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Localization, quantification and interaction with host factors of endogenous HTLV-1 HBZ protein in infected cells and ATL.

Raval GU, Bidoia C, Forlani G, Tosi G, Gessain A, Accolla RS - Retrovirology (2015)

Bottom Line: The calculated number of endogenous HBZ molecules varies between 17.461 and 39.615 molecules per cell, 20- to 50-fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein.HBZ interacts in vivo with p300 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p300 and JunD but also with CBP and CREB2.The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role of HBZ during HTLV-1 infection and cellular transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical and Morphological Sciences, School of Medicine, University of Insubria, Via Ottorino Rossi n.9, 21100, Varese, Italy. goutham.uthamraval@uninsubria.it.

ABSTRACT

Background: Human T cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of a severe form of neoplasia designated Adult T cell Leukaemia (ATL). It is widely accepted that the viral transactivator Tax-1 is the major viral product involved in the onset, but not in the maintenance, of neoplastic phenotype, as only 30-40% of ATL cells express Tax-1. It has been recently demonstrated that HBZ (HTLV-1 bZIP factor), a protein encoded by the minus strand of HTLV-1 genome, constantly expressed in infected cells and in ATL tumor cells, is also involved in the pathogenesis of leukaemia. The full role played by HBZ in oncogenesis is not clarified in detail also because of the limited availability of tools to assess quantitative expression, subcellular location and interaction of HBZ with host factors in ATL.

Results: By the use of the first reported monoclonal antibody against HBZ, 4D4-F3, generated in our laboratory it has been possible to carefully assess for the first time the above parameters in HTLV-1 chronically infected cells and, most importantly, in fresh leukemic cells from patients. Endogenous HBZ is expressed in speckle-like structures localized in the nucleus. The calculated number of endogenous HBZ molecules varies between 17.461 and 39.615 molecules per cell, 20- to 50-fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein. HBZ interacts in vivo with p300 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p300 and JunD but also with CBP and CREB2.

Conclusions: The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role of HBZ during HTLV-1 infection and cellular transformation.

No MeSH data available.


Related in: MedlinePlus

Co-localization of endogenous HBZ with intracellular factors in PH961 cells. PH961 patient cells were reacted in a pairwise combination with the 4D4-F3 anti HBZ mAb antibody, and either polyclonal rabbit anti-CBP, anti-JunD, anti-p300, anti-CREB2, or anti-nucleolin. For detection and other relevant information see the legend to Figure 6.
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Fig8: Co-localization of endogenous HBZ with intracellular factors in PH961 cells. PH961 patient cells were reacted in a pairwise combination with the 4D4-F3 anti HBZ mAb antibody, and either polyclonal rabbit anti-CBP, anti-JunD, anti-p300, anti-CREB2, or anti-nucleolin. For detection and other relevant information see the legend to Figure 6.

Mentions: To further extend the study on the subcellular location of possible interaction between HBZ and cellular factors in HTLV-1 infected and ATL tumor cells, an extensive analysis by confocal microscopy was carried out. Moreover, confocal investigation was extended, besides CBP and JunD, also to p300 and CREB-2, as well as nucleolin a marker of nucleoli. Importantly, CREB-2 was one of the first identified cellular nuclear proteins interacting with HBZ via its bZIP domain and this interaction was shown to inhibit the CREB-2/Tax-1 interaction instrumental for the activation of HTLV-1 LTR and the subsequent viral replication. The results of such analysis are shown in Figure 6 for C5MJ, in Figure 7 for ATL-2s and in Figure 8 for patient PH961. In all figures, the first series of vertical panels depicts the distribution of HBZ protein (in red fluorescence), the second series of vertical panels the staining pattern of the various nuclear markers listed in each panel (in green fluorescence) and in the third series of vertical panels the merged fluorescence. The fourth series of vertical panels depicts the peak fluorescence distribution along a single line section (ROI) of the corresponding markers to better evidence specific co-localizations. Irrespective of the cell analyzed, a common feature was the relatively low co-localization of HBZ with each one of the various markers analyzed. Within this frame, co-localization between HBZ and CREB-2 in C5MJ (Figure 6) and ATL-2s (Figure 7) cell lines seemed to be more pronounced as compared to the one found in patients PH961 tumor cells (Figure 8). A second important feature was related to PH961 cells in which all markers showed a relatively lower expression compared to the two cell lines, probably reflecting the fact the immortalization and in vitro tumor growth induce a generalized overexpression of genes involved in the homeostasis of cell growth. Within the above-mentioned limits, HBZ co-localized with CBP and p300 better in C5MJ than in ATL-2s. JunD was diffusely expressed in ATL-2s with respect to both C5MJ and patient PH961 cells, rendering difficult the appreciation of HBZ-JunD co-localization in ATL-2s cells. Instead, a detectable co-localization between HBZ and JunD was observed in both C5MJ and patient PH961 cells. HBZ did not co-localize with nucleolin in any of the cells under study, indicating that the HTLV-1 viral protein is not a nucleolar protein.Figure 6


Localization, quantification and interaction with host factors of endogenous HTLV-1 HBZ protein in infected cells and ATL.

