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Previous Article | Next Article 
Molecular and Cellular Biology, March 2003, p. 1885-1895, Vol. 23, No. 6
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.6.1885-1895.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
and Thomas Shenk*
Molecular Biology Department, Princeton University, Princeton, New Jersey 08544-1014
Received 31 October 2002/ Returned for modification 2 December 2002/ Accepted 6 December 2002
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ABSTRACT |
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INTRODUCTION |
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HCMV is a ubiquitous human pathogen, and a new infection or the reactivation of a latent infection in immunocompromised individuals can result in life-threatening disease. HCMV infection may also play a role in proliferative diseases such as atherosclerosis (27) and restenosis (38). Thus, we and others have been exploring how HCMV alters cell cycle progression (reviewed in reference 23). Infection of cells brought to quiescence either by contact inhibition or serum starvation results in an abortive mitogenic response with an increase in the level of cyclin E protein and kinase activity (4, 18). However, cyclin A is not induced, and cells do not synthesize host genomic DNA (4, 24). Thus, infection of quiescent cells stimulates their reentry into the cell cycle and progression through the G1 phase, with an eventual cell cycle arrest at the G1/S border. This cell cycle compartment is presumably favorable for viral replication, since the precursors for DNA replication are available but are not being consumed in the synthesis of the host cell's genome.
Here, we show that the HCMV UL82 gene product pp71 stimulates quiescent cells to enter the cell cycle. pp71 is a component of HCMV virions and is delivered to the cell at the very start of infection, a time when altering the host's cell cycle may be important to establishing a successful infection. pp71 targets the hypophosphorylated forms of the Rb family for proteasome-dependent degradation. Like the DNA tumor virus proteins E1A, T antigen, and E7, which contain LXCXE motifs (reviewed in reference 30), pp71 employs an LXCXD motif to attack the Rb pathway and induce cell cycle progression. However, unlike the DNA tumor virus oncoproteins, pp71 does not transform cells and does not induce apoptosis. pp71 sends a proliferative signal to cells that is strong enough to induce entry into the cell cycle and progression into S phase in the absence of other viral proteins but mild enough not to override the eventual cell cycle arrest imposed by the virus at the G1/S border during an infection. Thus, pp71 provides a new means with which to study the complexities of progression through the G1 phase of the cell cycle and the Rb/E2F pathway.
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MATERIALS AND METHODS |
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Plasmids. pCGN71 (C. J. Baldick and T. Shenk, unpublished data) encodes an XbaI-BamHI PCR fragment corresponding to the HCMV strain AD169 UL82 gene cloned in frame with the hemagglutinin (HA) epitope tag of the vector pCGN (41). This N-terminal HA-tagged pp71 allele was employed as the template to make all of the additional clones by PCR amplification. BamHI PCR fragments encoding N-terminal HA- and His-tagged pp71 alleles were cloned into pSG5 (Stratagene) to generate pSG5-pp71 and pSG5-His-pp71, respectively. The HA-pp71 point mutants were created by PCR amplification with mutagenic primers. pGSTpp71 was constructed by inserting a PCR fragment containing amino acids 1 to 407 of pp71 into pGTK (H. Zhu and T. Shenk, unpublished data). All clones generated by PCR amplification were confirmed by complete sequencing. pSG5L-HApRb (provided by J. DeCaprio) encodes HA-tagged Rb. pCMV-Rb, pCMV-p107, pHAp107, pCMV-p130, pHAp130, pCMVE2F-1, pCGNpp65, pSG5-T, pGSTpRb, pGSTp107, pGSTp130, pCMVE1A, pCMV19K, pCGNIE1, pCGNIE2, pCBSFlag-myc, and pH-rasEJ have been described previously (1, 8, 10, 19, 20, 25, 26, 34, 40, 43, 45, 46, 47).
Recombinant adenoviruses.
