Recruitment of Human Cytomegalovirus Immediate-Early 2 Protein onto Parental Viral Genomes in Association with ND10 in Live-Infected Cells^▿

Oligonucleotides and plasmids.

Oligonucleotides used for cloning, site-directed mutagenesis, and generation of recombinant BACs were obtained from Biomers (Ulm, Germany); the sequences are listed in Table 1. Plasmid pHM990, coding for IE2 fused to EGFP, was constructed as described elsewhere (50). For generation of expression plasmid pHM2626, which encodes a DNA-binding-deficient IE2 mutant fused to EGFP, nucleotide exchanges were introduced via site-directed mutagenesis, using the primer pair delDNAbinIE2-5′ and delDNAbinIE2-3′, resulting in the substitution of two histidines (amino acids 446 and 452 of IE2) of a putative zinc finger motif within IE2 with leucines, thus abolishing the DNA binding of IE2 (32). Site-directed mutagenesis was performed as described previously (29). Plasmid pHM2397, expressing Sp100 isoform A fused with the monomeric red fluorescent protein mCherry, was generated by PCR amplification of the mCherry coding sequence, using primers 5′- and 3′mCherry together with plasmid pRSET-B-mCherry as the template (44). In parallel, the coding sequence of Sp100 was amplified with primers 5′Sp100-mCherry and 3′FL-Sp100. Finally, primers 5′mCherry/BstXI-Sp100 and 3′Sp100/ApaI-mCherry were used for a hybrid PCR to generate the mCherry-Sp100 fusion gene, which was then inserted via BstXI and ApaI into the pLenti6/V5-D-Topo vector (Invitrogen). The recombinant vector pHM1471 was constructed as follows for generation of a recombinant BAC expressing IE2 fused to EGFP. First, the 5′ end of the genomic sequence required for homologous recombination was amplified, using plasmid pRR47 as the template and oligonucleotides FragA-5 and FragA-3mul. The resulting PCR product was ligated with plasmid pHM134 via HindIII and MluI, resulting in plasmid pHM1438. Next, the 3′ homologous region was amplified by PCR, using cosmid pCM1058 as the template and oligonucleotides FragB-5 and FragB-3, followed by insertion into pHM1438, using StuI and SphI, giving rise to plasmid pHM1469. Finally, the recombination cassette was released from vector pHM1469 by restriction with Asp718 and SphI and inserted into pST76K-SR to yield recombination vector pHM1471.

BAC mutagenesis.

For construction of the IE2-EGFP BAC, a two-step replacement procedure was performed as described previously (8). Starting with a BACmid (ΔkΔex5) already lacking the exon 5 sequence of the ie1/2 gene locus (20), an exon 5-EGFP hybrid gene was reinserted, using a recombination strategy involving cointegrate formation. For this, an 800-nucleotide fragment comprising 3′ genomic flanking sequences of exon 5 required for homologous recombination was amplified, using oligonucleotides 5′ and 3′Red1-N1-IE2/Frag.B and plasmid pHM1471 as templates. The resulting PCR product was inserted into plasmid pHM990 via NotI, giving rise to plasmid pHM2290. Thereafter, the IE2-EGFP/Frag.B fusion sequence was recloned into pHM1471, using restriction enzymes SphI and MluI, finally resulting in recombination vector pHM2291. In the next step, pHM2291 was transformed into Escherichia coli (strain DH10B) cells already harboring BACmid ΔkΔex5. Transformants were selected at 30°C on Luria-Bertani (LB) agar plates containing chloramphenicol (15 mg/ml) and kanamycin (15 mg/ml) to allow for cointegrate formation. Clones with cointegrates were subsequently identified by incubating the bacteria on chloramphenicol- and kanamycin-positive agar plates at 43°C. To achieve a resolution of the cointegrates, clones were streaked on LB plates containing chloramphenicol only and incubated at 30°C. Finally, to select for clones with a loss of the cointegrate, the bacteria were subjected to chloramphenicol agar plates additionally supplemented with 5% sucrose.

Viral nucleic acid isolation and analysis.

