The Carboxyl-Terminal Region of Human Cytomegalovi
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病菌学杂志 2005年第1期
Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California
Department of Biochemistry, Drexel University College of Medicine, Philadelphia, Pennsylvania
ABSTRACT
The human cytomegalovirus major immediate-early () protein IE1491aa plays an important role in controlling viral gene expression at low multiplicities of infection. With a transient complementation assay, full-length IE1491aa enhanced the growth of ie1 mutant virus CR208 20-fold better than a deletion mutant lacking 71 carboxyl-terminal amino acids (IE11-420aa). A 16-amino-acid domain between amino acids 476 and 491 was both necessary and sufficient for chromatin-tethering activity; however, this domain was completely dispensable for complementation of CR208 replication. The proximal 55-amino-acid acidic domain (amino acids 421 to 475) was found to be most important for function. A deletion mutant lacking only this domain retained chromatin-tethering activity but failed to complement mutant virus. Interestingly, serine phosphorylation (at amino acids 399, 402, 406, 423, 428, 431, 448, 451, and 455) was not required for complementation. These results show that IE1491aa is composed of at least two domains that support replication, a region located between amino acids 1 and 399 that complements ie1 mutant virus replication to low levels and an acidic domain between amino acids 421 and 479 that dramatically enhances complementation.
INTRODUCTION
Human cytomegalovirus (HCMV), the prototype of the betaherpesviruses, is a widespread opportunistic pathogen (40) causing neurological damage as a result of congenital infection as well as significant disease in organ transplant recipients and AIDS patients. Like all herpesviruses, the replication cycle of HCMV is temporally regulated by viral functions. The , or immediate-early (IE), phase immediately following viral entry is activated by virion-associated regulators independent of viral gene expression; the ?, or delayed-early, phase is activated by gene products but is independent of viral DNA replication; and the , or late, phase is activated by gene products as well as functions associated with viral DNA replication (33).
Infection has a dramatic impact on cellular gene expression as well (5, 19). The timing and intensity of these phases are also influenced by host cell type, differentiation state, and cell cycle status (6, 7, 11, 33). Undifferentiated human cells and cells from other animal species except chimpanzees (41) are nonpermissive for HCMV replication and are generally restricted at a step following entry, uncoating, and gene expression (28). The two major gene products, IE1491aa and IE2579aa, control the progression of HCMV replication in permissive cells by regulating later classes of viral gene expression (33). Ribozymes targeted to an ie1/ie2 common exon inhibit replication (55). Viral mutants that lack IE1491aa but express IE2579aa show a multiplicity of infection (MOI)-dependent block to replication associated with a failure to express critical viral gene products (12, 15, 35). Though less abundant than IE1491aa, IE2579aa is widely believed to be the key viral regulatory gene product based on several lines of evidence: (i) strong transactivation of TATA box-containing promoters from viral or cellular sources in transient assays (33), (ii) association with a wide range of cellular transcription factors and cell cycle-related proteins similar to adenovirus E1A (6, 7, 33), and (iii) failure of viral mutants in this gene or its murine CMV homologue (ie3) to replicate or to express viral ? or gene products (3, 30, 50).
IE1491aa (also called IE1 p72, IE72, and IE1), the major (immediate-early) gene product of CMV, is a 72- to 75-kDa phosphoprotein that accumulates in the nucleus throughout HCMV infection. Early evidence from transient assays revealed an adjunct role for IE1491aa in cooperation with IE2579aa to transactivate viral and cellular promoters (33). In the absence of IE2579aa, IE1491aa transactivates the ie1/ie2 promoter-enhancer (9) as well as promoters from selected cellular genes, including heat shock protein 70 (16), interleukin (IL)-1? (20), IL-6 (13), IL-8 (37), DNA polymerase (18), origin recognition complex I protein (47), and dihydrofolate reductase (32). In addition to its possible role in activation of host cell gene expression, IE1491aa represses expression of the IL-1 receptor type 1 gene in transient assays (24). IE1491aa induces transcription factors, including NF-B (9, 44), activator protein 1 (23), and E2F1 (32), and may participate in stimulation of the host cell toward an S-phase-like state (6, 11, 33).
IE1491aa is sumoylated on Lys450 (49, 52) and disrupts nuclear domain 10 (PML bodies) (2, 26), which is dependent on Leu174 (36, 51). IE1491aa-mediated disruption of nuclear domain 10 plays no apparent role in viral replication because ie1 mutant viruses fail to disrupt nuclear bodies (1) at high MOIs, where replication proceeds unimpeded. IE1491aa is a phosphoprotein (38) and is phosphorylated in the presence or absence of other viral proteins (7, 39) at a number of sites (Ser399, Ser402, Ser406, Ser423, Ser428, Ser431, Ser448, Ser451, and Ser455) near or within a carboxyl-terminal acidic domain (AD) (Himmelheber et al., 28th International Herpesvirus Workshop, abstract 5.32). IE1491aa may undergo autophosphorylation and may influence the phosphorylation state of other proteins, such as E2Fs and retinoblastoma protein family members (7, 39). The contribution of individual functional domains to these various IE1491aa functions has not yet been fully evaluated, with the exception that sumoylation (at Lys450) has been shown to be dispensable for viral growth (49).
IE1491aa may alter host cell behavior (46) such as susceptibility to apoptosis (54), suppress p107-mediated block in cellular proliferation (22), and activate cyclin E/cdk2 kinase activity (53), although these phenomena have only been observed in transient assays. The available ie1 mutant viruses show no obvious signs of altered host cell behavior during infection in permissive human fibroblasts (HFs) (12, 15; Dittmer and Mocarski, unpublished observations). Furthermore, constitutive expression of IE1491aa in normal or immortalized HFs has little overall impact on cell viability, growth properties, cell cycle control, or susceptibility to apoptosis (16; Dittmer and Mocarski, unpublished observations).
Unambiguous evidence supporting a role of IE1491aa in viral replication emerged from studies on ie1-deficient viruses (15, 35), which are replication deficient at low MOIs. The defect has been ascribed to poor expression of viral ? and genes, including UL44, UL57, UL69, UL98, and UL112 (12, 15). Although the mechanism underlying the MOI-dependent growth of this mutant is not understood, the results are consistent with an accessory role of IE1491aa acting together with IE2579aa to enhance the expression of essential viral ? and genes. Constitutive (15) or transient (this study) expression of IE1491aa fully complements mutant viral replication.
IE1491aa tethers to host metaphase chromatin during mitosis via an acidic carboxyl-terminal region (29, 51); however, no role for tethering in viral replication or in modifying host cell behavior has been reported. Two well-known gammaherpesvirus proteins tether as a part of their natural function, Epstein-Barr virus nuclear antigen 1 (EBNA-1) (31) and Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) (42). Tethering to metaphase chromatin is part of the genome maintenance function of these proteins during latency and collaborates with latent origin-specific binding to control replication of the viral genome. IE1491aa has no known role in HCMV latency and has not been found to bind directly to viral or host DNA. IE1491aa expression has not been detected during natural latency and becomes undetectable within a few days in experimental models of latency in myeloid progenitors (17, 25). Thus, any role of this regulatory protein in latent genome maintenance remains enigmatic.
We used the MOI-dependent growth properties of ie1 mutant virus CR208 as an assay system to study the role of IE1491aa domains during infection. A portion of IE1491aa (amino acids 1 to 420) was found to retain partial function, with the carboxyl-terminal acidic region (amino acids 420 to 479) necessary for full trans-complementation of the growth of CR208. This assay provided a simple and direct means to separate the roles of the chromatin-tethering domain (CTD; amino acids 476 to 491) and the adjacent AD (amino acids 421 to 479) without introducing the adventitious mutations that result in growth defects independent of the targeted gene that are found in HCMV mutants made by transfection of plasmids, cosmids, or bacmids (34). Our results also indicate that the acidic character of rather than serine phosphorylation in the AD region is important for complementation.
