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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.

Wagenknecht N, Reuter N, Scherer M, Reichel A, Müller R, Stamminger T - Viruses (2015)

Bottom Line: Characterization of THP-1 cells by immunofluorescence andWestern blot analysis confirmed the expression of all major ND10 components.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany. nadine.wagenknecht@viro.med.uni-erlangen.de.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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viruses-07-02751-f003: 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.

Mentions: Since the results of previous studies suggested that PML may be critical for the differentiation of myeloid progenitor cells to macrophages [48], it was important to investigate whether the knockdown of PML, hDaxx or Sp100 affects the differentiation of THP-1 monocytes to THP-1 derived macrophages. For this, the generated siC, siPML, siDaxx and siSp100 cells were treated with PMA for 48 h to induce terminal differentiation. Generally, effective differentiation is characterized by adherence of the cells and by morphological changes towards a macrophage-like phenotype. Whereas undifferentiated THP-1 cells exhibited the morphology of non-adherent cells (Figure 3a, subpanels a–d), treatment of the cells with PMA resulted in the adherence of all generated knockdown and control cell lines (Figure 3a, subpanels e–h) implying that loss of PML, hDaxx or Sp100 does not prevent differentiation of THP-1 monocytes to THP-1 derived macrophages. Furthermore, the knockdown of PML, hDaxx and Sp100 was also verified in the THP-1 derived macrophages by Western blotting (Figure 3b), ensuring that the knockdown was maintained during differentiation. Taken together, this experiment clearly showed that PML, hDaxx or Sp100 are not involved in the differentiation of THP-1 monocytes to THP-1 derived macrophages.


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.

Wagenknecht N, Reuter N, Scherer M, Reichel A, Müller R, Stamminger T - Viruses (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488718&req=5

viruses-07-02751-f003: 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.
Mentions: Since the results of previous studies suggested that PML may be critical for the differentiation of myeloid progenitor cells to macrophages [48], it was important to investigate whether the knockdown of PML, hDaxx or Sp100 affects the differentiation of THP-1 monocytes to THP-1 derived macrophages. For this, the generated siC, siPML, siDaxx and siSp100 cells were treated with PMA for 48 h to induce terminal differentiation. Generally, effective differentiation is characterized by adherence of the cells and by morphological changes towards a macrophage-like phenotype. Whereas undifferentiated THP-1 cells exhibited the morphology of non-adherent cells (Figure 3a, subpanels a–d), treatment of the cells with PMA resulted in the adherence of all generated knockdown and control cell lines (Figure 3a, subpanels e–h) implying that loss of PML, hDaxx or Sp100 does not prevent differentiation of THP-1 monocytes to THP-1 derived macrophages. Furthermore, the knockdown of PML, hDaxx and Sp100 was also verified in the THP-1 derived macrophages by Western blotting (Figure 3b), ensuring that the knockdown was maintained during differentiation. Taken together, this experiment clearly showed that PML, hDaxx or Sp100 are not involved in the differentiation of THP-1 monocytes to THP-1 derived macrophages.

Bottom Line: Characterization of THP-1 cells by immunofluorescence andWestern blot analysis confirmed the expression of all major ND10 components.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany. nadine.wagenknecht@viro.med.uni-erlangen.de.

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.

No MeSH data available.


Related in: MedlinePlus