Collagen IV (COL4A1, COL4A2), a Component of the Viral Biofilm, Is Induced by the HTLV-1 Oncoprotein Tax and Impacts Virus Transmission

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for Adult T-Cell Leukemia/Lymphoma (ATLL) and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 infects CD4⁺ T-cells via cell-to-cell transmission requiring reorganization of the cytoskeleton and expression of the viral transactivator and oncoprotein Tax. Viruses spread at the virological synapse (VS), a virus-induced specialized cell-cell contact, by polarized budding into synaptic clefts, and by cell surface transfer of viral biofilms (VBs). Since little is known about Tax's role in formation of the VB, we asked which component of the VB is regulated by Tax and important for HTLV-1 transmission. Collagens are not only structural proteins of the extracellular matrix and basal membrane but also represent an important component of the VB. Here, we report that among the collagens known to be present in VBs, COL4 is specifically upregulated in the presence of HTLV-1 infection. Further, we found that transient expression of Tax is sufficient to induce COL4A1 and COL4A2 transcripts in Jurkat and CCRF-CEM T-cells, while robust induction of COL4 protein requires continuous Tax expression as shown in Tax-transformed T-cell lines. Repression of Tax led to a significant reduction of COL4A1/A2 transcripts and COL4 protein. Mechanistically, luciferase-based promoter studies indicate that Tax activates the COL4A2 and, to a less extent, the COL4A1 promoter. Imaging showing partial co-localization of COL4 with the viral Gag protein in VBs at the VS and transfer of COL4 and Gag to target cells suggests a role of COL4 in VB formation. Strikingly, in chronically infected C91-PL cells, knockout of COL4A2 impaired Gag transfer between infected T-cells and acceptor T-cells, while release of virus-like particles was unaffected. Taken together, we identified COL4 (COL4A1, COL4A2) as a component of the VB and a novel cellular target of Tax with COL4A2 appearing to impact virus transmission. Thus, this study is the first to provide a link between Tax's activity and VB formation by hijacking COL4 protein functions.

Keywords: COL4A1; COL4A2; HTLV-1; Tax-1; collagen 4; collagen IV; viral biofilm; virus transmission.

FIGURE 1

COL4 is specifically upregulated in HTLV-1 positive T-cell lines as well as COL4A2 in ex vivo HAM/TSP patient samples. (A–D) Analysis of COL4 expression in HTLV-1 positive (C8166-45, MT-2, C91-PL, HuT-102) and HTLV-1 negative T-cell lines (Jurkat, HuT-78, CCRF-CEM, Molt-4). (A–C) mRNA of (A) COL4A1, (B) COL4A2, and (C) Tax was quantified by qPCR in the respective HTLV-1 positive and negative T-cell lines. The mean relative copy numbers (rcn), normalized on ACTB, of three individual experiments ± SE are depicted. Values were compared using two-tailed Mann–Whitney U test (^∗∗p < 0.01). (D) COL4 protein was detected in HTLV-1 positive and negative T-cell lines by means of Western Blot. Staining of Tax and Hsp90 α/β served as expression or loading control, respectively. (E) COL4A1, (F) COL4A2, and (G) Tax transcripts were quantified by qPCR comparing mRNA isolated from patient samples of HTLV-1 asymptomatic carriers (AC), slowly progressing HAM/TSP patients (TSP-L), rapidly progressing HAM/TSP patients (TSP-R), and uninfected controls (CD4⁺-NI). Statistical analysis was performed applying two-tailed Mann–Whitney U test (^∗p < 0.05; ^∗∗p < 0.01; ^****p < 0.0001; n.s., not significant).

FIGURE 2

COL4 protein localizes to extra- and intracellular compartments in HTLV-1 positive T-cells. (A,B) Immunofluorescence staining was performed in the HTLV-1 positive T-cell lines MT-2, HuT-102 and C91-PL and in the HTLV-1 negative T-cell line Jurkat. Cells were spotted on epoxy-resin coated glass slides. The plasma membrane was visualized in green by using a mouse primary antibody against CD98, together with the secondary antibody anti-mouse Alexa Fluor^® 488. COL4 protein was depicted in red, employing a COL4 targeting rabbit primary antibody that was recognized by the secondary antibody anti-rabbit Alexa Fluor^® 647. DAPI staining of nuclei (blue) and transmitted light (TL) served as controls. Cells in (Aa–t) were permeabilized before antibody treatment, cells in (Ba–t) were left without permeabilization showing extracellular COL4 protein only. The scale bars represent 10 μm.

