Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription

<div><p>The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5’LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-<i>N</i>-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: <i>O</i>-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and <i>O</i>-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.</p></div

OGA and OGT status in HTLV-1-transformed T cells and control transformed T cells.

<p>(<b>A, B</b>) RT-qPCR experiments comparing the amount of OGA (<b>A</b>) or OGT (<b>B</b>) mRNA in non-HTLV-1 (white bars) or in HTLV-1-transformed T cells (black bars) normalized to the level of HPRT mRNA. Results are means ± SEM of two independent experiments performed in triplicates. Statistical analyses are shown in right panels. (<b>C</b>) Western blot experiments showing the amount of OGA, OGT, Tax and tubulin in non-HTLV-1 or in HTLV-1-transformed T cells extracts. (<b>D</b>) OGA activity in non-HTLV-1 (empty circle) or HTLV-1 (black circle) transformed T cell extracts. Total OGA activity (left panel) was normalized to the OGA protein level in the extract determined by densitometric analysis of the OGA band detected on western-blot (middle panel), in order to estimate OGA specific activity (right panel). Results correspond to 3 independent experiments. Statistical significance was analyzed using the student’s t test (ns: not significant; *: p<0.05).</p

Effect of Tax on OGA activity and O-GlcNAcylation.

<p>(<b>A</b>) Total OGA activity was measured at two different time-points in extracts from TL-om1 T cells transfected with either the control or Tax plasmid. Results are means ± SEM of 4 independent experiments. The insert shows the expression of OGA, OGT and Tax in the cell extracts. (<b>B</b>) The BRET O-GlcNAc-biosensor is composed of Rluc8 fused to the fimbrial adhesin lectin domain GafD, a known OGT substrate peptide derived from casein kinase II placed between two flexible linkers (GGSGG), followed by the Venus variant of the yellow fluorescent protein (Adapted from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006518#ppat.1006518.ref027" target="_blank">27</a>]). (<b>C</b>) TL-om1 cells were transfected with the O-GlcNAc BRET biosensor and either the control or Tax plasmid. BRET experiments were performed 24h after transfection. BRET measurements were started 5 min after the addition of coelenterazine. Left panel: typical BRET experiment. Middle panel: mean delta BRET (increased BRET signal induced by Tax expression). Results are means ± SEM of 3 independent experiments. Right panel: level of O-GlcNAc proteins evaluated by western-blotting with an anti-O-GlcNAc antibody. (<b>D</b>) Total OGA activity was measured in extracts from HEK-293T cells transfected with either the control or Tax plasmid. Cells were extracted and OGA activity was measured at 30 min. Results are means ± SEM of 3 independent experiments. The insert shows the expression of OGA, OGT and Tax in the cell extracts. (<b>E</b>) HEK-293T cells were transfected with the O-GlcNAc BRET biosensor and either the control or Tax plasmid. BRET experiments were performed 48h after transfection. BRET measurements were started 5 min after the addition of coelenterazine. Left panel: typical BRET experiment. Middle panel: mean delta BRET (increased BRET signal induced by Tax expression). Results are means ± SEM of 3 independent experiments. Right panel: level of O-GlcNAc proteins evaluated by western-blotting with an anti-O-GlcNAc antibody. Statistical significance was calculated using the student’s t test (**: p<0.01; ***: p<0.001; ****: p<0.0001).</p

Effect of OGA inhibition on LTR activation and CREB O-GlcNAcylation in Tax expressing cells.

