Cellular and Viral Control over the Initial Events of Human Cytomegalovirus Experimental Latency in CD34⁺ Cells

Cells and viruses.Normal human dermal fibroblasts (NHDFs), TERT-immortalized human fibroblasts (TERT-HFs), human foreskin fibroblasts (HFFs), THP-1 cells, and differentiated THP-1 macrophage were cultured as previously described (61). Human CD34⁺ hematopoietic progenitor cells derived from cord blood (Lonza) were maintained in HPGM medium (Lonza) supplemented with recombinant human stem cell factor (SCF) (25 ng/ml), recombinant human thrombopoietin (TPO) (50 ng/ml), and recombinant human Fms-related tyrosine kinase 3 ligand (Flt3) (50 ng/ml), all from PeproTech Inc. CD34⁺ cells were maintained in culture for no more than 10 days (three passages). The percentages of cells from two separate and representative lots that remained CD34⁺ at different periods of maintenance with these culture conditions were determined by flow cytometry by using a phycoerythrin (PE)-conjugated anti-CD34⁺ antibody essentially as described previously (14). The differentiation status of these cells was maintained well for 7 days (99.5%, 99.1%, and 80.2% CD34⁺ cells on days 1, 4, and 7, respectively) but declined after longer periods of cell culture (34.7% CD34⁺ cells on day 10). Thus, HCMV IE gene expression experiments were conducted with cells that had been cultured 7 days or less. CD34⁺ cells were differentiated in HPGM medium as previously described (57). Briefly, CD34⁺ cells were grown for seven days in HPGM medium supplemented with transforming growth factor beta (TGF-β) (0.5 ng/ml), Flt3 (100 ng/ml), SCF (20 ng/ml), tumor necrosis factor alpha (TNF-α) (50 units/ml), and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (100 ng/ml; Fitzgerald). After seven days, lipopolysaccharide (LPS) (50 ng/ml) was added for an additional three days. Viruses used were AD169 and green fluorescent protein (GFP)-expressing FIX and TB40/E strains. Cells were infected in minimal volume for 60 min, followed by the addition of medium to normal culture volumes. Note that the titers used represent “fibroblast infectious units” and do not necessarily represent the efficiency with which the virus entered or infected other cell types, most notably CD34⁺ cells. Multiplicities of infection (MOIs) were selected in an attempt to equalize the potentials to synthesize IE genes in the different cell types. Transduction with recombinant adenoviruses has been described previously (61).

Inhibitors, antibodies, and Western blots.Valproic acid (VPA) (1 mM; Sigma) dissolved in water was added 18 h before infection (HFFs) (37), 3 h before infection (THP-1- and THP-1-derived macrophage cells) (61), or at the time of infection (CD34⁺) with HCMV. The following antibodies were from commercial sources: anti-Daxx (D7810) and antitubulin (DM 1A) (both from Sigma), anti-PML (H-238 and sc-966; Santa Cruz), anti-Sp100 (AB1380; Chemicon), anti-UL44 (CA006-100; Virusys), and anti-CD34 (555822; BD Pharmingen). Antibodies against pp71 (IE-233), IE1 (1B12), and pp28 (CMV157) and secondary antibodies have been previously described (61, 62). For Western blot analysis, either equivalent numbers of cells lysed in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (see Fig. 6D) or equivalent protein concentrations from cell lysates prepared in RIPA buffer with protease inhibitors (all other Western analyses) were analyzed as previously described (62).