BACmid DNA was isolated from bacteria by a standard alkaline lysis minipreparation procedure from 5-ml liquid cultures and characterized by restriction digestion using the enzyme BamHI. Thereafter, DNA fragments were separated via electrophoresis at 30 V overnight, using a 0.7% agarose gel. For Southern blot analysis, the DNA was transferred onto a nylon membrane (Biodyne B transfer membrane; PALL, Portsmouth, United Kingdom) and probed with biotinylated DNA spanning the IE2 cDNA sequence of the ie1/2 gene region of HCMV (exons 2, 3, and 5) according to the instructions of the manufacturer (NEBlot Phototope kit; Biolabs, Frankfurt, Germany).

Cells and viruses.

HFF cells were maintained in Eagle's minimal essential medium (GIBCO/BRL, Eggenstein, Germany) supplemented with 5% fetal calf serum. HFFs with a small interfering RNA-mediated kd of PML (PML-kd cells) were cultured in Dulbecco's minimal essential medium (GIBCO/BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum and 5 μg/ml puromycin (50). HFFs stably expressing Sp100 fused to the monomeric red autofluorescent protein mCherry were generated via retrovirus transduction, using the ViraPower lentiviral system according to the manufacturer's protocol (Invitrogen, Karlsruhe, Germany), and maintained in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum plus 2 μg/ml blasticidin. For transfection experiments, HFFs or mCh-Sp100 cells were seeded either on coverslips or into 4-well, chambered coverglass units with coverslip quality glass bottoms (Lab-Tek; Nunc). Transfection was performed using a TransPEI transfection reagent (Eurogentec, Belgium) according to the manufacturer's instructions. Viral stocks of wild-type (wt) AD169 and the recombinant AD169/IE2-EGFP virus were prepared and titrated via IE1p72 fluorescence as described previously (31). Quantification of viral input DNA by real-time PCR was also performed exactly as described previously (31, 50).

Reconstitution of recombinant cytomegaloviruses.

One day before transfection, HFFs were seeded into 6-well dishes at a density of 3.5 × 10⁵ cells/well. About 1 μg of BACmid DNA and 1 μg of the pp71 expression plasmid pCBpp71 were cotransfected, using SuperFECT transfection reagent (QIAGEN, Hilden, Germany) according to the instructions of the manufacturer. One week after transfection, the cells were transferred into 25-cm² flasks and incubated until plaques appeared.

Antibodies, indirect immunofluorescence analysis, and Western blot analysis.

The polyclonal antisera raised against exon 5 of IE2p86 (referred to as anti-pHM178) or pUL84 of HCMV were generated by immunizing rabbits with the respective prokaryotically expressed proteins. MAb-UL44 BS 510, MAb-MCP 28-4, and MAb-pp65 65-33 were kindly provided by B. Plachter (University of Mainz, Germany) and W. Britt (University of Birmingham, AL). MAb p63-27 and MAb 41-18 directed against IE1p72 and pp28 (UL99) were described previously (5, 39). MAb-UL69 69-66 (55) was used for detection of pUL69. For detection of endogenous PML and hDaxx, respectively, the monoclonal antibodies PG-M3 (Santa Cruz Biotechnology, Santa Cruz, CA) and MCA2143 (Serotec, Oxford, United Kingdom) were used. The monoclonal antibody Ac-15, which recognizes β actin, was purchased from Sigma (Deisenhofen, Germany). Anti-mouse as well as anti-rabbit horseradish peroxidase-conjugated secondary antibodies were obtained from Dianova (Hamburg, Germany), while Alexa 488-, Alexa 555-, and Cy3-conjugated secondary antibodies were purchased from Molecular Probes and GE Healthcare.

For indirect immunofluorescence analysis, primary HFF cells (1 × 10⁵) were grown on coverslips for transient transfection or HCMV infection. The conditions for transfection of the HFFs and the fixation and immunodetection of viral and cellular proteins were as previously described (22).

For Western blotting, extracts from infected cells were prepared in sodium dodecyl sulfate loading buffer, separated on 8 to 15% polyacrylamide gels containing sodium dodecyl sulfate, and transferred to nitrocellulose membranes. Western blotting and chemiluminescence detection were performed as described previously (30).

Time-lapse microscopy of live cells.

