Limits...
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

Epitope assignment of 4D4-F3 anti-HBZ mAb. a Western blot analysis of full length GFP-tagged HBZ (HBZ) and a series of GFP-tagged HBZ fragments listed in the top of the left and rightpanels. Cos cells were transfected with the various encoding GFP-HBZ cDNAs, with empty plasmid (mock) or with GFP cDNA (GFP). Twenty-four hours after transfection cell lysates were prepared and analyzed by western blotting using the 4D4-F3 anti HBZ mAb (left panel) or the anti-HBZ antiserum prepared from the same animal used for somatic cell fusion (right panel). b Schematic representation of the same results depicted in a. The various HBZ constructs are listed on the left side followed by bars representing size and regions of the specific fragment analyzed. Numbers indicate the specific aminoacid position in the various fragments. Plus (+) and minus (−) symbols indicate positive or negative reactivity, respectively, of the 4D4-F3 mAb. AD activation domain, BR1 basic region 1, BR2 basic region 2, DBD DNA binding domain, bZIB b zipper domain.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Epitope assignment of 4D4-F3 anti-HBZ mAb. a Western blot analysis of full length GFP-tagged HBZ (HBZ) and a series of GFP-tagged HBZ fragments listed in the top of the left and rightpanels. Cos cells were transfected with the various encoding GFP-HBZ cDNAs, with empty plasmid (mock) or with GFP cDNA (GFP). Twenty-four hours after transfection cell lysates were prepared and analyzed by western blotting using the 4D4-F3 anti HBZ mAb (left panel) or the anti-HBZ antiserum prepared from the same animal used for somatic cell fusion (right panel). b Schematic representation of the same results depicted in a. The various HBZ constructs are listed on the left side followed by bars representing size and regions of the specific fragment analyzed. Numbers indicate the specific aminoacid position in the various fragments. Plus (+) and minus (−) symbols indicate positive or negative reactivity, respectively, of the 4D4-F3 mAb. AD activation domain, BR1 basic region 1, BR2 basic region 2, DBD DNA binding domain, bZIB b zipper domain.

Mentions: In order to delineate the HBZ epitope region recognized by 4D4-F3, an epitope mapping was carried out. For this purpose lysates of Cos cells transfected with either Green Fluorescent Protein (GFP)-tagged HBZ or a series of GFP-HBZ deletion mutants were analyzed by western blot. 4D4-F3 mAb recognized all fragments including the basic region 1 (BR1) fragment of HBZ, the isolated diagnostic BR1 fragment but not the isolated DBD fragment (Figure 1a, left panel). The presence of the DBD fragment in the corresponding lysate and its immunogenicity was confirmed by the reactivity of the serum of the mouse whose spleen cells were used for fusion (Figure 1a, right panel). Based on the above results the epitope recognized by 4D4-F3 mAb was mapped to the region between amino acids 97–133 (Figure 1b) including the BR1 region. Figure 1b summarizes the results of epitope mapping, listing all the different HBZ deletion mutants used and their size.Figure 1


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)

Epitope assignment of 4D4-F3 anti-HBZ mAb. a Western blot analysis of full length GFP-tagged HBZ (HBZ) and a series of GFP-tagged HBZ fragments listed in the top of the left and rightpanels. Cos cells were transfected with the various encoding GFP-HBZ cDNAs, with empty plasmid (mock) or with GFP cDNA (GFP). Twenty-four hours after transfection cell lysates were prepared and analyzed by western blotting using the 4D4-F3 anti HBZ mAb (left panel) or the anti-HBZ antiserum prepared from the same animal used for somatic cell fusion (right panel). b Schematic representation of the same results depicted in a. The various HBZ constructs are listed on the left side followed by bars representing size and regions of the specific fragment analyzed. Numbers indicate the specific aminoacid position in the various fragments. Plus (+) and minus (−) symbols indicate positive or negative reactivity, respectively, of the 4D4-F3 mAb. AD activation domain, BR1 basic region 1, BR2 basic region 2, DBD DNA binding domain, bZIB b zipper domain.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Epitope assignment of 4D4-F3 anti-HBZ mAb. a Western blot analysis of full length GFP-tagged HBZ (HBZ) and a series of GFP-tagged HBZ fragments listed in the top of the left and rightpanels. Cos cells were transfected with the various encoding GFP-HBZ cDNAs, with empty plasmid (mock) or with GFP cDNA (GFP). Twenty-four hours after transfection cell lysates were prepared and analyzed by western blotting using the 4D4-F3 anti HBZ mAb (left panel) or the anti-HBZ antiserum prepared from the same animal used for somatic cell fusion (right panel). b Schematic representation of the same results depicted in a. The various HBZ constructs are listed on the left side followed by bars representing size and regions of the specific fragment analyzed. Numbers indicate the specific aminoacid position in the various fragments. Plus (+) and minus (−) symbols indicate positive or negative reactivity, respectively, of the 4D4-F3 mAb. AD activation domain, BR1 basic region 1, BR2 basic region 2, DBD DNA binding domain, bZIB b zipper domain.
Mentions: In order to delineate the HBZ epitope region recognized by 4D4-F3, an epitope mapping was carried out. For this purpose lysates of Cos cells transfected with either Green Fluorescent Protein (GFP)-tagged HBZ or a series of GFP-HBZ deletion mutants were analyzed by western blot. 4D4-F3 mAb recognized all fragments including the basic region 1 (BR1) fragment of HBZ, the isolated diagnostic BR1 fragment but not the isolated DBD fragment (Figure 1a, left panel). The presence of the DBD fragment in the corresponding lysate and its immunogenicity was confirmed by the reactivity of the serum of the mouse whose spleen cells were used for fusion (Figure 1a, right panel). Based on the above results the epitope recognized by 4D4-F3 mAb was mapped to the region between amino acids 97–133 (Figure 1b) including the BR1 region. Figure 1b summarizes the results of epitope mapping, listing all the different HBZ deletion mutants used and their size.Figure 1

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