MATERIALS AND METHODS
Plasmids. All ie1 mutants were constructed by PCR-driven mutagenesis with plasmid pON2202 (21) as a template. pCMV IE1full-length and pCMV IE1full-lengthGFP were constructed from a cDNA generated by PCR with oligonucleotides 1 and 2 (Table 1). pCMV IE11-420 and pCMV IE11-420GFP were generated by PCR with oligonucleotides 1 and 3. pCMV IE11-479 and pCMV IE11-479GFP were generated by PCR with oligonucleotides 1 and 4. The resulting PCR products for these nontagged cDNAs were cloned into HindIII- and XhoI-digested pcDNA3.1 (Invitrogen) and subsequently cloned into pCMV(4x)GFP after digestion with NheI and XbaI as green fluorescent protein (GFP)-tagged derivatives.
Plasmid pCMV(4x)GFP, a gift of Irina Conboy, expresses GFP under the control of an HCMV promoter in which the four NF-B sites were destroyed with point mutations (Conboy and Jones, personal communication). pCMV IE1421-475GFP was generated by ligating annealed oligonucleotides 5 and 6 to XhoI- and XbaI-digested pCMV IE11-420GFP, thereby adding two amino acids (threonine and methionine) between codons 420 and 476. To construct pCMV IE1476-491GFP, annealed oligonucleotides 5 and 6 were ligated to Xhol- and Xbal-digested pCMV(4x)GFP, thereby placing codons 476 to 491 immediately downstream of the natural IE1491aa initiation codon. To construct pCMVIE11-442GFP, pCMV IE11-414GFP, and pCMV IE11-399GFP, PCR products generated with oligonucleotides 2 and 8, 2 and 9, 2 and 10, respectively, were cloned into NheI- and XhoI-digested pCMV(4x)GFP. The pSG5-IE1 plasmid (provided by John Sinclair) was used as a template to make ie1 mutants with serine modifications at amino acids 399, 402, 406, 423, 428, 431, 448, 451, and 455. These serines were exchanged in groups of three to either alanine (pSG5-IE1SA) or glutamate (pSG5-IE1SE) with the QuikChange site-directed mutagenesis kit (Strategene) according to the manufacturer's protocol. The sequences of all primers are shown in Table 2 (48).
To generate the construct in which all nine serines were mutated, mutation reactions were performed sequentially with single and then double group mutations as templates. The original constructs were transferred into the pCMV-GFP backbone for comparison with the deletion mutants by PCR to amplify the region representing amino acids 350 to 491 followed by BglII and XhoI digestion and ligation into BglII- and XhoI-digested pCMV-IE1491GFP.
Cell and virus culture. All cells were cultivated at 37°C in a 5% CO2 atmosphere. HFs prepared locally from pooled newborn foreskins and immortalized ihfie1.3 cells stably expressing IE1491aa (15) were maintained in Dulbecco's modified Eagle's medium (Gibco-BRL) supplemented with 10% NuSerum I (Collaborative Research Inc.), 100 U of penicillin/ml, 100 U of streptomycin sulfate/ml, 0.66 mM arginine, 1.48 mM glutamine, and 0.24 mM asparagine (complete medium). The ie1-deficient HCMV strain CR208 and the repaired strain CRQ208 (15) were cultured in ihfie1.3 or in HF cells, respectively, as described (15). Cell supernatants were clarified by sedimentation at 1,500 x g for 20 min, disrupted with a probe type sonicator, and stored in aliquots at –80°C as virus stock. Infections were carried out with virus diluted in DMEM-0.2% NuSerum, and cells were rinsed once and incubated in complete medium supplemented with 0.16% pooled human gamma globulin (Baxter Corp.) to prevent viral spread through the medium. Virus titrations were performed in six-well dishes incubated for 10 days postinfection, at which time cells were methanol fixed and stained with Giemsa.
Cell transfection. Transfection of HFs was carried out with calcium phosphate precipitation (12, 15) modified as follows. HFs were split at a ratio of 1:2, incubated for 1 day, and split so that 2.2 x 105 cells were seeded into each well of a six-well dish. One day later, the medium was replaced with 2 ml of complete medium. To 15 μg of plasmid DNA and 31 μl of 2 M CaCl2, sterile H2O was added to a volume of 219 μl. Subsequently, 250 μl of 2x HBS (280 mM NaCl, 1.4 mM Na2HPO4, 10 mM KCl, 5.6 mM glucose, 20 mM HEPES, pH 7.05) was added dropwise, and this mixture was added to cells. After incubation for 4 to 6 h, the medium was removed and the cells were incubated for 90 s with 15% (vol/vol) glycerol in 1x HBS followed by four washes and addition of complete medium. GFP-based fluorescence 24 h later was used to assess transfection efficiency. For transfection-infection assays, the transfected cells were infected at 2 days posttransfection.
Immunoblot. Cells were harvested at 48 h posttransfection, lysed in 2x Laemmli buffer, and separated on a 10% polyacrylamide-sodium dodecyl sulfate gel (27). Proteins transferred to a nitrocellulose membrane were detected with anti-IE1/IE2 monoclonal antibody 810 (Chemicon) and anti-?-actin antibody A5441 (Sigma) with the enhanced chemiluminescence kit (Amersham Corp.) following the manufacturers' instructions.
Sequence analysis. The sequence of HCMV Towne strain IE1491aa (accession number gi:73633) amino acids 421 to 491 was aligned to that of full-length chimpanzee CMV IE1 (gi:19881132), rhesus CMV IE1 (gi:332085), and simian CMV IE1 (gi:1616980) with ClustalW of MacVector 7.1.1 (Accelrys, San Diego, Calif.).
RESULTS
Complementation of CR208 by transiently expressed IE1491aa and IE11-420. Replication of ie1-deficient CR208 is severely compromised on normal HFs at MOIs of <1, and this defect is complemented on IE1491aa-expressing ihfie1.3 cells (12, 15). We took advantage of this MOI-dependent growth phenotype to investigate the ability of full-length and mutant forms of IE1491aa to complement CR208 plaque formation following transient transfection. We first evaluated the relationship between MOI and plaque formation by plating serial threefold dilutions of CR208 on either normal HFs or complementing ihfie1.3 cells (Fig. 1A). Maximal differences in efficiency of plaque formation on 2.2 x 105 HFs were observed at MOIs below 0.01, where CR208 yielded few or no plaques and transfection of full-length IE1491aa into 10% of the cells resulted in the formation of up to 103 plaques (Fig. 1B). Therefore, MOIs in this range were employed in experiments to compare IE1491aa and mutants by a transient complementation assay.
We compared complementation of CR208 by full-length IE1491aa and a mutant lacking the carboxyl-terminal region (amino acids 421 to 491). This region has a dense concentration of 26 acidic amino acids (29), a chromatin localization domain (51), and nine sites of potential serine phosphorylation (Himmelheber et al., 28th International Herpesvirus Workshop, abstract 5.32). We transiently expressed pCMV IE1full-lengthGFP or mutant pCMV IE11-420GFP in HFs and subsequently infected these cells with CR208 at an MOI of 0.005 (determined by plaque assay on ihfie1.3 cells and depicted by the open bar in Fig. 1). Transient expression of the mutant resulted in 30-fold fewer plaques than with the full-length construct (Fig. 1B), even though the transfection efficiency of the plasmids was similar, based on the number of GFP-positive cells (data not shown). HFs transfected with the full-length construct complemented CR208 at levels that were comparable to infection of ihfie1.3 cells (Fig. 1B), so long as the transfection efficiency was taken into consideration. Plaquing efficiency following transfection by the method used here was consistently 10 to 20% of that observed in stably expressing cells.