FIGURE 3

Transient expression of Tax is sufficient to induce COL4A1 and COL4A2 transcripts, but not COL4 protein. (A) COL4A1 and (B) COL4A2 transcript levels were quantified by qPCR upon transient expression of 100 μg Tax expression plasmid (pEFneo-Tax1) or empty vector (pEFneo) in 10^∗10⁶ Jurkat and CCRF-CEM T-cells. The mean relative copy numbers (rcn), normalized on ACTB, of three independent experiments ± SE are depicted. Student’s t-test was conducted for statistical analysis (^∗p < 0.05; ^∗∗p < 0.01). (C) Immunoblotting for COL4 protein was performed in mock- (pEFneo) and Tax-transfected T-cell lines Jurkat and CCRF-CEM (100 μg DNA, 10^∗10⁶ cells) as well as for MT-2 cells as positive control. Tax and GAPDH or α-Tubulin staining were carried out as controls. (D) COL4A1 and COL4A2 transcript levels were quantified by qPCR upon transient expression of 50 μg Tax expression plasmid (pcTax), 17.5 μg of the HTLV-1 packaging plasmid pCMV-HT1-ΔEnv (HTLV) supplemented with 32.5 μg empty vector (pcDNA), or 50 μg empty vector (pcDNA) alone in 5^∗10⁶ Jurkat cells. The mean relative copy numbers (rcn) ± SE, normalized on ACTB, of one representative experiment is depicted. (E,F) mRNA of (E) COL4A1 and (F) COL4A2 transcripts was measured by qPCR in the Tax-inducible T-cell lines JPX9 and JPX9M at the indicated time points after induction of Tax wildtype protein (JPX9) or Tax mutant protein (JPX9M) by addition of 20 μM CdCl₂. The mean relative copy numbers (rcn), normalized on ACTB, of three independent experiments ± SE are depicted. (G) For qualitative analysis, Tax and ACTB transcripts were amplified by RT-PCR from JPX9 and JPX9M cell lysates at the indicated time points after induction of Tax wildtype (JPX9) or Tax mutant protein (JPX9M). (H) Western Blot analysis was carried out staining COL4 protein as well as Hsp90 α/β as loading control at the indicated time points after induction of Tax wildtype (JPX9) or Tax mutant (JPX9M) protein expression. MT-2 cells served as positive control for COL4 protein expression.

FIGURE 4

Tax transactivates the COL4A1 and COL4A2 promoter. (A) Schematic representation of the COL4A1/2 genomic organization. In the shared and bidirectional COL4A1/2 promoter region, putative genetic sites of GC-Box factors Specificity Protein 1 (SP1)/GC, CCCTC-binding factors and Nuclear Factor 1 (NF1)/CCAAT-Box binding transcription factors are indicated. Bioinformatic analysis was performed using the MatInspector software tool. (B–D) Conduction of luciferase-based reporter assays. (B,C) 5^∗10⁶ Jurkat T-cells were co-transfected with a mock (pEFneo) or Tax expression vector (pEFneo-Tax1; 30 μg DNA) together with (B) a luciferase reporter vector for the U3R promoter from the HTLV-1 5′LTR region (U3R-Luc; 20 μg DNA) as a positive control, or (C) luciferase reporter vectors specific for the COL4A1 or COL4A2 promoter region (COL4A1-Luc, COL4A2-Luc; 20 μg DNA). Values were normalized on protein content and the luciferase background activity measured by co-expression of a promoter-less luciferase vector (pGL3-Basic). The mean of five independent experiments ± SE is depicted and Student’s t-test was performed (^∗∗p < 0.01) comparing the different reporter vectors with their respective mock control. (D) Immunoblot staining of Tax and Hsp90 α/β protein served as expression control.

FIGURE 5

Continuous expression of Tax is necessary to induce and maintain COL4 protein expression. (A,C) Immunoblot of COL4 protein was carried out in (A) Tax-transformed T-cell lines (Tri, Tesi, TAXI-1) and (C) T-cell lines transformed by the oncoprotein Tio, encoded by Herpesvirus ateles (1765, 1766). The HTLV-1 positive T-cell line MT-2 served as positive control. In contrast, negative controls were the Tax and Tio negative T-cell lines Jurkat and 1851/1, as well as peripheral blood mononuclear cells (PBMC), that were either left unstimulated (unstim.) or stimulated (stim.) with 50 U/ml IL-2 together with 2 μg/ml PHA overnight followed by prolonged incubation with 50 U/ml IL-2 only. Staining of Tax, Tio and GAPDH were performed as control. (B) qPCR was performed to analyze abundance of Tax transcripts in Tri, Tesi, TAXI-1 and MT-2 T-cell lines. The mean relative copy numbers (rcn), normalized on ACTB, of one representative experiment ± SE are depicted. (D–F) Transcript levels of (D) Tax, (E) COL4A1, and (F) COL4A2 were detected in the Tax-transformed T-cell line Tesi in the presence of Tax (Tesi) or after repression of Tax for 10 days by addition of 1 μg/ml tetracycline (Tesi/Tet). The mean relative copy numbers (rcn), normalized on ACTB, of three independent experiments ± SD are shown. Student’s t-test was conducted for statistical analysis (^∗∗p < 0.01). (G) COL4 and Tax protein were detected in Tax-transformed T-cell lines expressing (Tesi) or repressing (Tesi/Tet) Tax protein and, as a control, in the HTLV-1 positive T-cell line MT-2. β-actin (ACTB) served as loading control.

