HBZ upregulates myoferlin expression to facilitate HTLV-1 infection

Figures

• 

  Fig 1. MyoF is expressed in HBZ-expressing cells and in HTLV-1-infected T-cells. (A) Relative MYOF mRNA levels in the indicated HeLa cell lines. The graph shows qRT-PCR results averaged from four independent experiments with values normalized to that of the empty-vector clone, pcDNA (set to 1), for each replicate. Error bars show standard deviations; ** p<0.01, *** p<0.001. (B) MyoF expression in empty vector (pcDNA) and HBZ-HeLa clones. Whole cell extracts (50 μg) were analyzed by Western blot using antibodies against MyoF, HBZ (6xHis) and β-actin. (C) Relative MYOF mRNA levels in resting and activated primary CD4+ T-cells and in T-cell lines. The graph shows qRT-PCR results averaged from two independent experiments in which values were normalized to those for the resting CD4+ T-cells (set to 1), error bars show standard deviations. Numbers above the bars indicate expression levels relative to the resting CD4+ T-cells. HTLV-1-negative lines are T-cell acute lymphoblastic leukemia lines (CEM, HSB-2, Jurkat, Molt-4 and SupT1) and cutaneous T-cell leukemia lines (HUT-78 and Myla); in vitro transformed HTLV-1-positive cells are 1185, C10/MJ, MT-2, MT-4 and SLB-1; ATL-derived cell lines are…

• 

  Fig 2. Expression of HBZ correlates with MyoF expression. (A) Deletion of HBZ in ST1 and KK1 ATL cells reduces MYOF expression. The graph was generated from published microarray data (GEO accession number GSE94409 [56]) and shows the percent reduction in MYOF transcript levels seven days after inducing CRISPR/Cas9-mediated knockout of HBZ in the ATL cell lines, ST1 and KK1, using two different guide RNAs (sgHBZ #1 and #2) compared to the guide RNA control. Data were obtained using GEO2R with calculations based on averaged values from the three array features probing for different regions of the MyoF transcript; ** p<0.01, *** p<0.001. (B) Relative MYOF mRNA levels in HTLV-1-immortalized human T-cell clones recently established from peripheral blood lymphocytes (PBL). The graph shows qRT-PCR results as follows: C3, value from one RNA extraction; ΔHBZ clones, values averaged from two extractions; all others, values averaged from three extractions. Values were normalized to those for the resting CD4+ T-cells (set to 1). Dark gray bars show clones derived from different PBL donors immortalized with HTLV-1-wt (CJ2, CJ4, C13, C15). Hatched bars show clones derived from different PBL donors…

• 

  Fig 3. HBZ binds the MYOF gene in conjunction with c-Jun and JunB. (A) HBZ binds to two sites within the MYOF gene in the ATL cell line, KK1. Peaks of enrichment for HBZ, the epigenetic mark, H3K27ac, and control (IgG) at the MYOF locus in KK1 cells are shown in the IGV Browser. Genomic coordinates are based on the NCBI36/hg18 assembly. Data were obtained from published ChIP-Seq data sets (GEO accession number GSE94732 [56]). (B) HBZ binds to the proximal peak in HeLa cells. The graph shows average values from three independent ChIP assays using empty vector (pcDNA) and HBZ (6x His C-terminal tag)-expressing HeLa cells. Shown are levels of HBZ-enrichment at the off-target site (1,796 bp from the end of the gene) and at the distal and proximal peaks with respect to the TSS; * p<0.05. (C) HBZ binds the distal and proximal peaks in SLB-1 cells. The graph shows fold enrichment relative to that for the off-target site (set to 1) averaged from three independent ChIP assays using SLB-1 cells transduced to express HBZ with a C-terminal 6x His tag; * p<0.05, *** p<0.001. (D) and (E) JunB and c-Jun are enriched at the distal and proximal peaks in ATL-2 cells. (F) and (G) JunD and MafG are not…