As expected, the failure of CR208 to form plaques at this MOI did not change when a control GFP expression vector was transfected into the cells (Fig. 1B). The pCMV IE1full-lengthGFP construct showed a transfection efficiency and complementation pattern similar to that of the nontagged full-length IE1full-length construct (data not shown). These results identified at least one potentially important functional domain in the carboxyl-terminal region of IE1491aa and revealed a low-level complementation activity of the truncated IE11-420 that was greater than that of negative controls. Furthermore, plaques formed with either full-length IE1 or the IE11-420 mutant were large, comparable in size to those formed by CR208 in ihfie1.3 monolayers or by parental virus on HFs (data not shown), suggesting that both retained full ability to complement cell-to-cell spread (12, 16) despite the fact that the transient transfection procedure did not deliver IE1 to all cells.
Conservation of two distinct domains within the carboxyl terminus of primate CMV homologs of IE1491aa. The carboxy-terminal 71 amino acids of IE1491aa include 26 acidic amino acids distributed in clusters containing as many as six consecutive acidic amino acids (Fig. 2A). The region is therefore strongly acidic (38%) and contains only one basic amino acid except for a short region located at the extreme carboxyl terminus. This 16-amino-acid region has a greater proportion of basic amino acids (Fig. 2A). The estimated isoelectric point of the extreme carboxyl-terminal 16 amino acids (476-491) is 9.81, compared with 3.64 for amino acids 420 to 491 and 4.54 for full-length IE1491aa. A comparison of this 71-amino-acid region from HCMV with homologues from chimpanzee CMV, simian CMV, and rhesus CMV revealed an AD with an adjacent highly conserved basic region towards the extreme carboxyl terminus in all (Fig. 2A). For example, the carboxyl terminus of HCMV and chimpanzee CMV IE1 exhibit 87% identity, compared to an overall 67% identity for the full-length proteins and 50% identity for the AD (data not shown). Parenthetically, neither rat nor murine CMV ie1 proteins have a domain that is homologous to this short carboxyl-terminal basic region (data not shown).
Chromatin tethering by the carboxyl-terminal 16 amino acids of IE1491aa. Previous studies showed that the IE1491aa protein tethers to metaphase chromosomes in transiently transfected cells (29, 51), although the importance of this activity to virus replication has not been addressed. A deletion affecting a large portion of the carboxyl-terminal 71 amino acids (amino acids 421 to 485), removing the entire acidic region along with part of the basic, highly conserved extreme carboxyl terminus, disrupted the ability of IE1491aa to tether (51). In order to more precisely identify the region responsible for chromatin tethering by IE1491aa, we constructed a series of deletion mutants, leaving amino acids 1 to 420 (pCMV IE11-420GFP), 1 to 479 (pCMV IE11-479), or 1 to 420 together with 476 to 491 (pCMV IE1421-475GFP) all fused to the amino terminus of GFP. In parallel, the extreme carboxyl-terminal IE1491aa amino acids 476 to 491 were placed downstream of the natural IE1491aa start codon and expressed as an amino-terminal fusion to GFP (pCMV IE1476-491GFP). The constructs were transiently transfected into HFs to assess chromatin-tethering activity in mitotic cells with either IE1491aa monoclonal antibody or GFP monoclonal antibody in immunofluorescence analysis (Fig. 2B) together with a DNA counterstain (Hoechst 33342 or propidium iodide; Fig. 2B). For comparison, the full-length IE1491aa protein was also visualized in stably expressing ihfie1.3 cells with monoclonal antibody.
While full-length protein associated with chromatin in dividing cells as expected (30, 51) (Fig. 2B), IE11-479 failed to bind to metaphase chromatin (Fig. 2B) (51). To directly assess the capacity of the different regions to mediate tethering, deletion of the 55-amino-acid acidic domain (pCMV IE1421-475GFP) did not disrupt the ability to associate with metaphase chromatin, and addition of the extreme carboxyl-terminal 16 amino acids (pCMV IE1476-491GFP) to GFP was sufficient for association (Fig. 2B). GFP alone stained the entire metaphase cell and was excluded from chromatin (data not shown). Thus, a CTD is located in a highly conserved, extremely carboxyl-terminal 16-amino-acid region of IE1491aa. This domain is both necessary and sufficient for binding to metaphase chromatin in transfected cells.
Role of the AD and CTD in complementation of CR208. In order to evaluate the role(s) of the 55-amino-acid AD and 16-amino-acid CTD in complementation of ie1 mutant virus, HFs were transfected with pCMV IE1full-lengthGFP, pCMV IE11-479, pCMV IE11-420GFP, pCMV IE1421-475GFP, and control pCMV-GFP. At 48 h posttransfection, cells were infected with CR208 at low MOIs, and plaque counts were performed 10 days later. Figure 3A shows complementation experiments at MOIs of 0.0004 and 0.002, which revealed the importance of the AD in complementation of ie1 mutant virus during replication at low MOIs. As expected, IE11-420 was defective in complementation, IE1491aa fully complemented plaque formation, and the GFP control plasmid failed to complement virus growth (Fig. 3A and data not shown). IE11-479 complemented CR208 as well as full-length IE1 at all MOIs tested (Fig. 3A and data not shown), consistent with a role for the AD in supporting replication.
Transfection of pCMV IE1421-475GFP, which lacked the 55-amino-acid AD but contained the 16-amino-acid CTD (Fig. 2B), complemented at about the same poor level as IE11-420. These results, which employed IE11-479 without a GFP fusion, were confirmed with pCMV IE11-479GFP (data not shown). Thus, the ability to tether chromatin did not contribute to IE1491aa function in viral replication in HFs, suggesting that this activity may be important in other cell types or, as suggested by the function of the other characterized herpesvirus tethering proteins LANA (42) and EBNA-1 (31), in latency. The AD is critical to the activity of IE1491aa during viral replication.
When the ratios of plaques obtained with these three ie1 mutants were calculated as a percentage of the plaques obtained with the full-length construct, differences were readily appreciated at MOIs of 0.0004, 0.002, and 0.01 (Fig. 3B). First, as expected, background replication in the absence of complementation was detected at the highest MOI, consistent with the established growth properties of CR208 (12, 15) and as shown in Fig. 1. Second, complementation appeared to be highly dependent on the AD at all MOIs tested, so that amino acids 1 to 420 complemented to consistently low levels. Finally, complementation by the 1 to 479 construct, which lacked the ability to tether chromatin, ranged from 180% of that of the full-length construct (MOI of 0.0004) to 130% (at an MOI of 0.002) to 60% (at an MOI of 0.01), all consistent with our interpretation that the CTD is dispensable for replication.
The behavior of the mutant with amino acids 1 to 420 fused to the CTD at amino acids 476 to 491 but lacking the AD (pCMV IE1421-475GFP) showed complementation similar to the region with amino acids 1 to 420 alone at low MOI (0.0004); however, complementation improved as the MOI was increased to 0.01, suggesting that the addition of the CTD may have had a modest positive impact on amino acids 1 to 420 in the absence of the 55-amino-acid AD. Taken together, these data emphasize the importance of this region of high negative charge as a critical functional domain for optimal viral replication.
To exclude any possibility that the differences in complementation resulted from different expression levels, we performed an immunoblot analysis of lysates of HFs transfected with IE1full-lengthGFP, IE11-420GFP, IE1421-475GFP, and IE11-479 (Fig. 3C). These four IE1 constructs did not differ in their expression levels. Therefore, the carboxyl-terminal 71 amino acids contribute to replication primarily through the presence of an AD and not because of variation in expression levels.
Role of phosphorylated serines in the acidic domain for complementation of CR208. IE1491aa was subjected to additional deletions and point mutations to investigate the role of acidic amino acids as well as other sequences in this region. pCMV IE11-442GFP lacks 37 amino acids of the AD, retaining only 13 of the 26 acidic amino acids; pCMV IE11-414GFP and pCMV IE11-399GFP lack the AD plus additional amino acids. When these three mutants were compared to pCMV IE1full-lengthGFP, pCMV IE11-420GFP, and pCMV GFP, the IE11-414 and IE11-399 constructs exhibited poor complementation of CR208, similar to IE11-420.
