Journal: Viruses

Article Title: Susceptibility of Primary HTLV-1 Isolates from Patients with HTLV-1-Associated Myelopathy to Reverse Transcriptase Inhibitors

doi: 10.3390/v3050469

Figure Lengend Snippet: RT activity in human T-lymphotropic virus type 1 (HTLV-1) isolates from peripheral blood mononuclear cell (PBMC) cultures of HTLV-1-associated myelopathy (HAM) patients. Centrifuge-enriched isolates from PBMC cultures of 5 patients with HAM (TAY, TAR, TAU, TBI, TAQ), before (T0) and after (T48) therapy with 3TC plus AZT, or from MT-2 cells, were assayed for p19. Serial dilutions of p19-equivalent isolates were added to the RT assay and amplified products of the template were visualized on a 2% agarose gel and quantitated by densitometry, as a measure of RT activity.

Article Snippet: p19 in supernatants or enriched isolates was assayed using an HTLV-1 p19 Gag antigen capture enzyme-linked immunosorbent assay (ELISA) (ZeptoMetrix, Buffalo, NY, USA).

Techniques: Activity Assay, Virus, Amplification, Agarose Gel Electrophoresis

Journal: Viruses

Article Title: Susceptibility of Primary HTLV-1 Isolates from Patients with HTLV-1-Associated Myelopathy to Reverse Transcriptase Inhibitors

doi: 10.3390/v3050469

Figure Lengend Snippet: In vitro human T-lymphotropic virus type 1 (HTLV-1) RT inhibitory concentrations (nM).

Article Snippet: p19 in supernatants or enriched isolates was assayed using an HTLV-1 p19 Gag antigen capture enzyme-linked immunosorbent assay (ELISA) (ZeptoMetrix, Buffalo, NY, USA).

Techniques: In Vitro, Virus, Sequencing

Journal: Viruses

Article Title: Susceptibility of Primary HTLV-1 Isolates from Patients with HTLV-1-Associated Myelopathy to Reverse Transcriptase Inhibitors

doi: 10.3390/v3050469

Figure Lengend Snippet: p19 release in culture supernatants from PBMCs of HAM patients before and after therapy. The p19 concentration in supernatants from PBMC cultures of 5 patients with HAM (TAY, TAR, TAU, TBI, TAQ), before (T0) and after (T48) therapy with 3TC plus AZT, was evaluated by ELISA. Values are expressed as the mean of quadruplicate samples ± standard deviation. Production of p19 (black column) in the supernatant of chronically infected cells MT-2 was used as a positive control.

Article Snippet: p19 in supernatants or enriched isolates was assayed using an HTLV-1 p19 Gag antigen capture enzyme-linked immunosorbent assay (ELISA) (ZeptoMetrix, Buffalo, NY, USA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Infection, Positive Control

Journal: PLOS Pathogens

Article Title: HBZ upregulates myoferlin expression to facilitate HTLV-1 infection

doi: 10.1371/journal.ppat.1011202

Figure Lengend Snippet: (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, Gag p19, and SU/Pr. (E) The graph shows quantification of band intensities of Pr (gp62) and SU (gp46) normalized to band intensities of β-actin averaged from three independent experiments. Error bars show standard deviations; ** p <0.01. (F) Ectopic co-expression of MyoF with Env in HEK293T cells increases SU (gp46) abundance. Cells (1 x 10 6 ) were transfected with pcDNA-GFP-HA-MyoF (3 μg) and pCMV-HTLV-1-Env (1 μg). Env was co-precipitated with Lens Culinaris Agglutinin lectin bound to agarose beads (P-lectin) from 879 μg of whole cell extracts (WCE) and analyzed along with 50 μg WCE by Western blot using antibodies against MyoF, and SU/Pr. (G) Inhibition of MyoF ectopically co-expressed with Env in HEK293T cells decreases SU/gp46 abundance. Cells (1 x 10 6 ) were transfected with pcDNA-GFP-HA-MyoF (3 μg) and pCMV-HTLV-1-Env-Flag-Myc (3 μg) expression vectors and treated with DMSO or WJ460 (1 μM) for 24h. Env was co-precipitated with Lens Culinaris Agglutinin lectin bound to agarose beads (P-lectin) from 768 μg of whole cell extracts (WCE) and analyzed along with 50 μg WCE by Western blot using antibodies against MyoF, β-actin and the Flag-epitope tag of Env. (H) Ectopic co-expression of MyoF with Env in HEK293T cells increases TM (gp21) abundance. Cells (1 x 10 6 ) were transfected with pcDNA-GFP-HA-MyoF (3 μg) and pCMV-HTLV-1-Env-Flag-Myc (3 μg). Env was co-precipitated with Lens Culinaris Agglutinin lectin bound to agarose beads (P-lectin) from 1190 μg of whole cell extracts (WCE) and analyzed along with 50 μg WCE by Western blot using antibodies against MyoF, and the Flag- and Myc-epitope tags of Env.