• 

  Fig 4. HBZ activates MYOF transcription through recruitment of p300/CBP. p300 (A) and CBP (B) bind the proximal MYOF peak in HeLa cells expressing HBZ. Graphs show average values from three independent ChIP assays using empty vector (pcDNA) and HBZ-expressing HeLa cells. Shown are levels of p300- and CBP-enrichment at the off-target site and at the distal and proximal peaks; * p<0.05. (C) siRNA-mediated depletion of p300 and CBP. HeLa cells were transfected with siRNAs targeting both p300 and CBP (p300/CBP) or an off-target siRNA (CT). Whole cell extracts (50 μg) were analyzed by Western blot using antibodies against p300, CBP and β-actin. (D) Depletion of p300 and CBP reduces HBZ-mediated MYOF expression. The graph shows qRT-PCR results averaged from four independent experiments with values normalized to those for the empty-vector clone (pcDNA) transfected with siRNA CT (set to 1); ** p<0.01. (E) Inhibition of p300/CBP KAT activity abrogates HBZ-mediated activation of MYOF transcription. HeLa cells expressing HBZ or carrying the empty vector (pcDNA) were treated with A485 (10 μM) or the carrier (DMSO) for 3h. The graph shows qRT-PCR results averaged from four independent experiments…

• 

  Fig 5. MyoF enhances HTLV-1 infection. (A) The flow diagram shows the co-culture/infection assay procedure using CHOK1-Luc or Jurkat-pminLUC-vCRE cells as target cells. (B) Inhibition of MyoF reduces HTLV-1 infection. ATL-2 cells were treated with DMSO or WJ460 (200nM or 1μM) prior to co-culture with CHOK1-Luc cells. (C) SLB-1 cells were treated with DMSO or 1 μM WJ460 prior to co-culture with Jurkat-pminLUC-vCRE cells. (D) C8166/45 and SLB-1 cells (both high Tax expression) and TL-Om1 cells (no Tax expression) were co-cultured with Jurkat-pminLUC-vCRE cells. (E) An HTLV-1-immortalized primary human T-cell clone (C13) was treated with DMSO or 1 μM WJ460 prior to co-culture with Jurkat-pminLUC-vCRE cells. Graphs (B), (C) and (D) show luciferase assay results averaged from at least three replicates for each condition of a single experiment and are representative of three independent experiments; * p<0.05, *** p<0.001. Graph (E) shows luciferase assay results average from three replicates for each condition of a single experiment and is representative of two independent experiments; ** p<0.01. (F) Knockdown of MyoF expression reduces HTLV-1 infection. MyoF expression in control (GFP) and…

• 

  Fig 6. MyoF regulates the intracellular abundance of HTLV-1 Env. (A) Inhibition of MyoF reduces levels of SU (gp46). ATL-2 cells were treated with DMSO or WJ460 (200 nM or 1 μM) for 24h. Whole cell extracts (50 μg) from uninfected CEM and the treated cells were analyzed by Western blot using antibodies against MyoF, β-actin and the viral proteins Tax, Gag p55, Gag p19, and SU/Pr. (B) ATL-2 and SLB-1 cells were treated with DMSO or 1 μM WJ460 for 24h. Whole cell extracts (100 μg for Tax; 50 μg for the others) were analyzed by Western blot using antibodies against β-actin and the viral proteins Tax, and SU/Pr. (C) The graph shows quantification of band intensities of Pr (gp62) and SU (gp46) normalized to band intensities of β-actin averaged from three and four independent experiments for ATL-2 and SLB-1 cells, respectively. Error bars show standard deviations; * p<0.05, ** p<0.01. (D) Knockdown of MyoF expression reduces levels of SU (gp46). Whole cell extracts were prepared from SLB-1 cells stably expressing an shRNA targeting GFP (negative control) or MYOF mRNA, and 50 μg from each cell line was analyzed by Western blot using antibodies against MyoF, β-actin and the viral proteins Tax,…