Live cells were observed using a Leica DMIRE2 inverted fluorescence microscope equipped with a Leica DFC300 FX digital camera. The excitation wavelength was controlled by mercury lamp illumination and a manual filter wheel equipped with filters suitable for EGFP, the monomeric red fluorescent protein mCherry, and DAPI (4′,6′-diamidino-2-phenylindole). Camera image acquisition was controlled by IM50 software (Leica). Single images or timed image sequences were exported as TIFF files from the IM50 software. Individual frames were prepared for presentation using Photoshop. An essential part of the system was the ability to maintain the cells for long periods in a controlled environment with constant temperature, CO₂, and humidity. The conditions within the box were maintained at 37°C, 5% CO₂, and 90% humidity to keep the cells healthy indefinitely. The cells were seeded into 4-well, chambered coverglass units with coverslip quality glass bottoms (Lab-Tek; Nunc) at a density of 1 × 10⁵ cells per well the day before each experiment, and the samples were examined with a ×63 dry objective lens.

Fluorescence in situ hybridization.

Fluorescence in situ hybridization was performed according to a protocol described by Everett et al. (15). HFF cells were grown on 13-mm-diameter glass coverslips and then infected with HCMV AD169 at an MOI of 0.5 PFU per cell. At various times thereafter, the cells were washed twice with phosphate-buffered saline (PBS) containing 1% fetal calf serum (FCS) and then fixed by incubation for 5 min at −20°C with precooled 95% ethanol-5% glacial acetic acid. The cells were then washed three times with PBS-1% FCS and stored at 4°C until further use. The probe for in situ hybridization was a BAC DNA containing the entire HCMV genome (8) (a gift from Ulrich H. Koszinowski, Munich, Germany). After a DNaseI treatment, the probe was labeled by nick translation with Cy3-dCTP (Amersham) according to the manufacturer's protocol. The cells were prehybridized by incubation for 30 min at 37°C with 20 μl of hybridization buffer (50% formamide, 10% dextran sulfate, 4× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) in a humidified microarray hybridization chamber (Camlab). The coverslips were then removed from the chamber and blotted dry. The probe was added to the hybridization buffer to a concentration of 1 ng/μl, after which each coverslip was incubated at 95°C for 2 min to denature the probe and the sample. The coverslips and hybridization mixture were then placed into the humidified chamber, and hybridization was continued overnight at 37°C. The cells were then washed for 5 min at 60°C with 2× SSC and once with 2× SSC at room temperature. After a further washing with PBS-1% FCS, the coverslips were incubated with the anti-IΕ2 polyclonal antibody anti-pHM178 for 1 h. After several washings, the cells were incubated with fluorescein isothiocyanate-conjugated sheep anti-mouse immunoglobulin G (Sigma) for another hour. The cells were washed several times, air dried, and then mounted on glass slides by using a glycerol-PBS mounting solution (CitiFluor). Images were analyzed using a Zeiss Axioplan-2 microscope and recorded with a cooled SPOT color digital camera (Diagnostic Instruments, Sterling Heights, MI) under image capture conditions that eliminated channel overlap. The images were exported as TIFF files and then processed with Photoshop.

Construction and verification of a recombinant HCMV expressing an IE2-EGFP fusion protein.

In order to investigate the in vivo dynamics of IE2 protein accumulation during infection, we generated a recombinant HCMV expressing EGFP fused to the C terminus of IE2. For construction of this recombinant virus, termed AD169/IE2-EGFP, BAC technology was employed (for a review, see reference 1). Starting with a BAC derived from the HCMV genome of strain AD169 carrying a deletion of the complete exon 5 sequence of the ie1/2 gene locus (designated ΔkΔex5) (20), we reintroduced the exon 5 sequence fused to EGFP via homologous recombination between up- and downstream-flanking regions of exon 5. For this purpose, a recombinant vector (pHM2291) based on plasmid pST76K-SR and containing the exon 5-EGFP fusion sequence enframed by the 5′- and 3′-ends of the homologous genomic sequences of ie2 exon 5 was used. The pST76K-SR-derived vector backbone also harbors a temperature-sensitive origin of replication, a kanamycin resistance gene, the recombinase gene recA, and the negative selection marker gene sacB, thus allowing a recombination strategy that involves the formation of a cointegrate (for a detailed description, see Materials and Methods).