Indirect immunofluorescence.Mock- or HCMV-infected THP-1 or CD34⁺ cells were collected by low-speed centrifugation, treated with trypsin (0.5 mg/ml) for 5 min at 37°C, again collected by low-speed centrifugation, and resuspended in cold phosphate-buffered saline (PBS). Cells were allowed to attach to water-washed coverslips as previously described (61). Additionally, CD34⁺ cells were differentiated on coverslips prior to infection. Cells were fixed with 1 to 4% paraformaldehyde in PBS and processed as described previously (61). Images were visualized and photographed using a Zeiss Axiovert 200 M deconvolution fluorescence microscope with a 60× objective.

siRNA, shRNA, and transfections.CD34⁺ cells were transfected using Amaxa Biosystems technology with small interfering RNA (siRNA) (1.25 μg/1.0 × 10⁶ cells) or with expression plasmids (1 μg/5.0 × 10⁵ cells, pSG5pp71 or pSG5pp71Did2-3) by using the human CD34 cell Nucleofector kit (VPA-1003; Amaxa Biosystems), following the manufacturer's protocol. Previously described synthetic, annealed, 21-base oligonucleotides (siRNA) were purchased from Dharmacon (61, 62). Cells transfected with siRNA were cultured for 48 h and then infected with HCMV AD169 (MOI of 1) for 24 additional hours. Cells transfected with pp71 expression plasmids were cultured for 36 h prior to collection for indirect immunofluorescence. The generation of TERT-immortalized HFs and THP-1 cells constitutively expressing a short hairpin RNA (shRNA) directed at Daxx was achieved using a retrovirus encoding the hDx2 sequence as previously described (61).

RT-PCR.Total RNA was isolated from CD34⁺ cells infected with HCMV at an MOI of 1 for 24 h after treatment with VPA or siRNA as described above by using the RNeasy minikit (catalog no. 74104; Qiagen). Isolated RNA was quantified, and equivalent amounts were treated with RNase-free DNase (M6101; Promega) following the manufacturer's protocol. Equivalent quantities of RNA were subsequently used in a reverse transcription (RT)-PCR (35 cycles), using the SuperScript III one-step RT-PCR system (catalog no. 12574-026; Invitrogen). Primer pairs included IE1 sense (5′-CGTCCTTGACACGATGGAGT) and antisense (5′-ATCTGTTTGACGAGTTCTGCC) primers (that span the intron between exon 2 and exon 3) and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as previously described (31). PCR products were separated on 1.5% agarose gels, and bands were quantified using Quantity-One software (Bio-Rad).

Q-PCR.DNA and RNA were isolated from 750,000 recombinant adenovirus (rAD)-transduced cells by using the DNeasy tissue kit (catalog no. 69504; Qiagen) and RNeasy minikit (catalog no. 74104; Qiagen), respectively, according to the manufacturer's instructions. RNA was treated with RNase-free DNase as described above. Subsequently, 1 μg of DNase-treated RNA was used for a first-strand cDNA synthesis procedure using the SuperScript III first-strand synthesis system (18080-051; Invitrogen) with provided oligo(dT) primers according to the manufacturer's specifications. Reactions without reverse transcriptase controlled for contaminating DNA (data not shown). DNA and cDNA were used in a quantitative real-time PCR (Q-PCR) to determine the relative activity of the MIEP assayed by expression of pp65 transcripts using previously described primer/probe sets (61) or that of the EF1α promoter expressing pp71. Primer pairs for pp71 were as follows: forward, 5′-CGTTCATTTGGAACACCGAC, and reverse, 5′-CTCTTCCTCTTCTTCCTCCTCTTC. The pp71 probe was 6-carboxyfluorescein (6FAM)-TCTCACACCCGGTAAACCGGACAAA-6-carboxytetramethylrhodamine (TAMRA) (Applied Biosystems). Expression levels of cellular β-actin quantitated with a primer pair and probe set previously described (61) were analyzed as a control and varied no more than 3-fold in all samples examined. Unknown sample values were determined based on a standard curve of known copy numbers of pp65, beta-actin (61), and pp71.

