The APC plays a key role in cell cycle regulation by targeting the degradation of specific cell cycle proteins in a timely manner. Not surprisingly, viruses target the APC as they manipulate the host cell cycle to facilitate their own replication (14
). Adenovirus E4orf4 has been implicated in targeting phosphatase PP2A to APC6 to inactivate the complex through dephosphorylation (17
). The chicken anemia virus apoptin protein also has been shown to target, and perhaps sequester, APC1, causing complex destabilization and G2
/M arrest (15
). Similarly, HCMV appears to specifically inhibit the APC in the process of creating a cellular environment more conducive to viral replication. Previous work indicated that Cdh1 no longer binds to APC3 as the infection progresses, suggesting that the loss in APC function is due to the lack of activation by Cdh1 (42
). Our studies show that this is only one of the multiple effects of the infection on the APC that could be responsible for its inactivation.
We find that Cdh1 becomes phosphorylated early after HCMV infection. In normal uninfected cells, Cdh1 phosphorylation and its subsequent dissociation from the complex are key mechanisms in mediating APC inactivation as the cells transition from G0
to S phase. The phosphorylation of Cdh1 in the infected cells would not be surprising given the heightened state of CDK activity during the infection. However, treatment with the CDK inhibitor roscovitine did not inhibit Cdh1 phosphorylation, although it did affect its accumulation. This implies that other kinases are involved in phosphorylating Cdh1 in infected cells. Alternatively, these results could be attributed to the indirect effects the drug has on the infection. We also noted that the addition of roscovitine at the beginning of the infection prevented the accumulation of not only Cdh1 but also two other APC substrates, geminin and Cdc6. Roscovitine had less effect on the accumulation of these proteins when administered at 4 or 8 h p.i. There are several explanations for this result, and they are not mutually exclusive. As our laboratory and others have shown, roscovitine severely reduces viral replication (3
). Addition of the drug at the time of infection alters IE gene expression such that IE2-86 expression is enhanced while that of IE1-72 is reduced. Early viral gene expression and viral DNA replication also are inhibited. However, if the drug is added at 6 h p.i., it no longer affects IE1-72 expression, and early gene expression along with viral DNA replication is restored (31
). Thus, some viral early gene expression may be necessary for inactivation of the APC. The kinetics of stabilization of the APC substrates (beginning around 8 h p.i.) provides support for this (1
). Alternatively, CDK activity may be required for the accumulation of some APC substrates due to direct effects on phosphorylation of other APC subunits or other proteins involved in the ubiquitin-proteasome degradation pathway. It also is possible that the drug affects the levels of RNA. The latter two possibilities may apply to both infected and uninfected cells, as the levels of geminin also were lower in the uninfected cells treated with the drug during all of the intervals. A small decrease in Cdc6 also was observed in the treated uninfected cells, although the levels were at the limit of detection in both the treated and untreated cells. Taken together, the results show that the phosphorylation of Cdh1 in infected cells is not CDK dependent, but the accumulation of the APC substrates may be partially affected, either directly or indirectly, by the inhibition of CDK activity.
While phosphorylation of Cdh1 in infected cells may contribute to its lack of association with the APC, we also found that exogenous TNT-synthesized Cdh1 had decreased binding affinity for APC3 in lysates obtained from infected cells as the infection progressed from 8 to 16 h p.i.; however, there was little change in binding to APC3 in uninfected cell lysates at any time point. APC3 from the uninfected cell lysates still was able to bind more 35S-Cdh1 despite potentially competing cellular Cdh1, whereas this would not have been a factor in the infected cells at 16 h p.i. based on the APC3 coimmunoprecipitation data. These results indicate that the core APC in infected cells is no longer capable of associating with the activator as the infection progresses. While unlikely, we cannot exclude the possibility that 35S-Cdh1 was modified by a factor in the infected-cell lysate.
In accord with the in vitro binding experiments using exogenous Cdh1, we demonstrate by coimmunoprecipitation assays that APC1 becomes dissociated from the TPR subunits with similar kinetics. Recent studies have further defined the intricate architecture of the APC (7
). The complex is composed of two main subcomplexes, one containing the catalytic core (i.e., APC2 and APC11) and the other containing the TPR subunits (i.e., APC3, APC6, APC7, and APC8), that are bridged by APC1, APC4, and APC5 (39
) (Fig. ). The binding between APC1, APC4, APC5, and the TPR subunit APC8 also is interdependent, in that each subunit is required for the association of the other three (38
). Without APC1, the overall structure of the APC would be greatly affected, as the two subcomplexes likely would be separated. Since full binding of Cdh1 to the APC is dependent on both APC3 and APC2 (38
), the dissociation of the core complex also could account for the inability of Cdh1 to bind the complex and for the lack of APC activity. Interestingly, previous studies have suggested that the APC contains multiple copies of the TPR subunits (7
) and that these TPR subunits remain assembled even in the absence of APC1 (38
). This correlates with our finding that APC3, APC7, and APC8 still remained in complex together despite the dissociation of APC1 upon HCMV infection.
FIG. 7. APC is inactivated through multiple mechanisms upon HCMV infection. (A) Schematic diagram of activated APC. Subunits are numbered accordingly. Unphosphorylated Cdh1 associates with and activates the complex, allowing the ubiquitination of the recruited (more ...)
We further showed by IFA that several APC subunits relocalized to the cytoplasm as the infection progressed, while APC1 remained nuclear. It is unclear whether the dissociation of APC1 causes the other subunits to disperse into the cytoplasm or whether it is the TPR subcomplex that is dissociating from the rest of the APC.
An important question raised by these studies is the following: why does HCMV destabilize the APC? There are at least three different possibilities. First, to inhibit host cell functions or promote viral replication, the virus may require high levels of cellular proteins that normally would be ubiquitinated by the APC and degraded by the proteasome. An example might be the premature accumulation of geminin, which inhibits the licensing of cellular origins of DNA replication. A second possibility is that there may be essential viral proteins that would be targeted for degradation by a functional APC. Finally, one or more of the individual APC subunits may need to be recruited for a specific role in the viral infection. Studies are currently in progress to address this question and to further elucidate the molecular mechanisms by which the APC is destabilized.
In summary, multiple mechanisms appear to be involved in inactivating the APC upon HCMV infection, including dissociation of the core APC and the relocalization of some subunits to the cytoplasm of the infected cells, beginning 8 to 12 h p.i. This time frame also correlates with the observed accumulation of APC substrates (1
) and loss of APC activity (42
). Although it is unknown at this time whether these events are interdependent or represent redundant pathways, they underscore the importance of disabling the APC during the infection.