplasm and underwent proteasome-dependent degradation. UL21a was specifically required for efficient viral replication, and the growth of a UL21a deletion virus was similar to that of a stop codon mutant, suggesting it is pUL21a which facilitates viral replication. To identify the role of pUL21a during virus infection, we analyzed fibroblasts infected with equal amounts of wild-type and UL21a deletion virus for defects in multiple steps of the viral lifecycle. The UL21a deletion virus entered cells and initiated viral gene expression efficiently; however, it synthesized viral DNA poorly and accumulated several immediate-early: IE) transcripts at reduced levels at late times of infection. The reduction in IE transcripts was dependent on the reduction in viral DNA synthesis, showing that multiple IE transcripts are dependent on viral DNA synthesis for their full expression. Finally, using complementing cells we show that it is the de novo synthesis of pUL21a which facilitates viral DNA synthesis. To determine the function(s) of pUL21a, we identified proteins that specifically interacted with pUL21a. We found that pUL21a interacts with the Anaphase-Promoting Complex: APC), a multi-subunit E3 ubiquitin ligase which targets multiple proteins for proteasome-dependent degradation. The APC is critical for progression through mitosis and the regulation of cellular DNA synthesis. We have found that pUL21a specifically binds to the APC, and is required for the accumulation of APC substrates, degradation of APC4 and APC5, and dissociation of the APC during HCMV infection. Finally, shRNA knockdown of the APC activator Cdh1 and to a lesser degree APC8, significantly restored late gene expression of the UL21a mutant virus, suggesting the APC has antiviral activity for HCMV. Thus, we propose that one mechanism for pUL21a to promote viral replication is to regulate the function of the APC