In order to summarize the complementation efficiency of the mutants, we related the ability of each to complement across a range of MOIs (0.004 to 0.02, based on the titer of CR208 on ihfie1.3 cells) in multiple experiments. From this depiction (Fig. 4B), the IE11-442 construct behaved like the wild-type and IE11-479 constructs, with complementation ranging from 50% to 110% of that with the full-length construct, indicating that the 22 amino acids from 421 to 442, including two clusters of acidic amino acids, retained relatively full activity. Complementation by IE11-414, ranging from 4 to 14%, and IE11-399, ranging from 1 to 8% of that of the full-length construct, was similar to that with IE11-420 (Fig. 4A), suggesting that deletion of amino acids adjacent to the AD did not further decrease function. This analysis clearly reinforces the importance of the AD as a functional domain supporting replication. This domain appears to act together with at least one additional functional domain in the 399 amino-terminal amino acids of IE1491aa because this region retains basal complementation activity above that of the GFP control.
The 55-amino-acid AD contains a number of hallmark components. A sumoylation site (that has been mapped to Lys450) is dispensable for viral growth in assays similar to those described here (49). The recent mapping of the phosphorylation sites on IE1 to serine residues 399, 402, 406, 423, 428, 431, 448, 451, and 455 (Himmelheber et al., 28th International Herpesvirus Workshop, abstract 5.32) prompted a final series of experiments. These nine serines were mutated to alanine (SA) to prevent phosphorylation and were changed to glutamate (SE) to mimic phosphorylation as well as add negative charge. Six of these serines were located within the 55-amino-acid AD that we determined to be important, so both the IE1SA and IE1SE mutant constructs were transiently expressed in HFs that were subsequently infected with CR208. The resulting plaque counts were compared to that of controls transfected with pCMV IE1full-lengthGFP, pCMV IE11-420GFP, or pCMV GFP and infected with CR208 in parallel. Both the IE1SA and IE1SE mutants complemented the growth of CR208 as well as the full-length, wild-type protein did (Fig. 4A). Similar results were obtained with both native and GFP-tagged expression constructs. These data further reinforce the importance of the acidic character of the AD and, curiously, demonstrate that phosphorylation of these serines plays little role in IE1 function during viral replication.
DISCUSSION
IE1491aa is the most abundant gene product made by HCMV immediately following entry into cells. The importance of this protein in the transition from the (immediate early) to the ? (delayed early) and (late) phases of infection has been well established from studies on the IE1-deficient virus CR208 (12, 15). CR208 displays impaired replication that correlates with poor expression of ? and viral gene products and failure to initiate DNA replication under low-MOI conditions. While the exact mechanism of action has not yet emerged from these studies, the primary role does not rely on disruption of nuclear domain 10 (1) and is independent of IE1491aa sumoylation (49).
To get a better understanding of the function of this protein within the context of the replication cycle, we evaluated ie1 mutants for their ability to complement the growth of CR208 at low MOIs and found a dominant role for the AD located between amino acids 421 and 475 acting in concert with a separate functional domain located in the amino-terminal 399 amino acids. Further analysis showed that independent expression of amino acids 420 to 479 was unable to complement CR208 replication (data not shown), indicating that the amino-terminal 399 amino acids provide a basal ability that is enhanced by the AD. The amino-terminal 85 amino acids of IE1491aa are identical to a region of IE2579aa and contain a mapped transactivation domain (43) that merits further analysis in the context of the IE1491aa replication function that we have described here. Thus, our study supports a role for IE1491aa in regulation of gene expression dependent on an AD in combination with at least one other region of the protein.
When the expression of the ? gene product ppUL44 was evaluated by immunofluorescence analysis, CR208-infected cells complemented with IE11-420 appeared to be less intensely stained than CR208-infected cells complemented with full-length protein (data not shown), consistent with previous analyses that focused IE2579aa function on the transition from the to ? phase of infection (12, 15). Interestingly, higher levels of expression of IE11-420 correlated with higher ppUL44 levels, suggesting that one component of AD-independent complementation depended on the level of expression of the mutant protein in individual cells. This threshold was apparently lowered during complementation with full-length IE1491aa, because ppUL44 expression could be readily detected in cells even with low levels of IE1491aa expression (data not shown). These two independent IE1491aa domains, defined by amino acids 1 to 399 and amino acids 421 to 475, appear to influence the transition from to ? gene expression. The mechanism of control should become better understood through the evaluation of mutant viruses that express mutant forms of IE1491aa.
We showed that the AD was more critical at low MOIs and that the acidic character appears to be responsible for this activity. The mechanism of IE1491aa AD function is unknown, although acidic domains play well-established roles in transactivation by many cellular and viral regulatory proteins (4). In some settings, such as herpes simplex virus VP16 (10) and Saccharomyces cerevisiae Gal4 (14), the transactivation activity may be conferred by the acidic quality of the domain rather than the primary amino acid sequence. The AD of IE1491aa appears substantially more acidic than either of these classic acidic transactivators. Consistent with this, our experiments showed that transactivation was only modestly affected when 13 of the 26 acidic amino acids were removed or when the acidic character was increased. Thus, the critical role of the AD was not entirely dependent on very high acidity, even though acidic amino acids make up a very high proportion of the domain (amino acids 421 to 475).
The structure of this region is predicted to be different from that of classical acidic transactivation domains, where acidic residues are exposed on the surface of an amphipathic helix and contact the transcription initiation machinery (4). Although preliminary attempts to substitute the S. cerevisiae transcription factor Gal4 acidic transactivation domain in place of the IE1491aa AD failed to reconstitute function in the assays described here (data not shown), additional experiments in this direction are warranted.
Comparison of the HCMV IE1491aa carboxyl-terminal region with the homologous regions from three other primate CMVs reveals similarities in the AD as well as a striking level of conservation in a 16-amino-acid domain that we define as a CTD. This conservation predicts that IE1491aa homologues will likely promote tethering to metaphase chromatin, as has already been observed for the homologue encoded by simian CMV (8). Although our experiments failed to detect any role for chromatin tethering during productive replication, conservation of the CTD suggests an important role in CMV biology, pathogenesis, or, particularly, latency. The IE1491aa CTD does not appear to exhibit sequence similarity with the tethering domains of gammaherpesvirus latency proteins EBNA-1 or Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen LANA, except in that all of these domains are small and contain basic amino acids. Also, the CTD maps to an amino-terminal region in both as a 14-amino-acid domain (amino acids 72 to 84) in EBNA-1 (31) and as an 18-amino-acid domain (amino acids 5 to 22) in LANA (42).
Chromatin tethering is important for latent genome maintenance in gammaherpesviruses and does not appear to be involved in transactivation functions in these systems (42, 45). How or whether IE1491aa plays a role in HCMV genome maintenance during latency is not known. In addition to their ability to tether to chromatin, both EBNA-1 and LANA bind specifically to viral DNA replication origins that control latent DNA replication (31, 42). Despite many attempts, IE1491aa has not shown the ability to directly bind viral or cellular DNA.
IE1491aa is phosphorylated on serines between amino acids 399 and 455 and is sumoylated at Lys450 (49, 52); however, substitution of glutamate or alanine at the nine known phosphorylated serines yielded a protein that retained full complementation ability. A mutation of Lys450 to arginine was previously shown to destroy the only sumoylation site but not the ability to complement CR208 (49). Therefore, it is not likely that sumoylation underlies AD function. Modifications such as sumoylation and phosphorylation may be important for protein stability or cell type-specific activity rather than the regulatory activity that we have studied here.
ACKNOWLEDGMENTS
We appreciate cell culture assistance by Yin Dong and discussions with Richard Greaves that led to this approach for analysis of CR208 defects.
This work was supported by grants from the PHS (AI20211 and AI30363 to E.S.M., CA71019 to J.A.-C.), and Medical Scientist Training Grant GM007365 supported G.B.S.
