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. 2015 Jun 5;7(6):2884-907.
doi: 10.3390/v7062751.

Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1

Nadine Wagenknecht  1 , Nina Reuter  2 , Myriam Scherer  3 , Anna Reichel  4 , Regina Müller  5 , Thomas Stamminger  6
Affiliations

Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1

Nadine Wagenknecht et al. Viruses. .

Abstract

Promyelocytic leukemia nuclear bodies, also termed nuclear domain 10 (ND10), have emerged as nuclear protein accumulations mediating an intrinsic cellular defense against viral infections via chromatin-based mechanisms, however, their contribution to the control of herpesviral latency is still controversial. In this study, we utilized the monocytic cell line THP-1 as an in vitro latency model for human cytomegalovirus infection (HCMV). Characterization of THP-1 cells by immunofluorescence andWestern blot analysis confirmed the expression of all major ND10 components. THP-1 cells with a stable, individual knockdown of PML, hDaxx or Sp100 were generated. Importantly, depletion of the major ND10 proteins did not prevent the terminal cellular differentiation of THP-1 monocytes. After construction of a recombinant, endotheliotropic human cytomegalovirus expressing IE2-EYFP, we investigated whether the depletion of ND10 proteins affects the onset of viral IE gene expression. While after infection of differentiated, THP-1-derived macrophages as well as during differentiation-induced reactivation from latency an increase in the number of IE-expressing cells was readily detectable in the absence of the major ND10 proteins, no effect was observed in non-differentiated monocytes. We conclude that PML, hDaxx and Sp100 primarily act as cellular restriction factors during lytic HCMV replication and during the dynamic process of reactivation but do not serve as key determinants for the establishment of HCMV latency.

Keywords: ND10; PML; cytomegalovirus; intrinsic immunity; latency; restriction factor.