Raval GU, Bidoia C, Forlani G, Tosi G, Gessain A, Accolla RS - Retrovirology (2015)

Co-localization of endogenous HBZ with intracellular factors in PH961 cells. PH961 patient cells were reacted in a pairwise combination with the 4D4-F3 anti HBZ mAb antibody, and either polyclonal rabbit anti-CBP, anti-JunD, anti-p300, anti-CREB2, or anti-nucleolin. For detection and other relevant information see the legend to Figure 6.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491271&req=5

Fig8: Co-localization of endogenous HBZ with intracellular factors in PH961 cells. PH961 patient cells were reacted in a pairwise combination with the 4D4-F3 anti HBZ mAb antibody, and either polyclonal rabbit anti-CBP, anti-JunD, anti-p300, anti-CREB2, or anti-nucleolin. For detection and other relevant information see the legend to Figure 6.
Mentions: To further extend the study on the subcellular location of possible interaction between HBZ and cellular factors in HTLV-1 infected and ATL tumor cells, an extensive analysis by confocal microscopy was carried out. Moreover, confocal investigation was extended, besides CBP and JunD, also to p300 and CREB-2, as well as nucleolin a marker of nucleoli. Importantly, CREB-2 was one of the first identified cellular nuclear proteins interacting with HBZ via its bZIP domain and this interaction was shown to inhibit the CREB-2/Tax-1 interaction instrumental for the activation of HTLV-1 LTR and the subsequent viral replication. The results of such analysis are shown in Figure 6 for C5MJ, in Figure 7 for ATL-2s and in Figure 8 for patient PH961. In all figures, the first series of vertical panels depicts the distribution of HBZ protein (in red fluorescence), the second series of vertical panels the staining pattern of the various nuclear markers listed in each panel (in green fluorescence) and in the third series of vertical panels the merged fluorescence. The fourth series of vertical panels depicts the peak fluorescence distribution along a single line section (ROI) of the corresponding markers to better evidence specific co-localizations. Irrespective of the cell analyzed, a common feature was the relatively low co-localization of HBZ with each one of the various markers analyzed. Within this frame, co-localization between HBZ and CREB-2 in C5MJ (Figure 6) and ATL-2s (Figure 7) cell lines seemed to be more pronounced as compared to the one found in patients PH961 tumor cells (Figure 8). A second important feature was related to PH961 cells in which all markers showed a relatively lower expression compared to the two cell lines, probably reflecting the fact the immortalization and in vitro tumor growth induce a generalized overexpression of genes involved in the homeostasis of cell growth. Within the above-mentioned limits, HBZ co-localized with CBP and p300 better in C5MJ than in ATL-2s. JunD was diffusely expressed in ATL-2s with respect to both C5MJ and patient PH961 cells, rendering difficult the appreciation of HBZ-JunD co-localization in ATL-2s cells. Instead, a detectable co-localization between HBZ and JunD was observed in both C5MJ and patient PH961 cells. HBZ did not co-localize with nucleolin in any of the cells under study, indicating that the HTLV-1 viral protein is not a nucleolar protein.Figure 6

Bottom Line: The calculated number of endogenous HBZ molecules varies between 17.461 and 39.615 molecules per cell, 20- to 50-fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein.HBZ interacts in vivo with p300 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p300 and JunD but also with CBP and CREB2.The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role of HBZ during HTLV-1 infection and cellular transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical and Morphological Sciences, School of Medicine, University of Insubria, Via Ottorino Rossi n.9, 21100, Varese, Italy. goutham.uthamraval@uninsubria.it.

ABSTRACT

Background: Human T cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of a severe form of neoplasia designated Adult T cell Leukaemia (ATL). It is widely accepted that the viral transactivator Tax-1 is the major viral product involved in the onset, but not in the maintenance, of neoplastic phenotype, as only 30-40% of ATL cells express Tax-1. It has been recently demonstrated that HBZ (HTLV-1 bZIP factor), a protein encoded by the minus strand of HTLV-1 genome, constantly expressed in infected cells and in ATL tumor cells, is also involved in the pathogenesis of leukaemia. The full role played by HBZ in oncogenesis is not clarified in detail also because of the limited availability of tools to assess quantitative expression, subcellular location and interaction of HBZ with host factors in ATL.

Results: By the use of the first reported monoclonal antibody against HBZ, 4D4-F3, generated in our laboratory it has been possible to carefully assess for the first time the above parameters in HTLV-1 chronically infected cells and, most importantly, in fresh leukemic cells from patients. Endogenous HBZ is expressed in speckle-like structures localized in the nucleus. The calculated number of endogenous HBZ molecules varies between 17.461 and 39.615 molecules per cell, 20- to 50-fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein. HBZ interacts in vivo with p300 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p300 and JunD but also with CBP and CREB2.

Conclusions: The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role of HBZ during HTLV-1 infection and cellular transformation.

No MeSH data available.


Related in: MedlinePlus