The PML-NB intrinsic defense is functional against low-passage clinical isolates of HCMV.Evidence supporting the presence of an intrinsic immune defense that blocks HCMV IE gene expression at the start of lytic and quiescent infections has been obtained in experiments utilizing high-passage laboratory-adapted viral strains such as AD169 (2, 56, 61, 62, 72, 73). If the PML-NB defense were to control the establishment of true HCMV latency in vivo, it would also have to be active against circulating viruses that are more similar to low-passage clinical viral strains than to AD169. We suspected that clinical isolates such as FIX (19) and TB40/E (69) would also be subject to the PML-NB-mediated intrinsic defense because the MIEPs (from −1140 to +970 [the ATG codon], where the major transcription initiation site is +1) of low-passage clinical viral strains are 98% identical to that of AD169 (data not shown). Our experiments proved this to be the case. Inhibition of HDAC activity by valproic acid (VPA) increased IE1 expression at the start of a low-multiplicity-of-infection (MOI) lytic infection of fibroblasts with laboratory (AD169) and clinical (FIX and TB40/E) viral strains (Fig. 1A). Likewise, telomerase-immortalized fibroblasts (TERT-HFs) constitutively expressing a short hairpin RNA directed at Daxx displayed higher levels of IE1 than the parental TERT-HFs when infected at a low MOI with AD169, FIX, or TB40/E (Fig. 1B). These results show that HDACs and Daxx negatively regulate the HCMV MIEP of clinical strains at the start of lytic infections in fibroblasts. Note that infection of fibroblasts with equal multiplicities of AD169 or clinical viral strains resulted in comparable steady-state levels of IE1 at 6 h postinfection (hpi) both in the absence and in the presence of the HDAC inhibitor (Fig. 1A) even though the particle-to-PFU ratios of clinical and laboratory strains are often found to differ dramatically.

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FIG. 1.

HCMV clinical isolates are susceptible to Daxx-mediated MIEP silencing. (Panel A) HFFs were mock infected (M) or treated without (−) or with (+) VPA ∼18 h prior to infection with AD169 (AD), TB40/E (TB40), or FIX at a multiplicity of infection (MOI) of 0.05. Lysates were harvested 6 h postinfection (hpi) and analyzed by Western blotting. (Panel B) TERT-HFs (T) or those constitutively expressing an shRNA to Daxx (D) were infected with AD169, TB40/E, or FIX at an MOI of 0.5. Lysates were harvested at 6 hpi and analyzed by Western blotting. (Panel C) THP-1 monocytes were mock infected or infected with AD169, TB40/E, or FIX in the absence (−) or presence (+) of VPA at an MOI of 1 for 12 h. Lysates were analyzed by Western blotting. (Panel D) THP-1 cells (T) or those expressing shRNA to Daxx (D) were infected with AD169, TB40/E, or FIX at an MOI of 1 for 24 h and analyzed by Western blotting. (Panel E) THP-1-derived macrophage (TDM) were mock infected or treated without (−) or with (+) VPA for 3 h prior to infection with AD169, TB40/E, or FIX at an MOI of 0.5. Lysates were harvested 12 hpi and analyzed by Western blotting. (Panel F) THP-1-derived macrophage (T) or those expressing shRNA to Daxx (D) were infected with AD169, TB40/E, or FIX at an MOI of 0.5 for 12 h and analyzed by Western blotting. For all experiments, tubulin (Tub) was used as a loading control. Short and long exposures are shown for IE1.

Using equivalent approaches, we made the similar observation that Daxx and HDACs also silence IE1 expression from infecting FIX and TB40/E viral genomes during quiescent infections of THP-1 cells. Specifically, treatment of THP-1 cells with VPA (Fig. 1C) or infection of Daxx-knockdown THP-1 cells (Fig. 1D) partially rescued IE gene expression for both AD169 and clinical viral strains. VPA treatment did not permit transit of tegument-delivered pp71 to the nucleus of undifferentiated THP-1 cells (data not shown). It was retained in the cytoplasm, as we previously demonstrated (61).