Present address: Station Biologique, Roscoff Cedex, France.
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Department of Biochemistry, Drexel University College of Medicine, Philadelphia, Pennsylvania
ABSTRACT
The human cytomegalovirus major immediate-early () protein IE1491aa plays an important role in controlling viral gene expression at low multiplicities of infection. With a transient complementation assay, full-length IE1491aa enhanced the growth of ie1 mutant virus CR208 20-fold better than a deletion mutant lacking 71 carboxyl-terminal amino acids (IE11-420aa). A 16-amino-acid domain between amino acids 476 and 491 was both necessary and sufficient for chromatin-tethering activity; however, this domain was completely dispensable for complementation of CR208 replication. The proximal 55-amino-acid acidic domain (amino acids 421 to 475) was found to be most important for function. A deletion mutant lacking only this domain retained chromatin-tethering activity but failed to complement mutant virus. Interestingly, serine phosphorylation (at amino acids 399, 402, 406, 423, 428, 431, 448, 451, and 455) was not required for complementation. These results show that IE1491aa is composed of at least two domains that support replication, a region located between amino acids 1 and 399 that complements ie1 mutant virus replication to low levels and an acidic domain between amino acids 421 and 479 that dramatically enhances complementation.
INTRODUCTION
Human cytomegalovirus (HCMV), the prototype of the betaherpesviruses, is a widespread opportunistic pathogen (40) causing neurological damage as a result of congenital infection as well as significant disease in organ transplant recipients and AIDS patients. Like all herpesviruses, the replication cycle of HCMV is temporally regulated by viral functions. The , or immediate-early (IE), phase immediately following viral entry is activated by virion-associated regulators independent of viral gene expression; the ?, or delayed-early, phase is activated by gene products but is independent of viral DNA replication; and the , or late, phase is activated by gene products as well as functions associated with viral DNA replication (33).
Infection has a dramatic impact on cellular gene expression as well (5, 19). The timing and intensity of these phases are also influenced by host cell type, differentiation state, and cell cycle status (6, 7, 11, 33). Undifferentiated human cells and cells from other animal species except chimpanzees (41) are nonpermissive for HCMV replication and are generally restricted at a step following entry, uncoating, and gene expression (28). The two major gene products, IE1491aa and IE2579aa, control the progression of HCMV replication in permissive cells by regulating later classes of viral gene expression (33). Ribozymes targeted to an ie1/ie2 common exon inhibit replication (55). Viral mutants that lack IE1491aa but express IE2579aa show a multiplicity of infection (MOI)-dependent block to replication associated with a failure to express critical viral gene products (12, 15, 35). Though less abundant than IE1491aa, IE2579aa is widely believed to be the key viral regulatory gene product based on several lines of evidence: (i) strong transactivation of TATA box-containing promoters from viral or cellular sources in transient assays (33), (ii) association with a wide range of cellular transcription factors and cell cycle-related proteins similar to adenovirus E1A (6, 7, 33), and (iii) failure of viral mutants in this gene or its murine CMV homologue (ie3) to replicate or to express viral ? or gene products (3, 30, 50).
IE1491aa (also called IE1 p72, IE72, and IE1), the major (immediate-early) gene product of CMV, is a 72- to 75-kDa phosphoprotein that accumulates in the nucleus throughout HCMV infection. Early evidence from transient assays revealed an adjunct role for IE1491aa in cooperation with IE2579aa to transactivate viral and cellular promoters (33). In the absence of IE2579aa, IE1491aa transactivates the ie1/ie2 promoter-enhancer (9) as well as promoters from selected cellular genes, including heat shock protein 70 (16), interleukin (IL)-1? (20), IL-6 (13), IL-8 (37), DNA polymerase (18), origin recognition complex I protein (47), and dihydrofolate reductase (32). In addition to its possible role in activation of host cell gene expression, IE1491aa represses expression of the IL-1 receptor type 1 gene in transient assays (24). IE1491aa induces transcription factors, including NF-B (9, 44), activator protein 1 (23), and E2F1 (32), and may participate in stimulation of the host cell toward an S-phase-like state (6, 11, 33).
IE1491aa is sumoylated on Lys450 (49, 52) and disrupts nuclear domain 10 (PML bodies) (2, 26), which is dependent on Leu174 (36, 51). IE1491aa-mediated disruption of nuclear domain 10 plays no apparent role in viral replication because ie1 mutant viruses fail to disrupt nuclear bodies (1) at high MOIs, where replication proceeds unimpeded. IE1491aa is a phosphoprotein (38) and is phosphorylated in the presence or absence of other viral proteins (7, 39) at a number of sites (Ser399, Ser402, Ser406, Ser423, Ser428, Ser431, Ser448, Ser451, and Ser455) near or within a carboxyl-terminal acidic domain (AD) (Himmelheber et al., 28th International Herpesvirus Workshop, abstract 5.32). IE1491aa may undergo autophosphorylation and may influence the phosphorylation state of other proteins, such as E2Fs and retinoblastoma protein family members (7, 39). The contribution of individual functional domains to these various IE1491aa functions has not yet been fully evaluated, with the exception that sumoylation (at Lys450) has been shown to be dispensable for viral growth (49).
IE1491aa may alter host cell behavior (46) such as susceptibility to apoptosis (54), suppress p107-mediated block in cellular proliferation (22), and activate cyclin E/cdk2 kinase activity (53), although these phenomena have only been observed in transient assays. The available ie1 mutant viruses show no obvious signs of altered host cell behavior during infection in permissive human fibroblasts (HFs) (12, 15; Dittmer and Mocarski, unpublished observations). Furthermore, constitutive expression of IE1491aa in normal or immortalized HFs has little overall impact on cell viability, growth properties, cell cycle control, or susceptibility to apoptosis (16; Dittmer and Mocarski, unpublished observations).
Unambiguous evidence supporting a role of IE1491aa in viral replication emerged from studies on ie1-deficient viruses (15, 35), which are replication deficient at low MOIs. The defect has been ascribed to poor expression of viral ? and genes, including UL44, UL57, UL69, UL98, and UL112 (12, 15). Although the mechanism underlying the MOI-dependent growth of this mutant is not understood, the results are consistent with an accessory role of IE1491aa acting together with IE2579aa to enhance the expression of essential viral ? and genes. Constitutive (15) or transient (this study) expression of IE1491aa fully complements mutant viral replication.
IE1491aa tethers to host metaphase chromatin during mitosis via an acidic carboxyl-terminal region (29, 51); however, no role for tethering in viral replication or in modifying host cell behavior has been reported. Two well-known gammaherpesvirus proteins tether as a part of their natural function, Epstein-Barr virus nuclear antigen 1 (EBNA-1) (31) and Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) (42). Tethering to metaphase chromatin is part of the genome maintenance function of these proteins during latency and collaborates with latent origin-specific binding to control replication of the viral genome. IE1491aa has no known role in HCMV latency and has not been found to bind directly to viral or host DNA. IE1491aa expression has not been detected during natural latency and becomes undetectable within a few days in experimental models of latency in myeloid progenitors (17, 25). Thus, any role of this regulatory protein in latent genome maintenance remains enigmatic.
We used the MOI-dependent growth properties of ie1 mutant virus CR208 as an assay system to study the role of IE1491aa domains during infection. A portion of IE1491aa (amino acids 1 to 420) was found to retain partial function, with the carboxyl-terminal acidic region (amino acids 420 to 479) necessary for full trans-complementation of the growth of CR208. This assay provided a simple and direct means to separate the roles of the chromatin-tethering domain (CTD; amino acids 476 to 491) and the adjacent AD (amino acids 421 to 479) without introducing the adventitious mutations that result in growth defects independent of the targeted gene that are found in HCMV mutants made by transfection of plasmids, cosmids, or bacmids (34). Our results also indicate that the acidic character of rather than serine phosphorylation in the AD region is important for complementation.