Recombinant adenoviruses were generated by using the AdEasy system (16) with pADEasy-1 and either pADtrack-CMV (16) or pACIC. ADTrack viruses express the indicated protein and enhanced green fluorescent protein (GFP), each from its own CMV promoter. To make pADIC, a PstI-BamHI internal ribosome entry site (IRES)-containing fragment from pIRES-EYFP (Clontech) was inserted into pECFP-N1 (Clontech) to make pIR-CFP. The IRES-enhanced cyan fluorescent protein (CFP) cassette was removed with XhoI and XbaI and cloned into the corresponding sites of pADtrack-CMV (the polylinker XhoI and a methylation-sensitive XbaI site after the enhanced GFP gene) to create pADIC. Adenovirus-IRES-CFP (ADIC) viruses express the gene of interest from the CMV promoter and the enhanced CFP gene from an IRES element. BamHI fragments encoding the desired gene were cloned into the BglII site of pADtrack-CMV or pADIC, and viruses were created as described previously (16). pADtrack-CMV and pADIC without inserts were used to make control viruses that express only the fluorescent protein. The ADtrack recombinant adenovirus expressing E2F-1 has been previously described (42). Purified adenovirus stocks were prepared as described previously (28), diluted with an equal volume of 2x storage buffer (50% glycerol, 4 mM MgCl2, 20 mM Tris [pH 8.0]), and stored at -20°C. Virus titers were determined by optical absorbance (28). Infections were at 
10,000 particles/cell.
Degradation analysis. For cotransfection assays, U-2 OS cells were transfected with the indicated plasmids by the calcium phosphate method. The DNA precipitate was removed 12 h later, and cells were rinsed and cultured in either 10% FBS-containing medium for 48 h or 0.1% FBS-containing medium for 24 h before protein lysates were harvested. Protein levels were quantitated by the Bradford assay, lysates separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blots were probed with the indicated antibodies. For the pulse-chase analysis, at 24 h after transfection U-2 OS cells cultured in 10% FBS-containing medium were incubated in cysteine- and methionine-free medium containing 10% dialyzed FBS for 1 h prior to a 30 min pulse-label period with 4 ml of medium containing 0.2 mCi of 35S-express (NEN)/ml. Cultures were chased in medium containing 10% FBS, and lysates were immunoprecipitated with the HA-specific antibody. Blots were quantitated with a phosphorimager. For the plots shown in Fig. 4A, arbitrary units for each time point were divided by the arbitrary units for the vector time-zero sample and the ratio is presented.
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Transformation assay. Primary rat embryo fibroblasts were a gift from M. Cole (Princeton University). Baby rat kidney cells were prepared essentially as described previously (37). Cells cultured on 10-cm-diameter plates were transfected with 3 µg of each of the indicated plasmids by the calcium phosphate method. Cultures were fed fresh media every 4 to 5 days, and transformed foci were counted after 18 to 21 days.
Antibodies and inhibitors. The following antibodies were from commercial sources: tubulin (for Western blots, DM 1A [Sigma]; for immunoprecipitations, H-300 [Santa Cruz]); HA (16B12; Babco); pp65 (1025; Rumbaugh-Goodwin Institute for Cancer Research, Inc., Plantation, Fla.); and E2F-1 (KH95), RB (C-20), p107(C-18), and p130 (C-20) (all from Santa Cruz). The E1A antibody was M73. The pp71 antibodies 10G11, 1E-233, 2H10-9, and 4F7 were identified in a screen of antibodies raised against HCMV virion proteins by P. Robinson. Mice were immunized with purified HCMV virions, and individual hybridomas that reacted in Western blots to virion proteins of approximately 70 kDa were screened for pp71 reactivity by Western blotting and immunoprecipitation of lysates from cells transfected with pCGN71. The pp71 antibody CMV355 has been described previously (31). For immunoprecipitations, a cocktail containing equal volumes of each of the five pp71 antibody tissue culture supernatants was employed. The following compounds were purchased from Calbiochem, dissolved in dimethyl sulfoxide (DMSO), and employed at the concentrations indicated in parentheses: lactacystin (20 µM), E64 (50 µM), proteasome inhibitor no. 1 (1 µM), ALLN (50 µM), epoxomicin (10 µM), and clasto-lactacystin ß-lactone (20 µM).
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RESULTS |
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We were also able to demonstrate a pp71-p107 in vivo interaction by immunoprecipitating pp71 and probing Western blots for bound p107 (Fig. 2D). p107 was captured by a pp71 antibody cocktail only when pp71 was coexpressed in cells, and it was not captured in pp71-coexpressing cells by an antibody to the adenovirus E1A protein. We were unable to detect an in vivo interaction between pp71 and either Rb or p130 by immunoprecipitation with the pp71 antibodies (data not shown). Although we could detect this complex by immunoprecipitation with antibodies to the Rb family member (Fig. 2C), the half-life of these complexes (see below) as well as the higher steady-state levels of pp71 in comparison to those of the Rb family members in these experiments (data not shown) probably prevent us from detecting all but the most-abundant pp71-p107 complexes with antibodies to pp71.