The successful and correct integration of the exon 5-EGFP fusion sequence into the BAC ΔkΔex5 was subsequently determined by PCR and sequencing reactions with primer pairs binding either within the exon 5 sequence or within exon 5 and the upstream- and downstream-flanking regions (data not shown). Furthermore, the structural integrity of the recombinant BACs was verified by restriction enzyme digestion and Southern blot analysis (Fig. 1). The cleavage of three independent IE2-EGFP BACs (Fig. 1B, lanes 3 to 5) with BamHI yielded complex restriction patterns identical to those of HCMV AD169 wt DNA and the parental ΔkΔex5 BAC, with the following predicted exceptions. The insertion of an additional BamHI restriction site together with the EGFP sequence in the recombinant BACs resulted in the replacement of a 5.2-kb fragment of the wt BAC (Fig. 1B, lane 1) with a 4.3-kb fragment in a positive IE2-EGFP BAC (Fig. 1B, lanes 3 to 5). The diagram in Fig. 1A indicates all existing BamHI restriction sites of the relevant ie2 gene region plus the additional BamHI cleavage site of the inserted EGFP sequence. Due to deletion of exon 5, the 5.2-kb wt fragment is also absent in the parental ΔkΔex5 BAC (Fig. 1B, lane 2). The correct integration of the IE2-EGFP recombination cassette was finally confirmed by Southern blot hybridization experiments using the BamHI-digested BAC DNAs together with a biotinylated probe representing the IE2 cDNA sequence, thus comprising exons 2, 3, and 5 of the MIE gene locus, as indicated in Fig. 1A. Consistent with the integration of an additional BamHI site into the IE2-EGFP BAC, successful recombination (Fig. 1C, lanes 3 to 5) resulted in a 1.6-kb fragment that migrates faster than the 5.2-kb fragment of the wt BAC (Fig. 1C, lane 1). The latter fragment was missing in the ΔkΔex5 BAC (Fig. 1C, lane 2). Taken together, the results of Southern blotting, PCR, and nucleotide sequence analysis showed that recombination events took place at the correct sites of the HCMV genome. Thus, we successfully generated a recombinant HCMV BAC with the insertion of an EGFP-tagged IE2 sequence.

Reconstitution and growth analysis of the AD169/IE2-EGFP virus.

Next, infectious viruses were reconstituted by transfection of HFF cells with purified AD169/IE2-EGFP BAC DNA. One week after being transfected, the cells were transferred to 25-cm² flasks and cultured for about 2 weeks, until plaques appeared. Thereafter, viral progeny particles were recovered and used to infect fresh HFF cultures for the preparation and titration of virus stocks via IE1p72 fluorescence (see Materials and Methods). In order to make sure that the particle-to-PFU ratios of the recombinant AD169/IE2-EGFP virus and the wild-type virus were comparable, we either quantified viral input DNA by real-time PCR (Fig. 2A) or investigated the uptake of viral particles via Western blot analysis using antibodies against the tegument protein pp65 (serving as the indicator protein for viral particle uptake; Fig. 2B) and the immediate-early protein IE1p72 (serving as the indicator protein for active viral transcription; Fig. 2C). As shown in Fig. 2A to C, no significant differences between the recombinant and wt viruses were observed in these experiments.

In order to characterize the growth properties of the AD169/IE2-EGFP virus, multistep growth curve analyses of AD169/IE2-EGFP and wt HCMV AD169 were then performed in parallel. For these analyses, virus replication was assayed after both high (MOI = 1) and low (MOI = 0.1) MOIs of HFF cells. The viral inocula used in these experiments were standardized for comparable expression levels of IE1p72 (5). At different time points postinoculation (3, 5, 7, 9, and 13 days), aliquots of supernatants of the infected cells were sampled for titration via IE1p72 staining. As Fig. 2E indicates, AD169/IE2-EGFP exhibited a slightly delayed kinetics of virus accumulation under conditions of low MOI compared to that for wt AD169, since a major release of infectious recombinant particles occurred after a retardation of about 3 days and the peak titers of AD169/IE2-EGFP remained 1 log below that of the wt virus. Under conditions of high MOI, however, no significant growth defect of AD169/IE2-EGFP could be detected (Fig. 2D), indicating that the production of infectious recombinant virus is not markedly impaired by the genomic insertion of the IE2-EGFP fusion sequence under the latter infection conditions.