MATERIALS AND METHODS
Plasmids. All ie1 mutants were constructed by PCR-driven mutagenesis with plasmid pON2202 (21) as a template. pCMV IE1full-length and pCMV IE1full-lengthGFP were constructed from a cDNA generated by PCR with oligonucleotides 1 and 2 (Table 1). pCMV IE11-420 and pCMV IE11-420GFP were generated by PCR with oligonucleotides 1 and 3. pCMV IE11-479 and pCMV IE11-479GFP were generated by PCR with oligonucleotides 1 and 4. The resulting PCR products for these nontagged cDNAs were cloned into HindIII- and XhoI-digested pcDNA3.1 (Invitrogen) and subsequently cloned into pCMV(4x)GFP after digestion with NheI and XbaI as green fluorescent protein (GFP)-tagged derivatives.
Plasmid pCMV(4x)GFP, a gift of Irina Conboy, expresses GFP under the control of an HCMV promoter in which the four NF-B sites were destroyed with point mutations (Conboy and Jones, personal communication). pCMV IE1421-475GFP was generated by ligating annealed oligonucleotides 5 and 6 to XhoI- and XbaI-digested pCMV IE11-420GFP, thereby adding two amino acids (threonine and methionine) between codons 420 and 476. To construct pCMV IE1476-491GFP, annealed oligonucleotides 5 and 6 were ligated to Xhol- and Xbal-digested pCMV(4x)GFP, thereby placing codons 476 to 491 immediately downstream of the natural IE1491aa initiation codon. To construct pCMVIE11-442GFP, pCMV IE11-414GFP, and pCMV IE11-399GFP, PCR products generated with oligonucleotides 2 and 8, 2 and 9, 2 and 10, respectively, were cloned into NheI- and XhoI-digested pCMV(4x)GFP. The pSG5-IE1 plasmid (provided by John Sinclair) was used as a template to make ie1 mutants with serine modifications at amino acids 399, 402, 406, 423, 428, 431, 448, 451, and 455. These serines were exchanged in groups of three to either alanine (pSG5-IE1SA) or glutamate (pSG5-IE1SE) with the QuikChange site-directed mutagenesis kit (Strategene) according to the manufacturer's protocol. The sequences of all primers are shown in Table 2 (48).
To generate the construct in which all nine serines were mutated, mutation reactions were performed sequentially with single and then double group mutations as templates. The original constructs were transferred into the pCMV-GFP backbone for comparison with the deletion mutants by PCR to amplify the region representing amino acids 350 to 491 followed by BglII and XhoI digestion and ligation into BglII- and XhoI-digested pCMV-IE1491GFP.
Cell and virus culture. All cells were cultivated at 37°C in a 5% CO2 atmosphere. HFs prepared locally from pooled newborn foreskins and immortalized ihfie1.3 cells stably expressing IE1491aa (15) were maintained in Dulbecco's modified Eagle's medium (Gibco-BRL) supplemented with 10% NuSerum I (Collaborative Research Inc.), 100 U of penicillin/ml, 100 U of streptomycin sulfate/ml, 0.66 mM arginine, 1.48 mM glutamine, and 0.24 mM asparagine (complete medium). The ie1-deficient HCMV strain CR208 and the repaired strain CRQ208 (15) were cultured in ihfie1.3 or in HF cells, respectively, as described (15). Cell supernatants were clarified by sedimentation at 1,500 x g for 20 min, disrupted with a probe type sonicator, and stored in aliquots at –80°C as virus stock. Infections were carried out with virus diluted in DMEM-0.2% NuSerum, and cells were rinsed once and incubated in complete medium supplemented with 0.16% pooled human gamma globulin (Baxter Corp.) to prevent viral spread through the medium. Virus titrations were performed in six-well dishes incubated for 10 days postinfection, at which time cells were methanol fixed and stained with Giemsa.
Cell transfection. Transfection of HFs was carried out with calcium phosphate precipitation (12, 15) modified as follows. HFs were split at a ratio of 1:2, incubated for 1 day, and split so that 2.2 x 105 cells were seeded into each well of a six-well dish. One day later, the medium was replaced with 2 ml of complete medium. To 15 μg of plasmid DNA and 31 μl of 2 M CaCl2, sterile H2O was added to a volume of 219 μl. Subsequently, 250 μl of 2x HBS (280 mM NaCl, 1.4 mM Na2HPO4, 10 mM KCl, 5.6 mM glucose, 20 mM HEPES, pH 7.05) was added dropwise, and this mixture was added to cells. After incubation for 4 to 6 h, the medium was removed and the cells were incubated for 90 s with 15% (vol/vol) glycerol in 1x HBS followed by four washes and addition of complete medium. GFP-based fluorescence 24 h later was used to assess transfection efficiency. For transfection-infection assays, the transfected cells were infected at 2 days posttransfection.
Immunoblot. Cells were harvested at 48 h posttransfection, lysed in 2x Laemmli buffer, and separated on a 10% polyacrylamide-sodium dodecyl sulfate gel (27). Proteins transferred to a nitrocellulose membrane were detected with anti-IE1/IE2 monoclonal antibody 810 (Chemicon) and anti-?-actin antibody A5441 (Sigma) with the enhanced chemiluminescence kit (Amersham Corp.) following the manufacturers' instructions.
Sequence analysis. The sequence of HCMV Towne strain IE1491aa (accession number gi:73633) amino acids 421 to 491 was aligned to that of full-length chimpanzee CMV IE1 (gi:19881132), rhesus CMV IE1 (gi:332085), and simian CMV IE1 (gi:1616980) with ClustalW of MacVector 7.1.1 (Accelrys, San Diego, Calif.).
RESULTS
Complementation of CR208 by transiently expressed IE1491aa and IE11-420. Replication of ie1-deficient CR208 is severely compromised on normal HFs at MOIs of <1, and this defect is complemented on IE1491aa-expressing ihfie1.3 cells (12, 15). We took advantage of this MOI-dependent growth phenotype to investigate the ability of full-length and mutant forms of IE1491aa to complement CR208 plaque formation following transient transfection. We first evaluated the relationship between MOI and plaque formation by plating serial threefold dilutions of CR208 on either normal HFs or complementing ihfie1.3 cells (Fig. 1A). Maximal differences in efficiency of plaque formation on 2.2 x 105 HFs were observed at MOIs below 0.01, where CR208 yielded few or no plaques and transfection of full-length IE1491aa into 10% of the cells resulted in the formation of up to 103 plaques (Fig. 1B). Therefore, MOIs in this range were employed in experiments to compare IE1491aa and mutants by a transient complementation assay.
We compared complementation of CR208 by full-length IE1491aa and a mutant lacking the carboxyl-terminal region (amino acids 421 to 491). This region has a dense concentration of 26 acidic amino acids (29), a chromatin localization domain (51), and nine sites of potential serine phosphorylation (Himmelheber et al., 28th International Herpesvirus Workshop, abstract 5.32). We transiently expressed pCMV IE1full-lengthGFP or mutant pCMV IE11-420GFP in HFs and subsequently infected these cells with CR208 at an MOI of 0.005 (determined by plaque assay on ihfie1.3 cells and depicted by the open bar in Fig. 1). Transient expression of the mutant resulted in 30-fold fewer plaques than with the full-length construct (Fig. 1B), even though the transfection efficiency of the plasmids was similar, based on the number of GFP-positive cells (data not shown). HFs transfected with the full-length construct complemented CR208 at levels that were comparable to infection of ihfie1.3 cells (Fig. 1B), so long as the transfection efficiency was taken into consideration. Plaquing efficiency following transfection by the method used here was consistently 10 to 20% of that observed in stably expressing cells.