Interestingly, IE1 expression was more robust in AD169-infected THP-1 cells in the presence of VPA (Fig. 1C) or in infected THP-1-shDaxx cells (Fig. 1D) than in cells infected with FIX or TB40/E clinical strains at the same multiplicity. This effect was not observed during lytic infection of fibroblasts (Fig. 1A and B). Such a difference may result from the different particle-to-PFU ratios of the viral stocks, because the PML-NB intrinsic defense is the sole or dominant repressive mechanism found in differentiated cells, or perhaps because of a clinical-strain-specific restriction to HCMV infection prior to IE gene expression in undifferentiated cells. Regardless of the mechanism, this observation may explain the different conclusions drawn about the contribution of Daxx to IE gene silencing during quiescent infection of NT2 cells between two independent studies, one of which used AD169 (61) while the other used the clinical strain Toledo (17).

An analysis of IE gene expression after infection of THP-1 cells previously differentiated into macrophage cells yielded results very similar to those observed in fibroblasts. Infections at identical MOIs resulted in similar levels of IE gene expression (Fig. 1E), which were enhanced by both VPA treatment (Fig. 1E) and Daxx knockdown (Fig. 1F). In total, these experiments show that the FIX and TB40/E clinical viral strains express more IE1 when the PML-NB-mediated intrinsic defense is inactivated in both differentiated (Fig. 1A, B, E, and F) and undifferentiated (Fig. 1C and D) cells. Thus, we conclude that the PML-NB intrinsic defense that silences the MIEP is active against low-passage clinical strains of HCMV during both lytic and quiescent infections.

CD34⁺ cells contain PML-NB proteins and structures.The susceptibility of clinical strains to the PML-NB-mediated intrinsic defense that silences HCMV IE gene expression (Fig. 1) and the indistinguishable chromatin structure found at the MIEP during early lytic, quiescent, and latent infections (25, 51, 57, 58, 79) led us to hypothesize that viral latency in CD34⁺ cells is controlled, in part, by the PML-NB intrinsic defense. Western blots indicated that Daxx, PML, and Sp100, all PML-NB constituents, were expressed in CD34⁺ cells (Fig. 2A). Multiple experiments consistently showed an increase in Daxx levels in CD34⁺ cells compared to fibroblasts. Conversely, PML levels were reduced in CD34⁺ cells, and the spectra of isoforms likely resulting from both alternative splicing and differential SUMOylation (30) present in the two cell types were different. However, all isoforms contain the RBCC/Trim motif that is required for the antiviral effects of PML (60). Sp100 levels were similar in the two cell types, but like for PML, differentially modified proteins and/or different protein isoforms were expressed. Similar to the case for PML, multiple Sp100 isoforms have been found to display antiviral activities (52).

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FIG. 2.

CD34⁺ cells contain PML-NBs. (Panel A) Fifty micrograms of protein lysate from NHDFs (F) or CD34⁺ cells (C) were analyzed by Western blotting. Approximate protein sizes in kDa are shown to the right. (Panel B) CD34⁺ cells were immobilized on coverslips and stained for PML and Sp100 (red and green, respectively). Nuclei were counterstained with Hoechst stain (blue). (Panel C) CD34⁺ cells stained for Daxx (green). (Panel D) CD34⁺ cells stained for Daxx (green) and PML (red) for which Daxx is punctate. (Panel E) CD34⁺ cells stained for Daxx (green) and PML (red) for which Daxx is diffuse. The magnification for all images in this figure is ×60.

Indirect immunofluorescent staining showed that PML and Sp100 were found to colocalize in punctate domains (Fig. 2B) in all cells examined. Interestingly, Daxx localization varied in different CD34⁺ cells (Fig. 2C), where it was found either in punctate domains that colocalized with PML (Fig. 2D) or dispersed throughout the nucleus, even though PML remained punctate (Fig. 2E). CD34⁺ cells contain less PML and more Daxx than fibroblasts (Fig. 2A). Because Daxx is recruited to PML-NBs specifically by the PML protein (27), perhaps Daxx binding sites on PML become saturated in some CD34⁺ cells, causing the extra Daxx to disperse throughout the nucleus. Alternatively, because cells at multiple stages of hematopoietic differentiation express CD34 (78), differing Daxx distributions may be due to the heterogeneous nature of CD34⁺ cell populations.