We also employed pp65 as a control in these binding studies since it is similar in sequence to pp71 but does not induce cell cycle progression in quiescent cells (Fig. 1). pp65 and pp71 are 26% identical and 44% homologous over the central 57% of the protein (data not shown) that contains the LXCXD motif of pp71. However, this sequence is altered to LXCXM in pp65, and despite their overall similarity, pp65 is unable to bind p107 in vivo (Fig. 2E). This finding agrees well with the inability of pp65 to stimulate the cell cycle and the importance of a proper LXCXE/D motif for interaction with the Rb family.
pp71 degrades the hypophosphorylated Rb family members. When we performed Western blots to analyze the lysates that were employed in the in vivo association experiments described above, we observed a decrease in the level of the Rb family proteins in cells expressing pp71 compared to E1A (Fig. 3A). We reexamined the effect of pp71 expression on the steady-state levels of each member of the Rb family in cotransfection assays by performing Western blot analysis with equal amounts of protein lysates that were separated on low-percentage polyacrylamide gels to distinguish between the differentially phosphorylated forms of the Rb family members. Under these conditions, hypophosphorylated forms migrate farther in the gels than hyperphosphorylated forms. Although only a small change in the levels of p107 was detected, we once again observed a dramatic decrease in the levels of Rb and p130 in the presence of pp71 (Fig. 3B). Interestingly, the growth-suppressive hypophosphorylated forms of the two proteins were preferentially missing. We also compared the effect of pp71 and pp65 expression on the levels of p130 (Fig. 3C) and found that, compared to pp65, which does not bind the Rb family (Fig. 2E) and does not stimulate the cell cycle (Fig. 1), pp71 expression caused a drop in the level of the p130 protein, with the hypophosphorylated forms preferentially missing. Thus, we speculated that pp71 might specifically bind to and subsequently degrade the hypophosphorylated forms of the Rb family members. Another possibility was that pp71 could phosphorylate the Rb family, but since it contains no consensus kinase or ATP binding motifs, we considered this unlikely.
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To test for degradation, we performed a pulse-chase analysis (Fig. 4), which demonstrated that the half-lives of Rb, p107, and p130 are all decreased by pp71. The half-life of Rb drops from >8 h to 3.9 ± 1.2 h, the half-life of p107 drops from 4.3 ± 0.3 h to 2.4 ± 0.4 h, and the half-life of p130 drops from 1.8 ± 0.1 h to <30 min. For p130, a significant amount of the protein was degraded during the 30-min pulse period before the chase was initiated. The proteins in labeled immunoprecipitates from the time course exhibited biphasic degradation curves in the presence of pp71 (Fig. 4A). The Rb family members are extremely susceptible to degradation by pp71 during the initial chase period, but at later time points they are more stable. This is the expected result if pp71 is degrading the hypophosphorylated forms of the proteins shortly after they are made, and once phosphorylated, they become more resistant to degradation. An autoradiograph of the first two time points of the Rb pulse-chase analysis supports this hypothesis (Fig. 4B). A qualitative examination indicates that, without pp71, there is more hypophosphorylated Rb at time zero than of the slower-migrating, hyperphosphorylated band. After 1 h of the chase period, a significant conversion of hypophosphorylated to hyperphosphorylated form is observed. In contrast, in the presence of pp71, there are similar amounts of hypo- and hyperphosphorylated Rb at time zero, and by 1 h, the hypophosphorylated form is substantially reduced with very little increase in the hyperphosphorylated form. This argues for a mechanism in which the hypophosphorylated form is lost by degradation not by conversion to the phosphorylated species. A quantitative examination of two independent experiments (Fig. 4C) confirms this interpretation. During the first hour of chase, a higher percentage of hypophosphorylated Rb is lost in the presence of pp71 than in its absence. However, only a small minority is converted to the hyperphosphorylated form in the presence of pp71 (much less than in its absence), and therefore, it must be degraded.
The pp71 LXCXD motif is required for the induction of DNA synthesis in quiescent cells and for degradation of the Rb family. We generated a mutation in the pp71 LXCXD motif (Fig. 5A) modeled after one that disrupted the ability of E1A to bind Rb and transform cells (7). This mutant protein (C219G), which contains a single cysteine-to-glycine change in the LXCXD motif, accumulates to levels similar to those of wild-type pp71 (Fig. 5C, bottom panel) and correctly localizes to the nucleus (data not shown). We made a recombinant adenovirus that expresses the C219G mutant and demonstrated by flow cytometry (Fig. 5B) and BrDU uptake (Fig. 5C, top panel) that it fails to induce DNA synthesis in quiescent cells.
