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Host-pathogen interactome mapping for HTLV-1 and -2 retroviruses.

Simonis N, Rual JF, Lemmens I, Boxus M, Hirozane-Kishikawa T, Gatot JS, Dricot A, Hao T, Vertommen D, Legros S, Daakour S, Klitgord N, Martin M, Willaert JF, Dequiedt F, Navratil V, Cusick ME, Burny A, Van Lint C, Hill DE, Tavernier J, Kettmann R, Vidal M, Twizere JC - Retrovirology (2012)

Bottom Line: Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively.Targets for HTLV-1 and HTLV-2 proteins implicate a diverse set of cellular processes including the ubiquitin-proteasome system, the apoptosis, different cancer pathways and the Notch signaling pathway.This study constitutes a first pass, with homogeneous data, at comparative analysis of host targets for HTLV-1 and -2 retroviruses, complements currently existing data for formulation of systems biology models of retroviral induced diseases and presents new insights on biological pathways involved in retroviral infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Ave,, Boston, MA 02215, USA.

ABSTRACT

Background: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL), whereas HTLV-2, highly similar to HTLV-1, causes no known overt disease. HTLV gene products are engaged in a dynamic struggle of activating and antagonistic interactions with host cells. Investigations focused on one or a few genes have identified several human factors interacting with HTLV viral proteins. Most of the available interaction data concern the highly investigated HTLV-1 Tax protein. Identifying shared and distinct host-pathogen protein interaction profiles for these two viruses would enlighten how they exploit distinctive or common strategies to subvert cellular pathways toward disease progression.

Results: We employ a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay. The final data set contained 166 interactions between 10 viral proteins and 122 human proteins. Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively. Targets for HTLV-1 and HTLV-2 proteins implicate a diverse set of cellular processes including the ubiquitin-proteasome system, the apoptosis, different cancer pathways and the Notch signaling pathway.

Conclusions: This study constitutes a first pass, with homogeneous data, at comparative analysis of host targets for HTLV-1 and -2 retroviruses, complements currently existing data for formulation of systems biology models of retroviral induced diseases and presents new insights on biological pathways involved in retroviral infection.

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HTLV-1 Rex and DLC2 co-localize in nucleolar foci. (A) HeLa cells were transfected with expression vectors for Rex1-GFP and Flag-DLC2 as indicated. Twenty-four hours post-transfection, cells were labeled with anti-flag M2 mouse antibody followed by alexa546-conjugated anti-mouse secondary antibody. Cells were stained with the far-red DNA marker DRAQ5 and analyzed by confocal microscopy. Merge corresponds to the simultaneous acquisition of all three fluorochromes. (B) Fluorescent intensities were plotted along the red line segments. The green and orange lines in the profile correspond to the relative intensities of GFP and Alexa 546.
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Figure 6: HTLV-1 Rex and DLC2 co-localize in nucleolar foci. (A) HeLa cells were transfected with expression vectors for Rex1-GFP and Flag-DLC2 as indicated. Twenty-four hours post-transfection, cells were labeled with anti-flag M2 mouse antibody followed by alexa546-conjugated anti-mouse secondary antibody. Cells were stained with the far-red DNA marker DRAQ5 and analyzed by confocal microscopy. Merge corresponds to the simultaneous acquisition of all three fluorochromes. (B) Fluorescent intensities were plotted along the red line segments. The green and orange lines in the profile correspond to the relative intensities of GFP and Alexa 546.