We counted the number of cells containing punctate or diffuse Daxx prior to or 24 h after HCMV AD169 infection (MOI of 3). We found that 81% ± 2% of mock-infected cells showed diffuse Daxx, whereas 68% ± 6% of HCMV-infected cells (identified by staining for tegument-delivered pp71) contained diffuse Daxx (punctate Daxx was observed in the remainder of the cells). Although small, this difference is statistically significant (P = 0.002). The biological significance of this observation (if any) is presently unclear, and this experiment is unable to discern between HCMV preferentially entering CD34⁺ cells with punctate Daxx and the virus somehow modulating PML-NB structure after viral entry.

Daxx proteins found outside the context of PML-NBs appear to be stronger transcriptional repressors than PML-NB-localized Daxx (38). Thus, the majority of CD34⁺ cells (those with diffuse Daxx) may provide an inherently more repressive environment for the viral MIEP (which is silenced by Daxx) than fibroblasts do (see below). From these observations, we conclude that CD34⁺ cells express PML, Sp100, and Daxx isoforms with potential antiviral activities (10) and have the capacity to assemble PML-NBs. Thus, the PML-NB-mediated intrinsic defense against HCMV could conceivably be active in CD34⁺ cells.

The HCMV MIEP is an inefficient promoter in CD34⁺ cells.The experiments described above show that PML-NB proteins that institute an intrinsic defense against HCMV by silencing the viral MIEP are present in CD34⁺ cells, and their steady-state levels and localizations indicate that CD34⁺ cells might be more restrictive to MIEP function than fibroblasts. To test this, we determined the activities, on a per-DNA-copy basis, of the MIEP and the cellular EF1α promoter in a series of cell types used to study HCMV infection. The EF1α promoter was selected because it is often used to express proteins in undifferentiated cells (6, 41, 61). In these experiments, we delivered the MIEP to cells by recombinant adenoviral (rAD) transduction. Serotype 5 adenoviruses enter CD34⁺ cells with an efficiency similar to that of HCMV, and in CD34⁺ cells successfully entered, they replicate with efficiencies approaching 100% (64), indicating that there are no substantial postentry blocks (e.g., inability to deliver genomes to the nucleus) to gene expression from adenoviral genomes in CD34⁺ cells. For these reasons, and because adenoviral genomes (26), like those of HCMV (28), associate with PML-NBs in infected cells, we chose adenoviral transduction (as opposed to retroviral transduction or plasmid transfection) for these experiments. Internally controlled quantitative (reverse transcriptase or straight) real-time PCR (Q-PCR) was used to determine the relative amounts of mRNA compared to DNA copies of the genes expressed from the MIEP or EF1α promoter in each cell type examined.

We found that the MIEP is over 100-fold less active in CD34⁺ cells than in THP-1 cells and over 1,000-fold less active in CD34⁺ cells than in THP-1-derived macrophage and in fibroblasts (which showed equivalent MIEP activities) (Fig. 3A). However, the EF1α promoter had similar activity, on a per-molecule basis, in CD34⁺ cells and fibroblasts (Fig. 3B). These experiments indicate that the HCMV MIEP is inherently less active in CD34⁺ cells than in other cell types routinely used to study HCMV infection. As mentioned above, the poor activity of the MIEP in CD34⁺ cells may be due in part to higher Daxx and lower PML levels in CD34⁺ cells (Fig. 2A), because Daxx is known to repress the MIEP (56, 62, 79) and PML is known to inhibit Daxx-mediated transcriptional repression (38). Alternatively or in addition, other cellular repressors (40, 42, 72, 80) or the proposed lack of activating transcription factors (17, 67) may also contribute to the poor function of the HCMV MIEP in CD34⁺ cells.

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FIG. 3.