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Figures

Figure 1
Figure 1
(a) Subnuclear localization pattern of major ND10 proteins in THP-1 monocytes; the percentages given at the right of panel a (subpanels e–h and i–l) indicate the fraction of cells (based on the analysis of approx. 70 individual cells) showing the respective colocalization pattern; (b) Subnuclear localization pattern of the major ND10 proteins in THP-1 derived macrophages. The following antibodies were used for staining of ND10 components: Anti-PML polyclonal antibody H238 (a, subpanel b, f and j; b, subpanel b and f), anti-Sp100 from Abnova (a and b, subpanel c), anti-hDaxx polyclonal antibody C20 (a, subpanel g and k; b, subpanel g). Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (a, subpanel a, e and i; b, subpanel a and e); (c) Detection of PML, hDaxx and Sp100 expression pattern in THP-1 monocytes compared to THP-1 derived macrophages by Western blotting. The upper panel shows hDaxx expression levels in both cell types assessed by staining with an anti-hDaxx rabbit monoclonal antibody. The middle panels show detection of various SUMOylated as well as non-SUMOylated PML as well as Sp100 isoforms using anti-PML antibodies A301-167A/A301-168A and anti-Sp100 rabbit serum GH3, respectively. In the lower panel, detection of β-actin was included as an internal loading control.
Figure 2
Figure 2
Detection of endogenous hDaxx, PML and Sp100 by Western blotting using cell lysates derived from THP-1 monocytes, which were transduced with the respective shRNA expression vectors as indicated. The upper panel shows hDaxx protein levels detected with an anti-hDaxx rabbit monoclonal antibody. The middle panels show staining of various SUMOylated and non-SUMOylated isoforms of PML using the rabbit polyclonal anti-PML antibodies A301-167A/A301-168A and the SUMOylated as well as non-SUMOylated isoforms of Sp100 with anti-Sp100 rabbit serum GH3. In the lower panel detection of β-actin served as an internal loading control. Lanes: 1, siC control cells; 2, siPML cells; 3, siDaxx cells; 4, siSp100 cells. Quantification of the reduction of protein expression via densitometry revealed a knockdown efficacy of 96% for siPML cells, of 68% for siDaxx cells and of 94% for siSp100 cells, respectively.
Figure 3
Figure 3
Depletion of PML, hDaxx or Sp100 does not prevent differentiation of THP-1 monocytes to THP-1 derived macrophages. (a) THP-1 monocytes were treated with PMA for 48 h followed by comparison of the morphology of THP-1 derived macrophages and THP-1 monocytes by transmitted light microscopy; (b) Verification of the knockdown of PML, hDaxx and Sp100 after differentiation of THP-1 monocytes. Cell lysates derived from the respective THP-1-derived macrophages were analyzed by Western blotting for PML, hDaxx and Sp100 expression. Upper panel: Staining of hDaxx protein levels of siC- (lane 1), siPML- (lane 2), siDaxx- (lane 3) and siSp100-transduced cells (lane 4) using a rabbit monoclonal anti-hDaxx antibody. Middle panels: Detection of various SUMOylated and non-SUMOylated isoforms of PML and Sp100 with the rabbit polyclonal anti-PML antibodies A301-167A + A301-168A and the anti-Sp100 rabbit serum GH3, respectively. Lower panel: β-actin detection was included as internal loading control.
Figure 4
Figure 4
Comparative analysis of viral protein accumulation after infection with wt TB40E and the recombinant viruses TB40E/IE1-mCherry and TB40E/IE2-EYFP. HFFs were infected in parallel with wt TB40E and TB40E/IE1-mCherry (a and b) or TB40E/IE2-EYFP (c and d) at an MOI of 0.5 (a and c) or 0.05 (b and d). Cell lysates were prepared at the indicated times after virus inoculation (24, 48, 72 and 96 hpi) and subjected to Western blot analyses. The protein levels of the viral immediate-early (IE) proteins IE1 and IE2, the early-late (E) protein UL44 as well as the true late (L) protein MCP were analyzed using specific antibodies against the respective proteins. Detection of β–actin served as an internal loading control.
Figure 5
Figure 5
HCMV infection of THP-1 cells results in a quiescent state with almost no detectable IE gene expression. (a) THP-1 monocytes were infected with TB40E/IE2-EYFP at an MOI of 0.1 or 4. At 24 hpi, IE2-EYFP positive cells were measured by flow cytometry; (b) THP-1 monocytes were differentiated to THP-1 derived macrophages by the addition of PMA for 48 h followed by infection of the obtained differentiated THP-1 cells with TB40E/IE2-EYFP at an MOI of 0.1. At 24 hpi, IE2-positive cells were detected by flow cytometry measuring EYFP autofluorescence.
Figure 6
Figure 6
Knockdown of PML, hDaxx or Sp100 in THP-1 monocytes does not permit HCMV IE gene expression. (a) Equal numbers of siC, siPML, siDaxx and siSp100 THP-1 cells were infected with TB40E/IE2-EYFP at an MOI of 0.1. EYFP expression was measured at 24 hpi by flow cytometry; (b) Mean values of three individual infection experiments according to (a). Statistical analyses using the paired t test for siC and siPML, siDaxx and siSp100, respectively, revealed no significant differences between knockdown and control cells siC.
Figure 7
Figure 7
Similar numbers of IE2-EYFP positive cells in PML-kd, hDaxx-kd, Sp100-kd and control THP-1 monocytes after infection of these cells with TB40E/IE2-EYFP at an MOI of 4. At 24 hpi, cells were harvested and IE2-EYFP expression was analyzed by flow cytometry.
Figure 8
Figure 8
Enhancement of HCMV IE gene expression in the absence of PML, hDaxx or Sp100 under low MOI conditions. THP-1 monocytes, retrovirally transduced as indicated, were differentiated to THP-1-derived macrophages by the addition of PMA for 72 h. Next, these cells were infected with TB40E/IE2-EYFP at an MOI of 0.01 (a), 0.05 (b) or 0.1 (c). At 24 hpi, cells were harvested and examined by flow cytometry for EYFP expression. Each infection was performed in triplicate and the standard deviations are depicted by error bars. Statistical significance (p-values) of the difference between control cells and knockdown cells was calculated using Student’s unpaired two-tailed t-test.
Figure 9
Figure 9
Depletion of PML, hDaxx or Sp100 affects the reactivation efficacy of latent HCMV. THP-1 monocytes, retrovirally transduced as indicated, were infected with TB40E/IE2-EYFP at an MOI of 0.1 and cultured for five days. Thereafter, aliquots of the latently infected THP-1 monocytes were fixed. The remainder of the cells were differentiated to THP-1-derived macrophages by the addition of PMA for 72 h. Then, cells were harvested and THP-1 monocytes as well as macrophages were examined by flow cytometry for EYFP expression. Each infection was performed in triplicate and the standard deviations are depicted by error bars. Statistical significance (p-values) of the difference between control cells and knockdown cells was calculated using Student’s unpaired two-tailed t-test.
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