Since the C219G mutant failed to induce DNA synthesis in quiescent cells, we reasoned that it would also be incapable of degrading the Rb family members. Western blotting of lysates from cotransfection assays demonstrated that the C219G mutant was indeed unable to degrade Rb (Fig. 6A) and p130 (see below) to the same extent as the wild-type protein. However, the C219G protein retained an unrelated function of pp71. When transfected into permissive cells, viral DNA produces live virus. This happens at a low efficiency that can be dramatically increased by cotransfection with a pp71 expression plasmid but not with plasmids expressing other tegument or immediate-early proteins (1). The C219G protein was able to enhance the infectivity of viral DNA as efficiently as wild-type pp71 (Fig. 6B). Consequently, we can conclude that the C219G mutation does not lead to a global inactivation of pp71. Rather, it specifically abolishes its cell cycle regulatory function.
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Unlike the DNA tumor virus oncoproteins, pp71 does not induce apoptosis and does not cooperate to transform cells. In many respects, pp71 appears to function in a fashion essentially identical to E1A, T antigen, and E7. These DNA tumor virus oncoproteins also share the ability to induce apoptosis (13, 32, 35) and to transform cells (reviewed in reference 30). We reasoned that if pp71 were to function exactly like these proteins, it would also retain these activities. Thus, we asked whether pp71 could induce apoptosis and transform cells.
We infected quiescent cells with recombinant adenoviruses expressing either pp71 or E2F-1 and assayed for apoptosis by determining the percentage of cells with a sub-G0 DNA content. Overexpression of E2F-1 has been shown to induce apoptotic cell death (9). Infection of quiescent cells with the recombinant adenovirus expressing E2F-1 led to apoptosis, as expected (Fig. 8A). However, pp71 induced little apoptosis at either time point examined, even though, in these same experiments, we observed significant cell cycle stimulation by pp71 (Fig. 8A). Although it is possible that pp71 induces and subsequently blocks apoptosis much like T antigen, we find this unlikely since pp71 was unable to prevent E2F-1-induced apoptosis (Fig. 8B). In this respect, the interaction of pp71 with the cellular pathways leading to apoptosis, and thus presumably the mechanism through which it induces cell cycle progression, is different from that of E1A, T antigen, and E7.
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DISCUSSION |
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Furthermore, proteasome inhibitors arrest cells in the G2 phase (reviewed in reference 29), where kinases that phosphorylate the Rb family are active and hyperphosphorylated forms of these proteins accumulate. If pp71 were to induce the phosphorylation of the Rb family, one would predict that proteasome inhibitors would either have no effect or perhaps enhance the ability of pp71 to direct the loss of hypophosphorylated Rb. However, these inhibitors stabilize the hypophosphorylated forms of the Rb family in the presence of pp71 (Fig. 7), opposing its function. Once again, this supports a model where pp71 works to degrade the Rb family members and not to directly or indirectly induce their phosphorylation. Furthermore, the E1A protein, which stimulates the cell cycle, does not affect the level of the Rb proteins in experiments in transiently transfected, asynchronous cells (Fig. 3A), where we have shown pp71 to have a dramatic effect (Fig. 3A to C, 4, 6A, and 7). Thus, the instability of the hypophosphorylated forms of the Rb proteins in these experiments is unique to pp71 and thus cannot be the result of cell cycle stimulation-induced phosphorylation.
pp71 and the DNA tumor virus oncoproteins E1A, T antigen, and E7 all contain an LXCXE/D motif that mediates binding in the pocket domains of the Rb family members. However, beyond this conserved motif, there are significant differences in the mechanisms with which these viral proteins attack the Rb pathway, in the eventual fate of the targeted Rb family member, and in the consequences of the cell cycle stimulation induced by these proteins.
E1A requires a CR1 domain that helps disrupt Rb-E2F complexes by competing for the E2F binding site on the Rb proteins (17). E1A does not degrade the Rb family members (2), but it does prevent the phosphorylation of p107 and p130, but not Rb, independently of its ability to bind them (33). T antigen prevents the phosphorylation of p107 and p130 through an unknown mechanism that requires binding (39). T antigen also degrades p130, but not Rb or p107 (40). The degradation is mediated by the proteasome and requires the DNA J domain of T antigen (40). E7 degrades all of the differentially phosphorylated forms of Rb in a reaction that requires proteasome function (3). Although this degradation was originally thought to be specific for Rb, recent evidence suggests that E7 also degrades p107 and p130 (2, 3, 14). An amino-terminal domain of unknown function is required for the E7-mediated degradation of Rb. We have no evidence that pp71 effects the phosphorylation status of the Rb family. It degrades Rb, p107, and p130 and has a strict preference for their hypophosphorylated forms (Fig. 3B and C, 4, and 7). As in each of the examples above, it is likely that a domain in addition to the LXCXD motif of pp71 is required for the degradation of the Rb family, and we are actively searching for such a sequence.
