Opposing effects of a tyrosine-based sorting motif and a PDZ-binding motif regulate human T-lymphotropic virus type 1 envelope trafficking

Abstract

Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein mediates binding of the virus to its receptor on the surface of target cells and subsequent fusion of virus and cell membranes. To better understand the mechanisms that control HTLV-1 Env trafficking and activity, we have examined two protein-protein interaction motifs in the cytoplasmic domain of Env. One is the sequence YSLI, which matches the consensus YXXPhi motifs that are known to interact with various adaptor protein complexes; the other is the sequence ESSL at the C terminus of Env, which matches the consensus PDZ-binding motif. We show here that mutations that destroy the YXXPhi motif increased Env expression on the cell surface and increased cell-cell fusion activity. In contrast, mutation of the PDZ-binding motif greatly diminished Env expression in cells, which could be restored to wild-type levels either by mutating the YXXPhi motif or by silencing AP2 and AP3, suggesting that interactions with PDZ proteins oppose an Env degradation pathway mediated by AP2 and AP3. Silencing of the PDZ protein hDlg1 did not affect Env expression, suggesting that hDlg1 is not a binding partner for Env. Substitution of the YSLI sequence in HTLV-1 Env with YXXPhi elements from other cell or virus membrane-spanning proteins resulted in alterations in Env accumulation in cells, incorporation into virions, and virion infectivity. Env variants containing YXXPhi motifs that are predicted to have high-affinity interaction with AP2 accumulated to lower steady-state levels. Interestingly, mutations that destroy the YXXPhi motif resulted in viruses that were not infectious by cell-free or cell-associated routes of infection. Unlike YXXPhi, the function of the PDZ-binding motif manifests itself only in the producer cells; AP2 silencing restored the incorporation of PDZ-deficient Env into virus-like particles (VLPs) and the infectivity of these VLPs to wild-type levels.

FIG. 1.

Two motifs in the C-terminal cytoplasmic domain of HTLV-1 Env modulate Env expression and functional activity. (A) Amino acid sequences of the C termini of WT and mutated HTLV-1 Envs. Amino acids comprising the tyrosine-based motif (YSLI) and the PDZ-binding motif (ESSL) are shown in bold. (B) Functional activities of WT and mutated versions of HTLV-1 Env were assessed in cell-cell fusion assays, cell-free infection assays, and cell-to-cell infection assays, as described in Materials and Methods. In cell-cell fusion assays, 293T cells were transfected with HTLV-1 Env plasmid and then cocultured with HeLa-Tat cells and HeLa-TZM cells; cell fusion resulted in luciferase expression. In cell-free infection assays, 293T cells were transfected with HTLV-1 pack aging (pCMVHT1M-ΔEnv) and reporter vectors and the indicated HTLV-1 Env expression plasmids; VLPs were then used to infect HeLa-P4 cells. In cell-to-cell infection assays, Jurkat cells were transfected with HTLV-1 vectors and then cocultured with Raji/CD4 cells. The results are presented as percentages relative to the value for the WT Env control and represent the means from at least three independent experiments; error bars show standard deviations. (C) 293T cells were transfected with pCMVHT1M-ΔEnv and either empty vector (mock) or the indicated HTLV-1 Env plasmids. Two days after transfection, cell lysates and VLP extracts were analyzed by Western blotting with anti-HTLV1 gp46 (SU) antibody. (D) 293T cells were transfected with pCMVHT1M-ΔEnv plus empty vector (mock) or the indicated myc-tagged HTLV-1 Env constructs. Two days later, cell lysates and VLP extracts were analyzed by immunoblotting. HTLV-1 Env SU (gp46) was detected with anti-HTLV-1 gp46 antibody, HTLV-1 TM (gp21) was detected with anti-myc antibody, and HTLV-1 MA was detected with anti-HTLV-1 p19 antibody. (E) 293T cells were transfected with HTLV-1 Env plasmids and a GFP-expressing plasmid; on the next day, cells were probed with anti-HTLV-1 gp46 (SU) for surface expression of Env. Samples were analyzed with a FACSCalibur instrument, and Env surface expression in GFP-positive cells was measured. These experiments were performed three times. Filled histogram, empty vector; dotted line, WT; solid line, Env with mutations. FL2-H, fluorescence in (585 ± 42 nm) channel.

FIG. 2.

Intracellular distribution of HTLV-1 envelope in HeLa-P4 cells. HeLa-P4 cells were transfected with the indicated HTLV-1 Env expression plasmids. Cells were permeabilized and stained with sera from HTLV-1-infected patients to detect Env (green) or with wheat germ agglutinin for the Golgi network (red). Distribution of Env with a truncated or substituted PDZ-binding ligand (ΔPDZ and QASS) in cells differed from that of other Env constructs and localized mainly in the Golgi network. Env with combined mutations (YSLK/ΔPDZ) was localized similarly to WT and YSLK Env. These experiments were performed three times.