Article Snippet: Antibodies used were as follows: anti-HTLV-1 Env (ARP-1578) and anti-Tax (hybridoma 168B17-46-92) were obtained from NIH AIDS Research and Reagent Program; anti-HBZ has been described [ ]; anti-6x His (ab9108) was purchased from Abcam; anti-MyoF (HPA014245) and anti-Myc (06–340) were purchased from MilliporeSigma; anti-HTLV-1 Env gp46 (1C11, sc-53890; 65/6C2.2.34, sc-57865), anti-Gag p19 (TP7, sc-57870) and anti-β-actin (C4, sc-47778) were purchased from Santa-Cruz; anti-clathrin (D3C6, #4796), anti-caveolin-1 (D46G3, #3267), anti-EEA1 (C45B10, #3288), anti-Rab5a (E6N8S, #46449), anti-Rab7 (D95F2, #9367), anti-Rab11 (D4F5, #5589), anti-LAMP-1 (D2D11, #9091) were purchased from Cell Signaling; anti-EHD2 (PA5-80576) was purchased from ThermoFisher Scientific.

Techniques: Inhibition, Western Blot, Knockdown, Expressing, Stable Transfection, shRNA, Negative Control, Transfection, FLAG-tag

Journal: PLOS Pathogens

Article Title: HBZ upregulates myoferlin expression to facilitate HTLV-1 infection

doi: 10.1371/journal.ppat.1011202

Figure Lengend Snippet: (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 values averaged from three replicates.

Article Snippet: Antibodies used were as follows: anti-HTLV-1 Env (ARP-1578) and anti-Tax (hybridoma 168B17-46-92) were obtained from NIH AIDS Research and Reagent Program; anti-HBZ has been described [ ]; anti-6x His (ab9108) was purchased from Abcam; anti-MyoF (HPA014245) and anti-Myc (06–340) were purchased from MilliporeSigma; anti-HTLV-1 Env gp46 (1C11, sc-53890; 65/6C2.2.34, sc-57865), anti-Gag p19 (TP7, sc-57870) and anti-β-actin (C4, sc-47778) were purchased from Santa-Cruz; anti-clathrin (D3C6, #4796), anti-caveolin-1 (D46G3, #3267), anti-EEA1 (C45B10, #3288), anti-Rab5a (E6N8S, #46449), anti-Rab7 (D95F2, #9367), anti-Rab11 (D4F5, #5589), anti-LAMP-1 (D2D11, #9091) were purchased from Cell Signaling; anti-EHD2 (PA5-80576) was purchased from ThermoFisher Scientific.

Techniques: Knockdown, Expressing, Stable Transfection, Transfection, shRNA, Western Blot, Staining, SDS Page, Positive Control, Inhibition, Virus, Enzyme-linked Immunosorbent Assay

Journal: Virology Journal

Article Title: Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

doi: 10.1186/1743-422X-4-66

Figure Lengend Snippet: The involvement of Vps4A and Vps4B in HTLV-1 Gag budding . A. 293T cells were cotransfected with pK30-Gag and the expression plasmid for Vps4AEQ or Vps4BEQ, or the empty vector as a control. Extracellular VLPs were pelleted from the culture fluids. VLP-associated or cell-associated Gag was detected by western blotting (WB) using anti-HTLV-1 p19 monoclonal antibody. C. 293T cells were cotransfected with pK30-Gag and the expression vector for wild-type Vps4A or Vps4B, or the empty vector as a control. The proteins were detected as described in A. B and D. Intensities of the bands corresponding to cell- and VLP-associated Gag in A and C were quantified using the LAS3000 imaging system (Fuji film). The efficiency of Gag-induced VLP budding in cells cotransfected with pK30-Gag and control vector (VLP/Cellular) was set to 1.0. The data represent averages and standard deviations (SD) of 3 independent experiments.

Article Snippet: The mouse anti-HTLV-1 p19 monoclonal antibody TP-7 (Abcam, Cambridge, UK) was used to detect K30Gag.