Protein expression kinetics during AD169/IE2-EGFP lytic infection.

During lytic replication, the IE2 gene region gives rise to three major protein isoforms, termed IE2p86, IE2p60, and IE2p40. Since these proteins share identical C-terminal sequences, fusion to EGFP was predicted to affect all IE2 protein isoforms. In order to confirm this and to compare the kinetics of viral protein accumulation of wt AD169 and AD169/IE2-EGFP, HFF cells were infected in parallel with wt and recombinant viruses (MOIs of 1 and 0.1), respectively. At various times postinfection (8, 12, 24, 48, 72, and 96 hpi), cells were harvested and viral proteins were examined by immunoblotting with antibodies directed against viral immediate-early (IE1, IE2), early-late (UL44, UL69, UL84), and true late (UL86, UL99) proteins (Fig. 3). As expected, all IE2 isoforms of the AD169/IE2-EGFP virus exhibited an increased molecular mass, indicating correct expression of the respective IE2 proteins in fusion with EGFP (Fig. 3A and B, upper panels, lanes 10 to 14). Interestingly, however, we observed a slight difference in the expression kinetics of the IE proteins of wt AD169- and AD169/IE2-EGFP-infected cells under low MOI conditions: both IE1p72 and IE2p86-EGFP could be detected in higher abundances at early times after infection with the AD169/IE2-EGFP virus (Fig. 3B, upper panels, compare lanes 2 and 3 and 10 and 11). In contrast, the increase in abundance observed for IE2 proteins during the late phase of the replication cycle with wt virus was less pronounced with the AD169/IE2-EGFP virus (Fig. 3B, upper panel, compare lanes 5 and 6 and 13 and 14). Similarly, late expression of UL84, UL69, UL86, and UL99 was diminished after infection with AD169/IE2-EGFP, whereas other early-late genes (e.g., UL44) were apparently not affected. This suggests that the C-terminal fusion of IE2 to EGFP may negatively affect specific functions of IE2, which correlates with the observation that the release of infectious viruses is somewhat diminished under conditions of low MOI with AD169/IE2-EGFP (Fig. 2B). However, since these effects were less pronounced under conditions of high MOI (Fig. 2D and 3A), we assumed that all major functions of IE2 are preserved.

Differential localization of IE2 and ND10 in live cells.

Previous indirect immunofluorescence studies with fixed cells have shown an intimate spatial association between the major regulatory protein IE2 and ND10 domains that is dependent on the mode of IE2 expression: while dot-like accumulations of transiently expressed IE2 display perfect ND10 colocalization (3, 25, 50), the viral IE protein can be found both colocalizing with and adjacent to this subnuclear structure during HCMV infection (3, 25, 26). Taking advantage of live-cell microscopy providing increased sensitivity, lower background signals, and the absence of potential artifacts caused by fixation and sample preparation, we investigated the subnuclear localization of IE2 in live cells. In order to visualize ND10 in live, HCMV-infected cells, primary human fibroblasts stably expressing Sp100 isoform A in fusion with the red autofluorescent protein mCherry (mCh-Sp100) were generated via retroviral transduction (see Materials and Methods). As shown in Fig. 4, the autofluorescent mCh-Sp100 fusion protein colocalized with both endogenous PML (Fig. 4, subpanels a to d) and hDaxx (Fig. 4, subpanels e to h) and could thus be used as a faithful marker for genuine ND10. Note that while most cells stably expressing mCh-Sp100 showed exclusive nuclear localization of the autofluorescent protein, we observed an additional cytoplasmic fluorescence (Fig. 4, subpanels i to m) in some cells (in particular those cells expressing high amounts of Sp100). This is not a novel observation, since cytoplasmic localization of Sp100 has been described previously and was correlated with a lack of SUMO modification of the respective Sp100 isoform (49).