• 

  Fig 7. MyoF restricts lysosomal degradation of Env. (A) Levels of endosomal proteins in HTLV-1 infected cells following WJ460 treatment. SLB-1 cells were treated with DMSO or 1 μM WJ460 for 24 h. Whole cell extracts (25 to 50 μg) were analyzed by Western blot using antibodies against the indicated proteins. (B) Knockdown of MyoF expression reduces the level of caveolin-1 (Cav1). Whole cell extracts (25 to 50 μg) were prepared from SLB-1 cells stably expressing an shRNA targeting GFP (negative control) or MYOF mRNA and analyzed by Western blot using antibodies against the indicated proteins. (C) Knockdown of MyoF expression does not affect the level of SU (gp46) at the cell-surface. SLB-1 cells stably expressing an shRNA targeting GFP (negative control) or MYOF mRNA were labeled with a primary antibody against SU/Pr followed by an APC-conjugated secondary antibody, fixed and analyzed by flow cytometry. Histograms are representative of two independent experiments and show relative cell surface labeling as follows: secondary antibody alone (CT) and SU-labeled shGFP cells, light gray and light blue, respectively; secondary antibody alone (CT) and SU-labeled shMYOF cells, dark gray and dark…

• 

  Fig 8. MyoF enhances HTLV-1-infected T-cell adhesion without affecting cell surface abundance of ICAM-1. (A) Knockdown of MyoF expression does not affect the level of ICAM-1 at the cell-surface. SLB-1 cells stably expressing an shRNA targeting GFP (negative control) or MYOF mRNA were labeled with an antibody against ICAM-1 followed by a FITC-conjugated secondary antibody, fixed and analyzed by flow cytometry. Histograms show relative cell surface labeling as follows: secondary antibody alone- (CT) and ICAM-1-labeled shGFP cells, light gray and light blue, respectively; secondary antibody alone- (CT) and ICAM-1-labeled shMYOF cells, dark gray and dark blue, respectively. (B) Knockdown or inhibition of MyoF reduces adhesion to CHO cells independent of LFA-1 expression. The flow diagram shows the co-culture/adhesion assay procedure using CHO or CHO-LFA-1 adherent cells. (C) Adhesion assays of HTLV-1 infected cells stably expressing shGFP or shMyoF to adherent cells. The graph shows binding of SLB-1 cells pretreated with DMSO or 1 μM WJ460, or SLB-1 cells stably expressing an shRNA targeting GFP (negative control) or MYOF mRNA, to CHO (grey) or CHO-LFA1 (black) cells. Values are the average…

• 

  Fig 9. MyoF increases levels of total extracellular and virion-associated SU (gp46). (A) Knockdown of MyoF expression reduces overall levels of SU in the culture medium of HTLV-1 cells. TCA-precipitated proteins from culture media of SLB-1 cells stably transfected with shRNA targeting GFP or MYOF mRNA were divided into equal fractions and analyzed by Western blot using an antibody against SU/Pr (upper panel) and, separately, by Coomassie blue-staining following SDS-PAGE (lower panel). Whole cell extract from SLB-1 cells served as a positive control. (B) Knockdown of MyoF expression reduces levels of SU in cell-free HTLV-1 virions. Culture media from SLB-1 cells stably expressing an shRNA targeting GFP or MYOF mRNA were filtered, ultracentrifuged and analyzed by Western blot using antibodies against SU/gp46 and Gag p19; * denotes a nonspecific band from the serum. (C) Knockdown or inhibition of MyoF does not affect levels of virus released from the cells. Clarified culture media from SLB-1 cells stably expressing an shRNA targeting GFP or MYOF mRNA and SLB-1 and ATL-2 cells treated with DMSO or 1 μM WJ460 for 24 h were analyzed by ELISA to quantify levels of Gag p19. The graph shows…

• 

  Fig 10. Proposed model of MyoF-mediated Env trafficking in HTLV-1-infected T-cells. Through its activation domain (AD), HBZ recruits p300/CBP to the MYOF gene enhancers to activate transcription. Env is processed to its mature form through the Trans-Golgi Network (TGN) and incorporated into the plasma membrane. The YXXΦ motif in Env mediates clathrin-dependent endocytosis and trafficking of Env to lysosomes. However, the presence of MyoF causes Env to be redirected to the endosomal recycling compartment from which Env traffics to sites of virion assembly. Apart from MyoF the ESSL domain separately reduces lysosomal-mediated degradation of Env. One component of this figure was drawn using a picture from Servier Medical Art, which are licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).