In addition to the subtle differences between pp71, E1A, T antigen, and E7 with regards to the effects on the phosphorylation and stability of the targeted Rb family member, as well as on the protein domains other than the LXCXD/E motif employed by these proteins, a more-profound difference separates pp71 from the others. Whereas E1A, T antigen, and E7 each induce apoptosis and transform cells, pp71 can perform neither of these functions (Fig. 8). Thus, the mechanism and consequences of the pp71-mediated degradation of the Rb family differs in important aspects from the way in which the DNA tumor virus proteins function.
There are several possible models for how pp71 attacks the Rb family and stimulates cell cycle progression without inducing apoptosis or transforming cells. For example, while conversion of the LXCXE in E1A to LXCXD did not abolish that protein's ability to bind Rb and transform cells (7), it did attenuate it. Perhaps because of the altered consensus sequence of the Rb-binding motif, pp71 is not as efficient as the DNA tumor virus oncoproteins in targeting the Rb family. If this were true, pp71 may deliver a signal that, while sufficient to induce cell cycle progression, may not be strong enough to trigger apoptosis or result in transformation. Alternatively, in addition to degrading the Rb family member, pp71 may degrade the E2F component of the Rb-E2F complex as well. This may serve to derepress the E2F-responsive genes but not activate them to a high degree, thus promoting an attenuated proliferative response.
A speculative yet provocative model is that pp71 may interact with Rb-E2F complexes in a manner that is fundamentally different from the way in which the DNA tumor virus proteins are thought to function. Although pp71 induces cell cycle progression, it does so less efficiently than E2F-1 (Fig. 5C and 8A and data not shown). Another member of the E2F family, E2F-4, also induces cell cycle progression, but it does so to a lesser extent than E2F-1 (9). Furthermore, E2F-1 is the only member of that family of transcription factors that induces apoptosis upon overexpression (9). Since the efficiency of cell cycle and apoptosis induction by pp71 more-closely mimics overexpression of E2F-4 than E2F-1, a mechanism in which pp71 were to target E2F-4-bound Rb family members more efficiently than those bound to other E2F family members is consistent with our cell cycle and apoptosis data. If this model is correct, then perhaps more E2F-4 is released than other family members, such as E2F-1. This in turn sends a moderate proliferative signal to the cell, one that HCMV can efficiently block before the infected cells enter the S phase (4, 24) and one that might not lead to apoptosis or transformation. This model is consistent with our data, since E2F-4 can bind to each of the Rb family members but is most often found in complexes with p130 (which we find to be degraded by pp71 with very rapid kinetics) and p107 (which we find binds most strongly to pp71). A comprehensive series of experiments will be required to test each of these possible models and to determine the molecular details of the mechanism employed by pp71 to disrupt the Rb pathway.
The inability of pp71 to induce apoptosis partially explains why inhibitors of apoptosis do not cooperate with pp71 to transform cells (Fig. 8C and D). However, unlike E1A, pp71 was also unable to cooperate with an activated ras allele in transformation assays. If pp71 does not interact as strongly with the Rb family members or in a way fundamentally different from that described above, this may explain why pp71 fails to transform cells. Alternatively pp71 may target Rb efficiently but lack an additional function required for transformation. For example, in addition to targeting the Rb family, E1A and T antigen also disrupt the function of the p300 tumor suppressor (reviewed in reference 15), and this activity is required for cellular transformation. Perhaps the ability to target p300 has not been conserved in pp71, and thus, it cannot transform cells. Further experiments are required to determine if pp71 fails to transform cells because of the way it targets Rb-E2F complexes or because it lacks an additional required activity, such as antagonizing p300 function.
Finally, pp71 represents the first example of a herpesvirus protein to utilize an LXCXD/E motif to target the Rb family. Although it shares many properties with the oncoproteins from the DNA tumor viruses, we believe the unique features of this interesting protein will provide an opportunity to further investigate the roles of the Rb proteins not only in HCMV infection but also in cellular growth control and carcinogenesis.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the NIH (CA82396).
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FOOTNOTES |
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Present address: Department of Microbiology, Immunology, and Medicine, University of California Medical Center, San Francisco, Calif.
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