Having generated the mCh-Sp100 cells, we first investigated the subcellular localization of transiently expressed IE2 in live cells. For this experiment, mCh-Sp100 cells were transfected with plasmid pHM990, coding for IE2p86 fused to EGFP. As shown in Fig. 5A, precise colocalization of transiently expressed IE2 dots and mCh-Sp100 was detected by live-cell imaging. In contrast, the subnuclear localization of IE2 was different in the context of viral infection. The IE2-EGFP protein expressed from the recombinant AD169/IE2-EGFP virus was predominantly distributed in dot-like structures which were partially colocalized with or juxtaposed to Sp100 in live-infected cells as early as 2.5 hpi (Fig. 5B and C). Even in cases where the IE2 and Sp100 signals appeared to be colocalizing, detailed analyses at high magnification revealed that the signals from the two proteins were rather distinct (Fig. 5B and C, insets). These images were remarkably similar to those showing the juxtaposition of the HSV-1 ICP4 transcriptional regulatory protein in foci associated with ND10 (15, 17, 18). In summary, these observations not only confirm previous results obtained with fixed cells but also prove that the AD169/IE2-EGFP virus can serve as a valuable tool to visualize IE2 in live cells.

The number of IE2 foci increases at higher-input MOIs and is independent of the presence of genuine ND10 domains.

An interesting observation after infection with the AD169/IE2-EGFP virus was that the number of IE2 foci in the nucleus of the infected cells was dependent on the input MOI. HFF cells were infected with the recombinant HCMV virus at either a low or high viral input, and images were captured 4 hpi. The number of IE2 foci at an MOI of 0.5 was significantly lower (Fig. 6A, subpanel a, and B) than the corresponding number of IE2 dots when the MOI was 2.5 (Fig. 6A, subpanels b, and B). Similar observations of the dependence of the number of IE2 foci on the input MOI were made when PML-kd cells lacking intrinsic ND10 were infected with the virus (Fig. 6A, subpanels c and d, B). This observation indicates that, in contrast to the dependence of the dot-like localization of transiently expressed IE2 on the presence of PML-associated nuclear bodies (50), the major regulatory IE2 protein can be recruited onto foci independently of the presence of ND10 during the course of a productive infection. Furthermore, since we did not observe an MOI-dependent increase in the size of IE2 dots, it seems unlikely that another preexisting cellular structure serves as the nucleation site for IE2 accumulations. Rather, the finding that the amount of formation of IE2 foci is proportional to the MOI suggests that viral factors might contribute. In fact, we could argue that a viral component that is imported during infection may be responsible for the formation of IE2 dot-like structures.

Dynamics of fluorescently labeled Sp100 and IE2 in live-infected cells.

Construction of the recombinant AD169/IE2-EGFP virus allowed us to visualize the viral IE2 protein even at very early times of infection and thus to monitor its accumulation in relation to ND10. Time-lapse microscopy of mCh-Sp100 cells infected with AD169/IE2-EGFP at an MOI of 0.5 revealed a dynamic association of IE2 dots with ND10 structures during the first hours postinfection. Figure 7 shows a sequence of images of a single cell starting 160 min after addition of the virus. Several of the IE2 foci were strikingly juxtaposed next to Sp100 (Fig. 7), but some of them rapidly fused to precise colocalizations (Fig. 7), followed by segregation of IE2 and Sp100 (Fig. 7), and finally, the dispersal of ND10 accumulations (Fig. 7). Once IE2 and Sp100 were associated, there was a limited amount of movement between them, but those present in close association remained so throughout the sequence, until the disruption of ND10 occurred. We concluded that the IE2 foci and ND10 are separate structures that associate rapidly at early stages of HCMV infection and that this association is very dynamic and exceptionally reminiscent of the dynamics of its HSV-1 functional homolog, the ICP4 protein (17).

Development of IE2 foci into viral replication compartments.