FIGURE 6

COL4 and Gag p19 partially co-localize and accumulate at the virological synapse and can be transferred to HTLV-1 negative T-cells in co-culture. (Aa–o) Immunofluorescence analysis was conducted in the HTLV-1 positive T-cell lines MT-2, C91-PL and C8166-45 spotted on poly-L-lysine coated coverslips. Permeabilized cells were stained with primary antibodies mouse anti-Gag p19 and the secondary antibody anti-mouse Alexa Fluor^® 488 (green), as well as rabbit anti-COL4 and the secondary antibody anti-rabbit Alexa Fluor^® 555 (red). DAPI staining of nuclei and transmitted light (TL) served as control. Insets show enlarged areas. A white arrow indicates partial co-localization of Gag and COL4 protein (yellow). The scale bar represents 10 μm. (Ba–e,Ca–j) Jurkat T-cells that had been prestained with CellTracker^TM Blue 7-Amino-4-Chlormethylcumarin (CMAC; 45 min, 20 μM, 37°C) were co-cultured with HTLV-1-infected MS-9 T-cells at a ratio of 1:1 for 20 or 50 min on poly-L-lysine coated coverslips. Following permeabilization, cells were stained with primary antibodies mouse anti-Gag p19 and the secondary antibody anti-mouse Alexa Fluor^® 488 (green), as well as rabbit anti-COL4 and the secondary antibody anti-rabbit Alexa Fluor^® 555 (red). Transmitted light served as control. Insets show enlarged areas. (Ba–e) A white arrow indicates partial co-localization of Gag and COL4 protein (yellow). (Ca–j) Black arrows indicate transferred COL4 protein from HTLV-1 positive MS-9 to Jurkat T-cells, a white arrow indicates partial co-localization of transferred Gag and COL4 protein (yellow). The scale bars represent 10 μm. (D) Manual counting was performed to quantify Gag and COL4 transfer from MS-9 donor cells to Jurkat acceptor cells. Gag only (black bars), COL4 only (gray bars) or Gag and COL4 (hatched bars) double positive Jurkat cells after 20 or 50 min of co-culture were determined and normalized on the number of Gag and/or COL4 positive MS-9 donor cells and on the ratio of Jurkat acceptor to MS-9 donor cells. Average values of counted cells ± SE are shown and Student’s t-test was conducted for statistical analysis (^∗p < 0.05).

FIGURE 7

Repression of COL4 protein impairs virus transmission to uninfected T-cells, but not Gag processing and virus release in chronically infected C91-PL cells. (A) Western Blot analysis to detect COL4 as well as Gag p55 precursor and processed Gag p19 matrix protein was carried out in C91-PL cells stably transduced either by a CRISPR control vector (scramble) or vectors to generate a knockout for COL4A1 or COL4A2. Hsp90 α/β served as loading control, lysates of Jurkat T-cells as negative staining control. (B) qPCR was performed to determine expression levels of COL4A1 and COL4A2 mRNA in transduced C91-PL cell lines from (A). The mean relative copy numbers (rcn), normalized on ACTB and the C91-PL scramble control cell line, of one representative experiment ± SD are shown. (C) The amount of Gag p19 protein in the supernatant of transduced C91-PL cell lines from (A) was assessed by Gag p19 ELISA. The mean of five independent experiments ± SD is depicted and was compared using Student’s t-test (n.s.; not significant). Jurkat T-cells were employed as negative control. (D,E) Jurkat T-cells, that had been prestained with CellTracker^TM Blue 7-Amino-4-Chlormethylcumarin (CMAC), were co-cultured with stably transduced C91-PL cell lines from (A) at a ratio of 1:1 for 1 h. The transfer of Gag p19 to Jurkat acceptor cells was measured by flow cytometry using the primary antibody mouse anti-gag p19 and the secondary antibody anti-mouse Alexa Fluor^® 647. (D) The representative gating strategy for co-cultures of scramble and COL4A2 knockout C91-PL with Jurkat cells reveals discrimination between CMAC-positive Jurkat acceptor T-cells and CMAC-negative C91-PL donor cells, further detecting Gag p19 in the distinct cell populations. (E) The amount of Gag p19 positive Jurkat acceptor cells normalized on the scramble C91-PL control cells of three independent experiments ± SD is depicted and was compared using Student’s t-test (n.s., not significant; ^∗p < 0.05; ^∗∗p < 0.01). Treatment of C91-PL scramble cells with 5 μM cytochalasin D (cyto D), an inhibitor of actin-polymerization, in comparison to the DMSO solvent control served as positive control for an impaired Gag p19 transfer.