The HCMV MIEP is less active in CD34⁺ cells than in other cell types. (Panel A) NHDF (HF), THP-1-derived macrophage (TDM), THP-1, and CD34⁺ cells were transduced with a recombinant adenovirus (rAD) expression system driven by the HCMV MIEP and harvested 24 h posttransduction. The relative quantity of expressed mRNA compared to input rAD genomic DNA was determined by Q-PCR (see Materials and Methods). Fold activity of the MIEP was compared to levels found in NHDFs, which were empirically set at 1. Averages and standard errors of the means for five independent experiments are shown. (Panel B) NHDF and CD34⁺ cells were transduced with an rAD expression system driven by the EF1α promoter and analyzed as described for panel A. Results from three independent experiments are shown, with the averages and standard errors of the means indicated.

The MIEP is repressed by the PML-NB defense in HCMV-infected CD34⁺ cells.We next asked if the HDAC and Daxx components of the PML-NB defense silence IE gene expression upon HCMV infection of CD34⁺ cells. We employed RT-PCR as a direct assay for MIEP function in CD34⁺ cells for multiple reasons, including the low infection efficiency of these cells reported previously (13, 34) and assayed here by tegument delivery of pp71 (Fig. 4A), the poor activity of the MIEP in these cells (Fig. 3A), the potential for microRNA-mediated silencing of the IE1 transcript (15, 49), and most importantly, to keep our experimental conditions consistent with the previous RT-PCR examinations of viral gene expression during latency (17, 44, 58, 76). In all experiments, mRNA levels for the glyceraldehyde phosphate dehydrogenase (GAPDH) housekeeping gene were used for normalization. We found a significant enhancement of IE1 mRNA production (∼15-fold over virus alone) in CD34⁺ cells treated with the HDAC inhibitor VPA after infection with HCMV AD169 (Fig. 4B and C). We also found that siRNA-mediated knockdown of Daxx in CD34⁺ cells (Fig. 4D) permitted a reproducible and statistically significant (P = 0.025) increase in AD169 IE1 expression compared to transfection of CD34⁺ cells with siRNA to a control protein Skp-1 (Fig. 4E and F). Although the magnitude of Daxx knockdown and the activation of IE gene expression are modest, five separate experiments using two different lots of CD34⁺ cells gave comparable results. Thus, HDACs and the PML-NB protein Daxx, which are part of an intrinsic immune defense against HCMV (63, 74), contribute to the silencing of viral IE gene expression when CD34⁺ cells are infected in vitro with a laboratory-adapted strain of HCMV. This finding is in agreement with previous data demonstrating a repressed chromatin structure at the MIEP during latency (57, 58) that is indistinguishable from that assembled by the PML-NB defense prior to lytic infection (79).

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FIG. 4.

Daxx and histone deacetylase activity control HCMV AD169 IE gene expression during infection of CD34⁺ cells. (Panel A) CD34⁺ cells were infected with AD169 (AD) or FIX at an MOI of 5 or 3, respectively. After 24 h, cells were harvested, treated with trypsin, immobilized on coverslips, and stained for pp71. The percentages of cells staining positive for pp71 are shown. Standard deviations are indicated with bars. (Panel B) CD34⁺ cells were mock treated (−) or treated with VPA (+) and either mock infected (−) or infected with AD169 (+) at an MOI of 1 as indicated. Cells were harvested 24 hpi, and total RNA was extracted and analyzed by RT-PCR for expression of IE1. Cellular GAPDH served as an internal control. (Panel C) Fold IE1 activation (normalized to GAPDH levels) for samples shown in panel B is shown. M, mock; V, AD169; VPA, AD169 + VPA. Bars indicate standard deviations. (Panel D) CD34⁺ cells were transfected without siRNA (Ø) or transfected with siRNA directed at Daxx (siD) or Skp-1 (siS). Forty-eight hours later, cells were infected with AD169 at an MOI of 1 for 24 h. Cells were harvested, and a fraction was used for Western blot analysis. Tubulin (Tub) was used as a normalization control. For the experiment shown, the Daxx level in the Daxx-knockdown cells was 42% of the untreated cells. In the Skp-1-knockdown cells, the Daxx level was 96% of the untreated cells. (Panel E) RNA was isolated from the remaining cells (treated as described for panel D above) and used in an RT-PCR assay for IE1 and GAPDH. Mock-infected (−) or AD169-infected (+) cells and cells transfected without siRNA (−), with siRNA directed at Daxx (D), or with siRNA directed at Skp-1 (S) were analyzed. (Panel F) Results from five independent experiments as outlined (panels D and E) were analyzed, and fold activation of IE1 in cells transfected with siRNA to Daxx over those transfected with siRNA to Skp-1 (siD/siS) is shown with the standard deviation (*, P value < 0.05).