Mentions: In an apoptotic pathway sub-network, KEGG analysis highlighted the tumor necrosis factor (TNF) receptor and the AKT/PI3K signaling pathways as potential targets for HTLV proteins. In this network HTLV Tax and Rex proteins are closely linked to the Akt/PI3K and mitochondrial apoptotic pathways. We identified interactions between HTLV Tax proteins and nitric oxide synthase 3 (NOS3), hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), Ewing sarcoma breakpoint region 1 (EWSR1) and glucose transporter-4 (SLC2A4) proteins. KEGG analysis indicated that phosphatidylinositol-3-kinase (PI3K), BCL2-antagonist of cell death (Bad), and DNA fragmentation factor alpha (DFFA) proteins are second-degree targets of HTLV Tax proteins (Figure 5). We also found that the HTLV Rex proteins interact with DLC2 (for dynein light chain 2), able to regulate cell death-inducing functions of pro-apoptotic proteins Bim (Bcl-2-interacting mediator of cell death) and Bmf (Bcl-2-modifying factor). HTLV Rex proteins are nuclear-localizing proteins well known to drive post-transcriptional export of viral mRNAs from the nucleus to the cytoplasm [36-38]. Besides its interaction with the cellular export factor CRM1 [39], functional relationship between Rex proteins and their cellular partners have not been fully investigated. Interaction between Rex proteins and DLC2 may shed light on a new role of Rex in the apoptotic pathway. To assess the subcellular localization of Rex1 and DLC2, we transfected HeLa cells with expression vectors for Rex1-GFP and Flag-tagged DLC2. Cells were stained by anti-flag antibody followed by Alexa546-conjugated secondary antibody and a far-red fluorescent DNA dye (DRAQ5) for nuclear staining. Consistent with previous reports [40-42], DLC2 was found exclusively in the cytoplasm (Figure 6A, DLC2); and Rex-GFP was localized in nucleolar foci (Figure 6A, Rex1-GFP). Co-expression of Rex1-GFP and Flag-DLC2 provoked a change in the localization of DLC2 with two patterns being observed. DLC2 was localized in the cytoplasm as well as in nuclear foci (Figure 6A, DLC2 + Rex1-GFP, Alexa546). It thus appeared that coexpression with Rex1 directs DLC2 in nucleolar foci as revealed by the good match of the green (Rex1-GFP) and orange (Flag-DLC2) fluorochromes. We conclude that HTLV Rex proteins might interfere with the anti-apoptotic activities of DLC2 in HTLV infected cells.


Host-pathogen interactome mapping for HTLV-1 and -2 retroviruses.

Simonis N, Rual JF, Lemmens I, Boxus M, Hirozane-Kishikawa T, Gatot JS, Dricot A, Hao T, Vertommen D, Legros S, Daakour S, Klitgord N, Martin M, Willaert JF, Dequiedt F, Navratil V, Cusick ME, Burny A, Van Lint C, Hill DE, Tavernier J, Kettmann R, Vidal M, Twizere JC - Retrovirology (2012)

HTLV-1 Rex and DLC2 co-localize in nucleolar foci. (A) HeLa cells were transfected with expression vectors for Rex1-GFP and Flag-DLC2 as indicated. Twenty-four hours post-transfection, cells were labeled with anti-flag M2 mouse antibody followed by alexa546-conjugated anti-mouse secondary antibody. Cells were stained with the far-red DNA marker DRAQ5 and analyzed by confocal microscopy. Merge corresponds to the simultaneous acquisition of all three fluorochromes. (B) Fluorescent intensities were plotted along the red line segments. The green and orange lines in the profile correspond to the relative intensities of GFP and Alexa 546.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351729&req=5