The IE2 protein is required for maximal efficiency of viral DNA replication in transient DNA cotransfection assays, while studies with fixed cells have also shown that it is incorporated into large nuclear structures corresponding to viral DNA replication compartments (4, 40). To investigate the fate of the initially formed IE2 foci during the entire course of a lytic HCMV infection, HFF cells were infected with the AD169/IE2-EGFP virus and examined thereafter at intervals comprising immediate-early, early, and late time points of the replication cycle. Time-lapse microscopy of individual live cells indicated that a proportion of the IE2 foci went on to expand and develop into larger nuclear structures (Fig. 8A). Most of the cells showed multiple separate structures within the nucleus, some of which became enlarged and some of which combined to form the fully developed oval or kidney-shaped viral DNA replication compartments at late times of infection. Additional evidence that the large nuclear structures represent viral replication compartments was obtained when 200 μg/ml phosphonoacetic acid was included in the medium: formation of viral DNA replication compartments but not of the initial IE2 foci was abrogated by this DNA replication inhibitor (data not shown). To further confirm the specificity of incorporation of IE2 into viral DNA replication compartments, we investigated the localization pattern of the protein along with the DNA polymerase processivity factor ppUL44, an essential HCMV replication protein known to be recruited into these compartments at late stages of the replication cycle (4). Infection of HFFs with AD169/IE2-EGFP and staining for ppUL44 demonstrated an accumulation of both proteins into large globular nuclear structures 30 hpi (Fig. 8B). These observations indicate that the IE2 protein can serve as a marker for monitoring the formation of viral replication compartments and suggest that the tiny IE2 accumulations at immediate-early times of infection may represent viral nucleoprotein complexes that develop into replication compartments as infection progresses.

IE2 foci are associated with viral genomes at early stages of HCMV infection.

The ability of IE2 to directly bind to viral DNA (12, 27) along with our observations that the number of IE2 foci increased with increasing viral input and that many of these initially formed IE2 dots later developed into replication compartments gave rise to the idea that at least a proportion of the IE2 foci must represent an association of the IE2 protein with parental viral genomes. To address this hypothesis directly, in situ hybridization for the detection of HCMV DNA in combination with antibody detection of IE2 was applied in fixed HCMV-infected cells. As shown in Fig. 9A, the majority of IE2 foci formed early during infection were strongly associated with foci of viral DNA that were detected by in situ hybridization. A large number of cells were examined, showing that all distinct nuclear DNA foci exhibited an IE2 signal, while a subset of the IE2 foci, in particular the smaller ones, were not associated with a DNA signal. The formation of larger complexes between viral DNA and IE2 was obvious at 24 hpi, when viral DNA started to replicate, giving rise to the development of replication compartments (Fig. 9A, lower subpanels). Thus, these experiments provide direct evidence for an association of IE2 foci with parental viral genomes at early times after infection.

DNA binding of IE2p86 is required for recruitment into viral replication compartments.

We next investigated whether the direct DNA-binding activity of IE2 is relevant for its association with viral DNA during the course of HCMV infection. Since previous studies demonstrated that recombinant cytomegaloviruses with mutations within the IE2 DNA binding domain are not viable (53), we chose a transient expression strategy in order to address this question. For this experiment, we first constructed a plasmid encoding a DNA-binding-deficient version of IE2 fused to EGFP. This was done via the substitution of two histidines (amino acids 446 and 452 of IE2) of a putative zinc finger motif within IE2 with leucines; this mutation has previously been shown to abrogate the DNA-binding activity of IE2 (32). Expression of the mutant pEGFP-ΔIE2 plasmid was tested by both Western blotting and immunofluorescence analysis, and no differences from wt protein could be detected in terms of expression levels or its subnuclear localization (data not shown). Next we investigated the capabilities of both wt and mutant IE2 proteins to be recruited into viral replication compartments in a transfection/superinfection approach. HFFs were initially infected with HCMV strain AD169 and subsequently transfected (2 hpi) with the plasmids expressing either wt EGFP-IE2 or the mutant EGFP-ΔIE2. Images were captured at different time points thereafter, starting at 13 hpi, when the EGFP signal was mature enough for visualization. As was expected, EGFP-IE2 foci appeared at early times of the replication cycle, and a proportion of them developed progressively into viral replication compartments (Fig. 9B, subpanels a to d). On the other hand, the mutant EGFP-ΔIE2 protein failed to form normal IE2 dot-like structures but was instead evenly distributed in the nuclei of infected cells. The mutant ΔIE2 protein remained in a diffuse form even at late times of the replicative cycle, when viral DNA replication compartments were formed, as indicated by the recruitment of ppUL44 into them (Fig. 9B, panels e to h). This finding suggests that the IE2 DNA-binding activity is a prerequisite for the formation of IE2 foci and recruitment into replication compartments. Based on the above observations and considering that both the wt EGFP-IE2 and its mutant version are able to colocalize with ND10 after transient expression, viral DNA appears to be an important determinant of IE2 subnuclear localization, suggesting that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.