Our demonstration that the PML-NB defense contributes to silencing of the MIEP in HCMV-infected CD34⁺ cells led us to ask why it is not neutralized by tegument-delivered pp71, as it is when lytic infection initiates (62). Identical to the cases for NT2 and THP-1 cells (61), we found tegument-delivered pp71 exclusively in the cytoplasm of CD34⁺ cells infected with either AD169 or FIX (Fig. 5A). In contrast, we found that prior differentiation of CD34⁺ cells into dendritic cells and subsequent infection with AD169 permitted both the nuclear localization of tegument-delivered pp71 and IE1 expression (Fig. 5B). Identical to the cases for other cell types we have examined (61), pp71 that is newly expressed by either adenoviral transduction or plasmid transfection efficiently localizes to the nuclei of CD34⁺ cells (Fig. 5C). In CD34⁺ cells in which Daxx had a punctate nuclear localization, newly expressed wild-type pp71 also appeared as punctate dots that colocalized with Daxx (Fig. 5D). pp71 was found throughout the nucleus in cells with a diffuse pattern of Daxx staining (data not shown). The Did2-3 pp71 mutant, unable to bind Daxx (20), showed a diffuse staining pattern (Fig. 5D) both in the nucleus and in the cytoplasm. Thus, while tegument-delivered pp71 is trapped in the cytoplasm, de novo-expressed wild-type pp71, presumably by binding to Daxx, is recruited to PML-NBs in CD34⁺ cells, as expected.

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FIG. 5.

Differentiation-dependent localization of tegument-delivered pp71 and IE1 expression during HCMV infection of CD34⁺ cells. (Panel A) CD34⁺ cells were infected with AD169 or FIX at an MOI of 5 or 3, respectively. After 24 h, cells were harvested, treated with trypsin, immobilized on coverslips, and stained with an antibody to pp71 (AD169, green; FIX, red). (Panel B) CD34⁺ cells were differentiated into dendritic cells and infected with AD169 at an MOI of 5 for 24 h. Adherent cells on coverslips were analyzed for the presence of pp71 or IE1 (both green). (Panel C) CD34⁺ cells were transduced with a recombinant adenovirus that expresses pp71 (rADpp71) at 1,000 particles per cell for 24 h or transfected with a pp71 expression plasmid (pSG5-pp71) for 36 h and analyzed for pp71 expression (red). (Panel D) CD34⁺ cells were transfected with expression plasmids for wild-type pp71 (pp71) or a pp71 mutant unable to bind Daxx (Did2-3) for 24 h and stained with antibodies to pp71 (red) and Daxx (green). In all panels, nuclei were counterstained with Hoechst stain (blue). The image magnification was ×60.

In addition to the PML-NB defense, clinical strains of HCMV employ a dominant means to silence IE gene expression during latency.Our data indicate that artificial inactivation of the PML-NB intrinsic defense allows for the expression of viral IE genes upon infection of CD34⁺ cells with the high-passage laboratory-adapted HCMV strain AD169. We next asked if this also held true for the FIX and TB40/E clinical strains of HCMV. In contrast to our results with AD169, we found that addition of VPA to CD34⁺ cells prior to infection with either FIX (Fig. 6A and B) or TB40/E (Fig. 6C) failed to induce IE1 gene expression. This result is unlikely to be due to differing infection efficiencies of the two viral strains, because the numbers of CD34⁺ cells that received tegument proteins upon HCMV infection were similar for representative laboratory and clinical virus strains (Fig. 4A). Thus, we conclude that clinical strains, in addition to the PML-NB defense, experience an additional postentry, HDAC-independent block to IE gene expression in CD34⁺ cells compared to a laboratory-adapted viral strain infecting these cells.

