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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

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.
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viruses-07-02751-f007: 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.

Mentions: As Saffert and Kalejta observed increased IE gene expression in THP-1 cells devoid of hDaxx upon HCMV infection at an MOI of 3 [30] and as high MOIs are used by many groups to establish latency in vitro, we also infected control and knockdown THP-1 cells with TB40E/IE2-EYFP at an MOI of 4. Detection of IE gene expression in the individual cell lines was achieved by measuring EYFP autofluorescence at 24 hpi using flow cytometry analysis. However, infection under high MOI conditions did not reveal any significant differences in the numbers of IE2-EYFP positive cells when comparing siC with siPML, siDaxx or siSp100 cells, respectively (Figure 7). In summary, these results suggest that neither PML, hDaxx nor Sp100 are involved in the regulation of IE gene expression in undifferentiated cells and that only differentiation of the cells enables IE gene expression.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

viruses-07-02751-f007: 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.
Mentions: As Saffert and Kalejta observed increased IE gene expression in THP-1 cells devoid of hDaxx upon HCMV infection at an MOI of 3 [30] and as high MOIs are used by many groups to establish latency in vitro, we also infected control and knockdown THP-1 cells with TB40E/IE2-EYFP at an MOI of 4. Detection of IE gene expression in the individual cell lines was achieved by measuring EYFP autofluorescence at 24 hpi using flow cytometry analysis. However, infection under high MOI conditions did not reveal any significant differences in the numbers of IE2-EYFP positive cells when comparing siC with siPML, siDaxx or siSp100 cells, respectively (Figure 7). In summary, these results suggest that neither PML, hDaxx nor Sp100 are involved in the regulation of IE gene expression in undifferentiated cells and that only differentiation of the cells enables IE gene expression.

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