Research Article: Cyclin A Degradation by Primate Cytomegalovirus Protein pUL21a Counters Its Innate Restriction of Virus Replication

Date Published: December 26, 2013

Publisher: Public Library of Science

Author(s): Nicolas Caffarelli, Anthony R. Fehr, Dong Yu, William J. Britt.


Cyclin A is critical for cellular DNA synthesis and S phase progression of the cell cycle. Human cytomegalovirus (HCMV) can reduce cyclin A levels and block cellular DNA synthesis, and cyclin A overexpression can repress HCMV replication. This interaction has only been previously observed in HCMV as murine CMV does not downregulate cyclin A, and the responsible viral factor has not been identified. We previously reported that the HCMV protein pUL21a disrupted the anaphase-promoting complex (APC), but a point mutant abrogating this activity did not phenocopy a UL21a-deficient virus, suggesting that pUL21a has an additional function. Here we identified a conserved arginine-x-leucine (RxL) cyclin-binding domain within pUL21a, which allowed pUL21a to interact with cyclin A and target it for proteasome degradation. Homologous pUL21a proteins from both chimpanzee and rhesus CMVs also contained the RxL domain and similarly degraded cyclin A, indicating that this function is conserved in primate CMVs. The RxL point mutation disabled the virus’ ability to block cellular DNA synthesis and resulted in a growth defect similar to pUL21a-deficient virus. Importantly, knockdown of cyclin A rescued growth of UL21a-deficient virus. Together, these data show that during evolution, the pUL21a family proteins of primate CMVs have acquired a cyclin-binding domain that targets cyclin A for degradation, thus neutralizing its restriction on virus replication. Finally, the combined proteasome-dependent degradation of pUL21a and its cellular targets suggests that pUL21a may act as a novel suicide protein, targeting its protein cargos for destruction.

Partial Text

Human cytomegalovirus (HCMV), a widespread β-herpesvirus that establishes a lifelong infection, is capable of causing severe complications in immuno-naïve populations and immuno-compromised patients [1]. HCMV is known to use a number of mechanisms to manipulate the host cell cycle so that infected cells are arrested at the G1/S boundary. Some of these mechanisms are inhibition of Rb and activation of the E2F family of transcription factors [2]–[6], modulation of the anaphase-promoting complex (APC) [7], [8], suppression of the mini-chromosome maintenance complex (MCM) [9], and alteration of cyclin/cyclin-dependent kinase (CDK) activity [10]–[15]. It has been postulated that these regulations provide essential nutrients and cellular enzymes needed for viral DNA synthesis while preventing cellular DNA synthesis from competing for these important resources.

Here we describe a novel function for the HCMV protein pUL21a to bind to the important cell cycle regulator cyclin A and direct it for proteasome degradation, which represents a novel viral mechanism to combat this host factor. This interaction is mediated by a highly conserved RxL cyclin-binding domain that is present in pUL21a proteins of all primate CMVs tested in this study. pUL21a from ChCMV and RhCMV not only similarly reduce cyclin A levels, they also rescued the growth defect of pUL21a-deficient HCMV virus. pUL21a is a highly unstable protein subject to rapid proteasome-mediated degradation [21]. Intriguingly, it binds to two major cell cycle regulators, the anaphase-promoting complex (APC) [7] and cyclin A, and targets both for degradation in a proteasome-dependent manner. Finally, this pUL21a-mediated cyclin A degradation bears an important consequence on virus replication. Mutation in the RxL cyclin-binding domain of pUL21a destroys the ability of HCMV to inhibit cyclin A, resulting in unchecked cellular DNA synthesis and severe attenuation of virus growth. Importantly, growth of the UL21a-deficient virus can be rescued by knocking down cyclin A, providing the definitive proof that pUL21a targets cyclin A for degradation in order to antagonize its innate restriction on HCMV replication.