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Imaging

Journal: Virology Journal

Article Title: Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

doi: 10.1186/1743-422X-4-66

Figure Lengend Snippet: Effects of AIP1/Alix DN mutants on HTLV-1 VLP production . A. 293T cells were cotransfected with pK30-Gag and the expression plasmid for AIP1 (1–628), AIP1 (424–628), or AIP1 WT, or the empty vector as a control. Extracellular VLPs were pelleted from the culture fluids. VLP- or cell-associated Gag was detected by WB using anti-p19 monoclonal antibody. B. Intensities of the bands corresponding to cell- and VLP-associated Gag in A were quantified using the LAS3000 imaging system (Fuji Film). The efficiency of Gag-induced VLP budding in cells cotransfected with pK30-Gag and the control vector (VLP/Cellular) was set to 1.0. The data represent averages and standard deviations (SD) of 3 independent experiments.

Article Snippet: The mouse anti-HTLV-1 p19 monoclonal antibody TP-7 (Abcam, Cambridge, UK) was used to detect K30Gag.

Techniques: Expressing, Plasmid Preparation, Control, Imaging

Journal:

Article Title: Reciprocal Interactions between Human T-Lymphotropic Virus Type 1 and Prostaglandins: Implications for Viral Transmission

doi: 10.1128/JVI.75.1.192-198.2001

Figure Lengend Snippet: PGE2 upregulates HTLV-1 LTR activity. (A) PGE2 upregulates HTLV-1 LTR activity in a dose-dependent manner. A total of 40 million PBMC were transfected with 10 μg of pU3R-luc and were left unstimulated or were stimulated with the indicated amount of PGE2, and luciferase activity of the transfected cell lysates was assayed 2 days posttransfection. Fold induction is the luciferase activity relative to that obtained without PGE2 treatment. Results are means ± standard errors of the means from three independent experiments. (B and C) PKA activity is involved in PGE2 induction of expression from the HTLV-1 LTR. A total of 40 million PBMC (B) or 20 million Jurkat cells (C) were transfected with 10 μg of pU3R-luc, and luciferase activity of the transfected cell lysates was assayed 2 days posttransfection. Where indicated, cells were treated with PGE2 (100 nM) in the presence or absence of H7 (1 μM) or HA-1004 (1 μM) for 8 h before harvest. Fold induction is the luciferase activity relative to that obtained in the absence of the reagents. Results are means ± standard errors of the means from six independent experiments.

Article Snippet: Cell-free culture supernatants were collected on day 7 for measurement of HTLV-1 p19 antigen by ELISA (Cellular Products Inc., Buffalo, N.Y.).

Techniques: Activity Assay, Transfection, Luciferase, Expressing

Journal:

Article Title: Reciprocal Interactions between Human T-Lymphotropic Virus Type 1 and Prostaglandins: Implications for Viral Transmission

doi: 10.1128/JVI.75.1.192-198.2001

Figure Lengend Snippet: PGE 2 induces HTLV-1 replication a

Article Snippet: Cell-free culture supernatants were collected on day 7 for measurement of HTLV-1 p19 antigen by ELISA (Cellular Products Inc., Buffalo, N.Y.).

Techniques:

Journal:

Article Title: Reciprocal Interactions between Human T-Lymphotropic Virus Type 1 and Prostaglandins: Implications for Viral Transmission

doi: 10.1128/JVI.75.1.192-198.2001

Figure Lengend Snippet: Expansion by PGE 2 of HTLV-1 infection to CBMC a

Article Snippet: Cell-free culture supernatants were collected on day 7 for measurement of HTLV-1 p19 antigen by ELISA (Cellular Products Inc., Buffalo, N.Y.).

Techniques: Infection

Journal:

Article Title: Reciprocal Interactions between Human T-Lymphotropic Virus Type 1 and Prostaglandins: Implications for Viral Transmission

doi: 10.1128/JVI.75.1.192-198.2001

Figure Lengend Snippet: HTLV-1 Tax transactivates the COX-2 promoter. Jurkat cells (A) or PBMC (B) were transfected with 10 or 40 μg of phPES(−1432/+59), respectively, along with 10 μg of pMT-2T or pMT-Tax, respectively. Where indicated, PBMC were stimulated with PMA (1 μM) plus ionomycin (1 μM) for 8 h. Fold induction is the luciferase activity relative to that obtained with phPES(−1432/+59) plus pMT-2T in the absence of PMA plus ionomycin. Results are means ± standard errors of the means from six independent experiments. (C) PBMC were transfected with 40 μg of phPES(−1432/+59) and treated with GST or GST-Tax (100 ng/ml) for 2 days. Results are means ± standard errors of the means from three independent experiments.

Article Snippet: Cell-free culture supernatants were collected on day 7 for measurement of HTLV-1 p19 antigen by ELISA (Cellular Products Inc., Buffalo, N.Y.).

Techniques: Transfection, Luciferase, Activity Assay