As expected, the failure of CR208 to form plaques at this MOI did not change when a control GFP expression vector was transfected into the cells (Fig. 1B). The pCMV IE1full-lengthGFP construct showed a transfection efficiency and complementation pattern similar to that of the nontagged full-length IE1full-length construct (data not shown). These results identified at least one potentially important functional domain in the carboxyl-terminal region of IE1491aa and revealed a low-level complementation activity of the truncated IE11-420 that was greater than that of negative controls. Furthermore, plaques formed with either full-length IE1 or the IE11-420 mutant were large, comparable in size to those formed by CR208 in ihfie1.3 monolayers or by parental virus on HFs (data not shown), suggesting that both retained full ability to complement cell-to-cell spread (12, 16) despite the fact that the transient transfection procedure did not deliver IE1 to all cells.
Conservation of two distinct domains within the carboxyl terminus of primate CMV homologs of IE1491aa. The carboxy-terminal 71 amino acids of IE1491aa include 26 acidic amino acids distributed in clusters containing as many as six consecutive acidic amino acids (Fig. 2A). The region is therefore strongly acidic (38%) and contains only one basic amino acid except for a short region located at the extreme carboxyl terminus. This 16-amino-acid region has a greater proportion of basic amino acids (Fig. 2A). The estimated isoelectric point of the extreme carboxyl-terminal 16 amino acids (476-491) is 9.81, compared with 3.64 for amino acids 420 to 491 and 4.54 for full-length IE1491aa. A comparison of this 71-amino-acid region from HCMV with homologues from chimpanzee CMV, simian CMV, and rhesus CMV revealed an AD with an adjacent highly conserved basic region towards the extreme carboxyl terminus in all (Fig. 2A). For example, the carboxyl terminus of HCMV and chimpanzee CMV IE1 exhibit 87% identity, compared to an overall 67% identity for the full-length proteins and 50% identity for the AD (data not shown). Parenthetically, neither rat nor murine CMV ie1 proteins have a domain that is homologous to this short carboxyl-terminal basic region (data not shown).
Chromatin tethering by the carboxyl-terminal 16 amino acids of IE1491aa. Previous studies showed that the IE1491aa protein tethers to metaphase chromosomes in transiently transfected cells (29, 51), although the importance of this activity to virus replication has not been addressed. A deletion affecting a large portion of the carboxyl-terminal 71 amino acids (amino acids 421 to 485), removing the entire acidic region along with part of the basic, highly conserved extreme carboxyl terminus, disrupted the ability of IE1491aa to tether (51). In order to more precisely identify the region responsible for chromatin tethering by IE1491aa, we constructed a series of deletion mutants, leaving amino acids 1 to 420 (pCMV IE11-420GFP), 1 to 479 (pCMV IE11-479), or 1 to 420 together with 476 to 491 (pCMV IE1421-475GFP) all fused to the amino terminus of GFP. In parallel, the extreme carboxyl-terminal IE1491aa amino acids 476 to 491 were placed downstream of the natural IE1491aa start codon and expressed as an amino-terminal fusion to GFP (pCMV IE1476-491GFP). The constructs were transiently transfected into HFs to assess chromatin-tethering activity in mitotic cells with either IE1491aa monoclonal antibody or GFP monoclonal antibody in immunofluorescence analysis (Fig. 2B) together with a DNA counterstain (Hoechst 33342 or propidium iodide; Fig. 2B). For comparison, the full-length IE1491aa protein was also visualized in stably expressing ihfie1.3 cells with monoclonal antibody.
While full-length protein associated with chromatin in dividing cells as expected (30, 51) (Fig. 2B), IE11-479 failed to bind to metaphase chromatin (Fig. 2B) (51). To directly assess the capacity of the different regions to mediate tethering, deletion of the 55-amino-acid acidic domain (pCMV IE1421-475GFP) did not disrupt the ability to associate with metaphase chromatin, and addition of the extreme carboxyl-terminal 16 amino acids (pCMV IE1476-491GFP) to GFP was sufficient for association (Fig. 2B). GFP alone stained the entire metaphase cell and was excluded from chromatin (data not shown). Thus, a CTD is located in a highly conserved, extremely carboxyl-terminal 16-amino-acid region of IE1491aa. This domain is both necessary and sufficient for binding to metaphase chromatin in transfected cells.
Role of the AD and CTD in complementation of CR208. In order to evaluate the role(s) of the 55-amino-acid AD and 16-amino-acid CTD in complementation of ie1 mutant virus, HFs were transfected with pCMV IE1full-lengthGFP, pCMV IE11-479, pCMV IE11-420GFP, pCMV IE1421-475GFP, and control pCMV-GFP. At 48 h posttransfection, cells were infected with CR208 at low MOIs, and plaque counts were performed 10 days later. Figure 3A shows complementation experiments at MOIs of 0.0004 and 0.002, which revealed the importance of the AD in complementation of ie1 mutant virus during replication at low MOIs. As expected, IE11-420 was defective in complementation, IE1491aa fully complemented plaque formation, and the GFP control plasmid failed to complement virus growth (Fig. 3A and data not shown). IE11-479 complemented CR208 as well as full-length IE1 at all MOIs tested (Fig. 3A and data not shown), consistent with a role for the AD in supporting replication.
Transfection of pCMV IE1421-475GFP, which lacked the 55-amino-acid AD but contained the 16-amino-acid CTD (Fig. 2B), complemented at about the same poor level as IE11-420. These results, which employed IE11-479 without a GFP fusion, were confirmed with pCMV IE11-479GFP (data not shown). Thus, the ability to tether chromatin did not contribute to IE1491aa function in viral replication in HFs, suggesting that this activity may be important in other cell types or, as suggested by the function of the other characterized herpesvirus tethering proteins LANA (42) and EBNA-1 (31), in latency. The AD is critical to the activity of IE1491aa during viral replication.
When the ratios of plaques obtained with these three ie1 mutants were calculated as a percentage of the plaques obtained with the full-length construct, differences were readily appreciated at MOIs of 0.0004, 0.002, and 0.01 (Fig. 3B). First, as expected, background replication in the absence of complementation was detected at the highest MOI, consistent with the established growth properties of CR208 (12, 15) and as shown in Fig. 1. Second, complementation appeared to be highly dependent on the AD at all MOIs tested, so that amino acids 1 to 420 complemented to consistently low levels. Finally, complementation by the 1 to 479 construct, which lacked the ability to tether chromatin, ranged from 180% of that of the full-length construct (MOI of 0.0004) to 130% (at an MOI of 0.002) to 60% (at an MOI of 0.01), all consistent with our interpretation that the CTD is dispensable for replication.
The behavior of the mutant with amino acids 1 to 420 fused to the CTD at amino acids 476 to 491 but lacking the AD (pCMV IE1421-475GFP) showed complementation similar to the region with amino acids 1 to 420 alone at low MOI (0.0004); however, complementation improved as the MOI was increased to 0.01, suggesting that the addition of the CTD may have had a modest positive impact on amino acids 1 to 420 in the absence of the 55-amino-acid AD. Taken together, these data emphasize the importance of this region of high negative charge as a critical functional domain for optimal viral replication.
To exclude any possibility that the differences in complementation resulted from different expression levels, we performed an immunoblot analysis of lysates of HFs transfected with IE1full-lengthGFP, IE11-420GFP, IE1421-475GFP, and IE11-479 (Fig. 3C). These four IE1 constructs did not differ in their expression levels. Therefore, the carboxyl-terminal 71 amino acids contribute to replication primarily through the presence of an AD and not because of variation in expression levels.
Role of phosphorylated serines in the acidic domain for complementation of CR208. IE1491aa was subjected to additional deletions and point mutations to investigate the role of acidic amino acids as well as other sequences in this region. pCMV IE11-442GFP lacks 37 amino acids of the AD, retaining only 13 of the 26 acidic amino acids; pCMV IE11-414GFP and pCMV IE11-399GFP lack the AD plus additional amino acids. When these three mutants were compared to pCMV IE1full-lengthGFP, pCMV IE11-420GFP, and pCMV GFP, the IE11-414 and IE11-399 constructs exhibited poor complementation of CR208, similar to IE11-420.