<p>(<b>A, B</b>) Effect of OGA inhibition on Tax-mediated LTR transactivation in (<b>A</b>) C8166 T cells or (<b>B</b>) HEK-293T cells. Left panels: Cells were transfected with the U3R-LTR-luciferase and pRL-TK plasmid and in the case of HEK-293T cells, the Tax plasmid, and were incubated or not with the OGA inhibitor Thiamet G for 24h. Luciferase production was then measured using the dual luciferase assay. Results are means ± SEM of 4 independent experiments performed in duplicates. Right panels: level of O-GlcNAcylation and Tax expression in each condition. (<b>C</b>) Effect of Tax on CREB O-GlcNAcylation in HEK-293T cells. HEK-293T cells were transfected with either the control or Tax plasmid and treated or not with the OGA inhibitor Thiamet G. Cell extracts were prepared two days post-transfection. Left panel: total proteins (lysates) or WGA-bound proteins (WGA) were separated by SDS-PAGE and blotted with either an anti-O-GlcNAc or an anti-CREB antibody. Tax expression in cell lysates is also shown. Middle and right panel: Total O-GlcNAc signal (middle panel) or WGA/lysate ratio for CREB signal (right panel) in two independents experiments. (<b>D</b>) Effect of Tax on CREB O-GlcNAcylation in TL-om1 T cells. TL-om1 T cells were transfected with either the control or Tax plasmid. Left panel: Total proteins (lysates) or WGA-bound proteins (WGA) were separated by SDS-PAGE and blotted with either an anti-O-GlcNAc or anti-CREB antibody. Tax expression in cell lysates is also shown. Middle and right panels: Total O-GlcNAc signal (middle panel) or WGA/lysate ratio for CREB signal (right panel) in two independents experiments.</p

Mutation of CREB Serine 40 inhibits both Tax-mediated increase in CREB O-GlcNAcylation and LTR activation.

<p>(<b>A</b>) HEK-293T cells were transfected with the control (- Tax) or Tax plasmid (+ Tax) and either the wild-type (wt) or S40A mutant YFP-CREB construct and lysed 48h post-transfection. The amounts of wt and S40A YFP-CREB in the lysate were evaluated by measuring fluorescence emission at 530 nm after excitation at 480 nm. After normalization for fluorescence, O-GlcNAcylated proteins were captured on wheat germ lectin agarose (WGA) beads. Total proteins (Lysates) or WGA-bound proteins (WGA) were separated by SDS-PAGE and blotted with an anti-CREB antibody. Tax expression in cell lysates is also shown. <b>(B)</b> HEK-293T cells were transfected and cell extracts were normalized as in (A), but wt and mutant YFP-CREB proteins were immunoprecipitated using an anti-GFP antibody. The level of O-GlcNAcylation of wt and S40A YFP-CREB was evaluated using an anti-O-GlcNAc antibody. The membranes were then striped and reprobed with an anti-CREB antibody. (<b>C</b>) Effect of wt or S40A mutant YFP-CREB on Tax-mediated LTR activation. Left panel: HEK-293T cells were transfected with U3R-LTR-luciferase and pRL-TK plasmids along with Tax and either a control plasmid or the plasmid coding for wt or S40A YFP-CREB. Luciferase production was then measured two days post-transfection using the dual luciferase assay. Results are from 4 independent experiments performed in duplicates. Statistical significance was analyzed using the Tukey’s multiple comparison test (*: p<0.05; ***: p<0.001; ns: not significant). Right panel: levels of Tax and CREB expression in the transactivation experiments.</p

Effect of O-GlcNAcylation on the HTLV-1 LTR.

<p>(<b>A</b>) Effect of Thiamet G on Ser133 phospho-CREB recruitment to the vCRE LTR region. C8166 T cells were cultured with or without Thiamet G for 48h before chromatin preparation. Chromatin was precipitated with either control IgG or an anti-Ser133 phospho-CREB and recovered DNA was amplified using a pair of primers specific for the vCRE sequence. Results correspond to means ± SEM of triplicate determinations obtained in a representative experiment out of 2. (<b>B-D</b>) Detection of OGA or OGT on the vCRE sequence by ChIP in C8166 (<b>B</b>) and MT2 (<b>C</b>) HTLV-1-transformed T cells or in HTLV-1-immortalized CIB T cells (<b>D</b>). Cells were treated as above and chromatin was precipitated using anti-OGT, anti-OGA or control (IgG) antibody. Recovered DNA was amplified using pairs of primers specific for the vCRE sequence or for alpha-satellite sequences as negative control. Results correspond to means ± SEM of triplicate determinations obtained in a representative experiment out of 2.</p