FIG. 3.

siRNA silencing of AP2 and AP3 μ subunits caused increased accumulation of WT and ΔPDZ Env in cells and VLPs. (A) HeLa-P4 cells were transfected with siRNAs directed against the μ subunit of either AP2 or AP3 and then transfected with pCMVHT1M-ΔEnv plus the indicated HTLV-1 Env expression plasmids. Cell lysates were examined by immunoblotting with antibodies to the μ chains of AP2 and AP3 (bottom), which showed the efficiency of siRNA targeting and transfection. Anti-HTLV-1 gp46 antibody was used to probe for Env expression, and anti-HTLV-1 p19 antibody revealed that equal amounts of VLP extracts were loaded onto the gel. This experiment was performed three times. (B) shRNA delivered by lentiviral vector constructs and directed against the μ subunit of AP2 was used to silence AP2 in 293T cells, and cells were then transfected with pCMVHT1M-ΔEnv plus the indicated HTLV-1 Env expression plasmids. Cell-free infection was performed as described in Materials and Methods. Cell lysates were examined by immunoblotting with antibodies to μ chains of AP2 (bottom). Error bars show standard deviations.

FIG. 4.

Dlg1 is not a binding partner for the PDZ-binding ligand of HTLV-1 Env. HeLa-P4 cells were transfected with pooled siRNA directed against Dlg1 and later transfected with the indicated HTLV-1 myc-tagged Env plasmids. Immunoblot analysis of cell lysates with antibody to Dlg1 confirmed the efficiency and specificity of Dlg1 silencing. Probing the blots with anti-myc antibody revealed bands corresponding to the Env precursor (gp63) and TM (gp21); anti-gp46 antibody detected HTLV-1 Env SU; and antibody to VDAC was used as a loading control. Silencing of Dlg1 did not cause significant changes in expression of WT* and ΔPDZ* Env. This experiment was repeated three times.

FIG. 5.

Substitution of the HTLV-1 tyrosine-based motif with different YXXΦ signals affected functions of Env. (A) Amino acid sequences of the C-terminal cytoplasmic domains of WT and variant Env proteins. The tyrosine-based motif is shown in bold. The YSLI (WT) element was replaced with the tyrosine-based motif from the transferrin receptor (YTRF), HIV-1 envelope (YSPL), or VSV-G protein (YTDI). (B) 293T cells were transfected with pCMVHT1M-ΔEnv plus the indicated Env expression plasmids. Two days later, cell and VLP lysates were examined by immunoblotting with antibody to HTLV-1 gp46 (SU). (C) 293T cells were transfected with pCMVHT1M-ΔEnv plus the indicated myc-tagged HTLV-1 Env expression plasmids. Two days later, cell and VLP lysates were examined by immunoblotting with antibody to HTLV-1 gp46 (SU); anti-myc, to detect gp21 (TM); or anti-HTLV-1 p19 (MA). (D) 293T cells were transfected with HTLV-1 Env plasmids; on the next day, cells were probed with anti-HTLV1 gp46 (SU) for surface expression of Env. Filled histogram, empty vector; dotted line, WT; solid line, Env with mutations. Samples were analyzed with a FACSCalibur instrument, and Env surface expression in GFP-positive cells was measured. These experiments were performed three times. (E) HTLV-1 Envs with WT or variant tyrosine-based motifs were tested in cell-cell fusion assays and infectivity assays as described for Fig. 1B. The results for the indicated Env variants are presented as percentages compared with the WT level. These experiments were performed at least three times; error bars show standard deviations.

FIG. 6.

siRNA silencing of AP2 and AP3 increased the cellular accumulation and VLP incorporation of Env with variant tyrosine-based motifs. (A and B) HeLa-P4 cells were transfected with siRNA to the μ subunit of AP2 or AP3. Immunoblot analysis of cell lysates with antibodies to the μ chains of AP2 and AP3 shows the efficiency and specificity of siRNA silencing (A, bottom). After transfection with siRNA, cells were again transfected with pCMVHT1M-ΔEnv and the indicated Env expression plasmids. Blots were probed with anti-HTLV-1 gp46 (SU); anti-VDAC, as a loading control for cell lysates; or anti-HTLV-1 p19 (MA), to normalize VLP loading. This experiment was performed three times. (C) shRNA directed against the μ subunit of AP2 was used to silence AP2 in 293T cells (as described for Fig. 3B), and cells were then transfected with pCMVHT1M-ΔEnv plus the indicated HTLV-1 Env expression plasmids. Cell-free infection was performed as described in Materials and Methods. Error bars show standard deviations.