Figure 6: HTLV-1 Rex and DLC2 co-localize in nucleolar foci. (A) HeLa cells were transfected with expression vectors for Rex1-GFP and Flag-DLC2 as indicated. Twenty-four hours post-transfection, cells were labeled with anti-flag M2 mouse antibody followed by alexa546-conjugated anti-mouse secondary antibody. Cells were stained with the far-red DNA marker DRAQ5 and analyzed by confocal microscopy. Merge corresponds to the simultaneous acquisition of all three fluorochromes. (B) Fluorescent intensities were plotted along the red line segments. The green and orange lines in the profile correspond to the relative intensities of GFP and Alexa 546.
Mentions: In an apoptotic pathway sub-network, KEGG analysis highlighted the tumor necrosis factor (TNF) receptor and the AKT/PI3K signaling pathways as potential targets for HTLV proteins. In this network HTLV Tax and Rex proteins are closely linked to the Akt/PI3K and mitochondrial apoptotic pathways. We identified interactions between HTLV Tax proteins and nitric oxide synthase 3 (NOS3), hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), Ewing sarcoma breakpoint region 1 (EWSR1) and glucose transporter-4 (SLC2A4) proteins. KEGG analysis indicated that phosphatidylinositol-3-kinase (PI3K), BCL2-antagonist of cell death (Bad), and DNA fragmentation factor alpha (DFFA) proteins are second-degree targets of HTLV Tax proteins (Figure 5). We also found that the HTLV Rex proteins interact with DLC2 (for dynein light chain 2), able to regulate cell death-inducing functions of pro-apoptotic proteins Bim (Bcl-2-interacting mediator of cell death) and Bmf (Bcl-2-modifying factor). HTLV Rex proteins are nuclear-localizing proteins well known to drive post-transcriptional export of viral mRNAs from the nucleus to the cytoplasm [36-38]. Besides its interaction with the cellular export factor CRM1 [39], functional relationship between Rex proteins and their cellular partners have not been fully investigated. Interaction between Rex proteins and DLC2 may shed light on a new role of Rex in the apoptotic pathway. To assess the subcellular localization of Rex1 and DLC2, we transfected HeLa cells with expression vectors for Rex1-GFP and Flag-tagged DLC2. Cells were stained by anti-flag antibody followed by Alexa546-conjugated secondary antibody and a far-red fluorescent DNA dye (DRAQ5) for nuclear staining. Consistent with previous reports [40-42], DLC2 was found exclusively in the cytoplasm (Figure 6A, DLC2); and Rex-GFP was localized in nucleolar foci (Figure 6A, Rex1-GFP). Co-expression of Rex1-GFP and Flag-DLC2 provoked a change in the localization of DLC2 with two patterns being observed. DLC2 was localized in the cytoplasm as well as in nuclear foci (Figure 6A, DLC2 + Rex1-GFP, Alexa546). It thus appeared that coexpression with Rex1 directs DLC2 in nucleolar foci as revealed by the good match of the green (Rex1-GFP) and orange (Flag-DLC2) fluorochromes. We conclude that HTLV Rex proteins might interfere with the anti-apoptotic activities of DLC2 in HTLV infected cells.

Bottom Line: Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively.Targets for HTLV-1 and HTLV-2 proteins implicate a diverse set of cellular processes including the ubiquitin-proteasome system, the apoptosis, different cancer pathways and the Notch signaling pathway.This study constitutes a first pass, with homogeneous data, at comparative analysis of host targets for HTLV-1 and -2 retroviruses, complements currently existing data for formulation of systems biology models of retroviral induced diseases and presents new insights on biological pathways involved in retroviral infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Ave,, Boston, MA 02215, USA.

ABSTRACT

Background: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL), whereas HTLV-2, highly similar to HTLV-1, causes no known overt disease. HTLV gene products are engaged in a dynamic struggle of activating and antagonistic interactions with host cells. Investigations focused on one or a few genes have identified several human factors interacting with HTLV viral proteins. Most of the available interaction data concern the highly investigated HTLV-1 Tax protein. Identifying shared and distinct host-pathogen protein interaction profiles for these two viruses would enlighten how they exploit distinctive or common strategies to subvert cellular pathways toward disease progression.

Results: We employ a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay. The final data set contained 166 interactions between 10 viral proteins and 122 human proteins. Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively. Targets for HTLV-1 and HTLV-2 proteins implicate a diverse set of cellular processes including the ubiquitin-proteasome system, the apoptosis, different cancer pathways and the Notch signaling pathway.

Conclusions: This study constitutes a first pass, with homogeneous data, at comparative analysis of host targets for HTLV-1 and -2 retroviruses, complements currently existing data for formulation of systems biology models of retroviral induced diseases and presents new insights on biological pathways involved in retroviral infection.

Show MeSH
Related in: MedlinePlus