Studies on the Contribution of Human Cytomegalovirus UL21a and UL97 to Viral Growth and Inactivation of the Anaphase-Promoting Complex/Cyclosome (APC/C) E3 Ubiquitin Ligase Reveal a Unique Cellular Mechanism for Downmodulation of the APC/C Subunits APC1, APC4, and APC5

Abstract

Human cytomegalovirus (HCMV) deregulates the cell cycle by several means, including inactivation of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Viral proteins UL97 and UL21a, respectively, affect the APC/C by phosphorylation of APC/C coactivator Cdh1 and by inducing the degradation of subunits APC4 and APC5, which along with APC1 form the APC/C platform subcomplex. The aim of this study was to further characterize the mechanism of APC/C inactivation and define the relative contributions of UL21a and UL97 to APC/C substrate accumulation and to viral growth. We show that in uninfected cells, UL21a but not UL97 can disrupt APC/C function, leading to the accumulation of substrates. We find that UL21a is necessary and sufficient to induce the degradation of APC1, in addition to the previously reported APC4 and APC5. We also demonstrate that there is a previously unreported cellular mechanism for a specific decrease in the levels of all three platform subunits, APC1, APC4, and APC5, upon the depletion of any one of these subunits or of subunit APC8. Finally, we show that at a low multiplicity of infection, either UL97 or UL21a can partially complement a growth-defective mutant virus lacking both UL21a and UL97, with significantly greater benefit afforded by the expression of both proteins. This double mutant also can be partially rescued by inactivation of the APC/C using small interfering RNAs against specific subunits. These results further our understanding of HCMV's interaction with the cell cycle machinery and reveal a new cellular pattern of APC/C subunit downmodulation. IMPORTANCEHCMV lytic infection subverts the host cell cycle machinery in multiple ways. A major effect is inactivation of the APC/C, which plays a central role in the control of cell cycle progression. This study provides further insight into the mechanism of inactivation. We discovered that the APC1 subunit, which along with APC4 and APC5 form the platform subcomplex of the APC/C, is an additional target of the degradation induced by HCMV protein UL21a. This study also shows for the first time that there is a unique cellular process in uninfected cells whereby depletion of APC1, APC4, APC5, or APC8 recapitulates the pattern of HCMV-mediated APC/C subunit degradation. H uman cytomegalovirus (HCMV) infects the majority of the human population, causing significant morbidity and mortality in immunocompromised individuals, such as transplant patients and those with HIV. HCMV is also the leading viral cause of birth defects. These manifest as neural developmental defects ranging from hearing loss to calcification of the developing brain and death at a rate of 1 or 2 per 1,000 newborns.HCMV lytic infection both modulates and is influenced by the host cell cycle. The virus preferentially infects cells in G 0 or G 1 . Infection in other phases of the cell cycle results in a delay of immediate early gene expression until completion of mitosis in the case of a G 2 infection. Infection during S phase remains unproductive i...

Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation

“…Overall, our data argue in favor of a model of the HCMV terminase as a multiprotein complex in which the individual subunits stabilize each other upon assembly. Such a setting was also proposed for other CMV protein complexes, namely, for the murine CMV (MCMV) US22 protein family (60) and for the HCMV major and small capsid proteins (18), as well as for the cellular anaphase-promoting complex, a multiprotein assembly targeted by HCMV (61).…”

Human cytomegalovirus protein pUL36: a dual cell death pathway inhibitor

“…From a comparison with our previous study of HCMV-induced protein degradation between 2-24 h (10), seven proteins were degraded with high confidence throughout early and late infection ( Figures 1C, 1D). These proteins included HLTF and Anaphase Promoting Complex subunits 1 and 5 (ANAPC1/5), whose degradation by HCMV has been well characterised (10,19). The effector of necroptosis MLKL was the most significantly downregulated protein at 48 h of HCMV infection and was among proteins most significantly rescued by addition of MG132 ( Figure 1B).…”

Network mechanisms and dysfunction within an integrated computational model of progression through mitosis in the human cell cycle

“…Entry of the infected cell into mitosis is regulated HCMV pUL21a which limits Cyclin A levels and subsequently CCNB1 levels ( Fig 9A) [55]. Expressions of pUL97 and pUL21a also inactivated APC/C during infection (Fig 9B and 9C) [56][57][58][59]. However, the reason for APC/C inactivation and how it contributes to HCMV replication remain unknown.…”

“…This perturbation resulted in increased oscillations with an eventual mitotic collapse defined by reduced MPF, LMNAP and PTTG1T and elevated APC/CT (Fig 9E). By a different mechanism, HCMV pUL21 also mediates the degradation of subunits APC1, APC4 and APC5 resulting in the inactivation of APC/C ( Fig 9C) [56,57]. For this mechanism, we increased the rate constant k d15.1 resulting in decreased oscillations and a mitotic collapse exhibiting elevated levels of MPF, CCNB1, PTTG1T and LMNAP ( Fig 9B).…”

“…It is important to note that the kinetics of mitotic entry are regulated by the HCMV protein pUL21a which suppresses Cyclin A levels ( Fig 9A) [55]. By simulating mechanisms of two viral proteins, pUL97 and pUL21a [56,57,59], we suggest that disrupting APC/C is necessary to induce a mitotic collapse supporting MPF kinase (CCNB1:CDK1) levels and nuclear lamina disruption ( Fig 9H) [69]. There are numerous experimental perturbations that result in a mitotic collapse exhibiting loss of MPF kinase activity (Figs 3F, 4D and 5E).…”