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FIG. 6.

HCMV clinical isolates institute a dominant block to IE gene expression. (Panel A) CD34⁺ cells were mock infected (−), infected (+) with AD169 (MOI of 1) or FIX (MOI of 1), or coinfected with both (each at an MOI of 1 added to the cells simultaneously [total MOI of 2]) in the absence (−) or presence (+) of VPA. Cells were harvested 24 hpi, and isolated RNA was used in an RT-PCR for IE1 and GAPDH expression. (Panel B) Fold activation of IE1 during infection in the presence of VPA compared to virus alone was determined for experiments described for panel A. (Panel C) Experiments were conducted as outlined above by using AD169 (AD; MOI of 1), clinical isolate TB40/E (TB; MOI of 1), or both (AD-TB; each at an MOI of 1, added to the cells simultaneously [total MOI of 2]). Fold IE1 activation with virus plus VPA over virus alone is shown. For panels B and C, results from four (B) or three (C) independent experiments with standard errors of the means shown. (Panel D) CD34⁺ cells left untreated (−) or treated at the time of infection with 1 mM VPA (+) as indicated were infected with AD169 (MOI of 1) or FIX (MOI of 1) or coinfected with a mixture of AD169 and FIX (each at an MOI of 1 for a total MOI of 2) for 24 h, after which the cells were washed free of virus and VPA and cultured for an additional 6 days. Equal cell numbers were lysed in RIPA buffer, and their protein contents were analyzed by Western blotting with the indicated antibodies. Tubulin was used as a loading control. Note that titers are based on fibroblast infection and do not represent the efficiency with which HCMV enters CD34⁺ cells (Fig. 4A).

We then asked if this additional restriction to IE gene expression was observed in mixed coinfections in which CD34⁺ cells were infected with equal multiplicities of the laboratory-adapted AD169 virus and a clinical strain of HCMV. We found that in CD34⁺ cultures coinfected with heterogeneous viral mixtures, the VPA-induced activation of IE1 gene expression from the AD169 genome was not observed (Fig. 6A, B, and C). Thus, the additional restriction to IE gene expression observed upon HCMV infection of CD34⁺ cells by clinical viral strains acts in trans and in a dominant fashion, as it was able to restrict the ability of VPA to permit IE gene expression from coinfecting AD169 genomes.

These experiments indicate that in VPA-treated CD34⁺ cells infected with laboratory (AD169) but not clinical (FIX or TB40/E) strains of HCMV, latency is not established, but rather lytic infection is initiated because the viral IE genes are expressed. We confirmed this by detecting the expression of IE1 at the protein level, as well as the expression of the UL44 protein (encoded by an early gene), by Western blot analyses in VPA-treated CD34⁺ cells infected with AD169, whereas IE1 and UL44 expression levels were substantially lower in those infected with FIX or coinfected with AD169 and FIX (Fig. 6D). However, this initiated lytic infection that progresses at least to the early phase appears to be abortive, because we were unable to detect expression of the pp28 protein encoded by the true late gene UL99 (9). This result is identical to what we observed in NT2 cells (a model for HCMV quiescence), in which IE1 and UL44 protein expression but not pp28 protein expression was observed upon infection of Daxx-knockdown cells (61).

In total, our data demonstrated that HCMV experimental latency in CD34⁺ cells infected in vitro is established in part by a cellular intrinsic immune defense mediated by Daxx and HDACs because tegument-delivered pp71 fails to enter the nucleus and inactive this defense by degrading Daxx. Furthermore, we demonstrated that an additional mechanism, revealed only by the comparison of laboratory and clinical viral strains, acts in an HDAC-independent, trans-dominant manner to silence the MIEP during experimental latency in CD34⁺ cells infected in vitro with clinical strains of HCMV.