In order to summarize the complementation efficiency of the mutants, we related the ability of each to complement across a range of MOIs (0.004 to 0.02, based on the titer of CR208 on ihfie1.3 cells) in multiple experiments. From this depiction (Fig. 4B), the IE11-442 construct behaved like the wild-type and IE11-479 constructs, with complementation ranging from 50% to 110% of that with the full-length construct, indicating that the 22 amino acids from 421 to 442, including two clusters of acidic amino acids, retained relatively full activity. Complementation by IE11-414, ranging from 4 to 14%, and IE11-399, ranging from 1 to 8% of that of the full-length construct, was similar to that with IE11-420 (Fig. 4A), suggesting that deletion of amino acids adjacent to the AD did not further decrease function. This analysis clearly reinforces the importance of the AD as a functional domain supporting replication. This domain appears to act together with at least one additional functional domain in the 399 amino-terminal amino acids of IE1491aa because this region retains basal complementation activity above that of the GFP control.
The 55-amino-acid AD contains a number of hallmark components. A sumoylation site (that has been mapped to Lys450) is dispensable for viral growth in assays similar to those described here (49). The recent mapping of the phosphorylation sites on IE1 to serine residues 399, 402, 406, 423, 428, 431, 448, 451, and 455 (Himmelheber et al., 28th International Herpesvirus Workshop, abstract 5.32) prompted a final series of experiments. These nine serines were mutated to alanine (SA) to prevent phosphorylation and were changed to glutamate (SE) to mimic phosphorylation as well as add negative charge. Six of these serines were located within the 55-amino-acid AD that we determined to be important, so both the IE1SA and IE1SE mutant constructs were transiently expressed in HFs that were subsequently infected with CR208. The resulting plaque counts were compared to that of controls transfected with pCMV IE1full-lengthGFP, pCMV IE11-420GFP, or pCMV GFP and infected with CR208 in parallel. Both the IE1SA and IE1SE mutants complemented the growth of CR208 as well as the full-length, wild-type protein did (Fig. 4A). Similar results were obtained with both native and GFP-tagged expression constructs. These data further reinforce the importance of the acidic character of the AD and, curiously, demonstrate that phosphorylation of these serines plays little role in IE1 function during viral replication.
DISCUSSION
IE1491aa is the most abundant gene product made by HCMV immediately following entry into cells. The importance of this protein in the transition from the (immediate early) to the ? (delayed early) and (late) phases of infection has been well established from studies on the IE1-deficient virus CR208 (12, 15). CR208 displays impaired replication that correlates with poor expression of ? and viral gene products and failure to initiate DNA replication under low-MOI conditions. While the exact mechanism of action has not yet emerged from these studies, the primary role does not rely on disruption of nuclear domain 10 (1) and is independent of IE1491aa sumoylation (49).
To get a better understanding of the function of this protein within the context of the replication cycle, we evaluated ie1 mutants for their ability to complement the growth of CR208 at low MOIs and found a dominant role for the AD located between amino acids 421 and 475 acting in concert with a separate functional domain located in the amino-terminal 399 amino acids. Further analysis showed that independent expression of amino acids 420 to 479 was unable to complement CR208 replication (data not shown), indicating that the amino-terminal 399 amino acids provide a basal ability that is enhanced by the AD. The amino-terminal 85 amino acids of IE1491aa are identical to a region of IE2579aa and contain a mapped transactivation domain (43) that merits further analysis in the context of the IE1491aa replication function that we have described here. Thus, our study supports a role for IE1491aa in regulation of gene expression dependent on an AD in combination with at least one other region of the protein.
When the expression of the ? gene product ppUL44 was evaluated by immunofluorescence analysis, CR208-infected cells complemented with IE11-420 appeared to be less intensely stained than CR208-infected cells complemented with full-length protein (data not shown), consistent with previous analyses that focused IE2579aa function on the transition from the to ? phase of infection (12, 15). Interestingly, higher levels of expression of IE11-420 correlated with higher ppUL44 levels, suggesting that one component of AD-independent complementation depended on the level of expression of the mutant protein in individual cells. This threshold was apparently lowered during complementation with full-length IE1491aa, because ppUL44 expression could be readily detected in cells even with low levels of IE1491aa expression (data not shown). These two independent IE1491aa domains, defined by amino acids 1 to 399 and amino acids 421 to 475, appear to influence the transition from to ? gene expression. The mechanism of control should become better understood through the evaluation of mutant viruses that express mutant forms of IE1491aa.
We showed that the AD was more critical at low MOIs and that the acidic character appears to be responsible for this activity. The mechanism of IE1491aa AD function is unknown, although acidic domains play well-established roles in transactivation by many cellular and viral regulatory proteins (4). In some settings, such as herpes simplex virus VP16 (10) and Saccharomyces cerevisiae Gal4 (14), the transactivation activity may be conferred by the acidic quality of the domain rather than the primary amino acid sequence. The AD of IE1491aa appears substantially more acidic than either of these classic acidic transactivators. Consistent with this, our experiments showed that transactivation was only modestly affected when 13 of the 26 acidic amino acids were removed or when the acidic character was increased. Thus, the critical role of the AD was not entirely dependent on very high acidity, even though acidic amino acids make up a very high proportion of the domain (amino acids 421 to 475).
The structure of this region is predicted to be different from that of classical acidic transactivation domains, where acidic residues are exposed on the surface of an amphipathic helix and contact the transcription initiation machinery (4). Although preliminary attempts to substitute the S. cerevisiae transcription factor Gal4 acidic transactivation domain in place of the IE1491aa AD failed to reconstitute function in the assays described here (data not shown), additional experiments in this direction are warranted.
Comparison of the HCMV IE1491aa carboxyl-terminal region with the homologous regions from three other primate CMVs reveals similarities in the AD as well as a striking level of conservation in a 16-amino-acid domain that we define as a CTD. This conservation predicts that IE1491aa homologues will likely promote tethering to metaphase chromatin, as has already been observed for the homologue encoded by simian CMV (8). Although our experiments failed to detect any role for chromatin tethering during productive replication, conservation of the CTD suggests an important role in CMV biology, pathogenesis, or, particularly, latency. The IE1491aa CTD does not appear to exhibit sequence similarity with the tethering domains of gammaherpesvirus latency proteins EBNA-1 or Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen LANA, except in that all of these domains are small and contain basic amino acids. Also, the CTD maps to an amino-terminal region in both as a 14-amino-acid domain (amino acids 72 to 84) in EBNA-1 (31) and as an 18-amino-acid domain (amino acids 5 to 22) in LANA (42).
Chromatin tethering is important for latent genome maintenance in gammaherpesviruses and does not appear to be involved in transactivation functions in these systems (42, 45). How or whether IE1491aa plays a role in HCMV genome maintenance during latency is not known. In addition to their ability to tether to chromatin, both EBNA-1 and LANA bind specifically to viral DNA replication origins that control latent DNA replication (31, 42). Despite many attempts, IE1491aa has not shown the ability to directly bind viral or cellular DNA.
IE1491aa is phosphorylated on serines between amino acids 399 and 455 and is sumoylated at Lys450 (49, 52); however, substitution of glutamate or alanine at the nine known phosphorylated serines yielded a protein that retained full complementation ability. A mutation of Lys450 to arginine was previously shown to destroy the only sumoylation site but not the ability to complement CR208 (49). Therefore, it is not likely that sumoylation underlies AD function. Modifications such as sumoylation and phosphorylation may be important for protein stability or cell type-specific activity rather than the regulatory activity that we have studied here.
ACKNOWLEDGMENTS
We appreciate cell culture assistance by Yin Dong and discussions with Richard Greaves that led to this approach for analysis of CR208 defects.
This work was supported by grants from the PHS (AI20211 and AI30363 to E.S.M., CA71019 to J.A.-C.), and Medical Scientist Training Grant GM007365 supported G.B.S.
Present address: Station Biologique, Roscoff Cedex, France.
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