Zika M Oligopeptide ZAMP Confers Cell Death-Promoting Capability to a Soluble Tumor-Associated Antigen through Caspase-3/7 Activation

Abstract

Mosquito-borne Zika virus (ZIKV) is an emerging flavivirus of medical concern associated with neurological disorders. ZIKV utilizes apoptosis as a mechanism of cell killing. The structural M protein may play a role in flavivirus-induced apoptosis. The death-promoting capability of M has been restricted to an oligopeptide representing the residues M-32/40. Here, we evaluated the apoptosis inducing ability of the residues M-31/41 of ZIKV. The ZIKV M oligopeptide was associated to a soluble form of GFP (sGFP) and the resulting sGFP-M31/41 construct was assessed in Huh7 cells. Expression of sGFP-M31/41 can trigger apoptosis in Huh7 cells through caspase-3/7 activation. The translocation of sGFP-M31/41 in the endoplasmic reticulum was a prerequisite for apoptosis induction. The residues M-33/35/38 may play a critical role in the death-promoting activity of sGFP-M31/41. The effect of ZIKV M oligopeptide defined as ZAMP (for Zika Apoptosis M Peptide) on expression of a tumor-associated antigen was assayed on megakaryocyte-potentiating factor (MPF). Expression of MPF-ZAMP construct resulted in caspase-associated apoptosis activation in A549 and Huh7 cells. ZIKV has been proposed as an oncolytic virus for cancer therapy. The ability of the Zika M oligopeptide to confer death-promoting capability to MPF opens up attractive perspectives for ZAMP as an innovative anticancer agent.

Keywords: M protein; Zika virus; anticancer agent; anticancer virotherapy; apoptotic cell death; arbovirus; caspase-3/7 activation; tumor cells; tumor-associated antigen; viral apoptosis inducer; viral oligopeptide.

Figure 1

Schematic representation of the GFP-M oligopeptide constructs. In (A), a schematic representation of mature prM protein that is structured into a “pr” polypeptide followed by the residues M-1/41 which compose the M ectodomain and ending in a transmembrane anchoring region with two transmembrane domains (TMDs). The residues M-1/41 of epidemic Brazilian ZIKV strain BeH819015, epidemic La Reunion 2018 DENV-2 strain RUJul and YFV 17D vaccine strain are listed. The last C-terminal residues of flavivirus M ectodomain are underlined. In (B), the GFP constructs are preceded at the N-terminus by the signal peptide (SP) of ZIKV prM protein followed by a FLAG epitope. The soluble sGFP-M oligopeptide constructs are ended by a glycine−serine spacer followed by the residues M-31/41 of ZIKV, DENV-2 or YFV. The sGFP construct is restricted to a glycine−serine spacer at its C-terminus. The GFP^ZIKV.M-31/41 construct is a mutant lacking in prM signal peptide. The mutGFP^ZIKV.M-31/41 construct was made by replacing the residues M-E33/W35/R38 with alanine. The three Ala mutations in mutGFP^ZIKV.M-31/41 are underlined.

Figure 2

Effect of sGFP-M31/41 constructs on cell membrane integrity. Huh7 cells were transfected 24 h (left) or 48 h (right) with plasmids expressing sGFP or sGFP-M31/41 constructs, or mock-transfected cells (control). LDH activity was measured and cell membrane permeability was expressed as signal intensity (O.D.). The results are the mean (±SEM) of three (24 h) or six independent experiments (48 h). Statistical analysis for comparing sGFP-M constructs with sGFP was performed and noted (* p < 0.01); differences that were not statistically significant are omitted.

Figure 3

Expression of sGFP-M31/41 constructs affects cell viability. Huh7 cells were transfected 24 h with plasmids coding for sGFP^ZIKV.M-31/41, sGFP^{DENV-2.M-31/41,} or sGFP^YFV.M-31/41 or mock-transfected (control). The sGFP plasmid construct served as a control. In (A), the level of cell metabolic activity was measured using MTT assay and expressed as signal intensity (O.D.). The results are the mean (±SEM) of six independent assays. Statistical analysis for comparing sGFP-M constructs with sGFP was performed and noted (* p < 0.0001). In (B), Caspase 3/7 enzymatic activity was expressed as the fold change of caspase enzymatic activity in assay relative to sGFP. The results are the mean (±SEM) of six independent assays. Statistical analysis for comparing sGFP-M constructs with sGFP was performed and noted (** p < 0.001; * p < 0.01).

Figure 4

Caspase-associated apoptosis activation requires the signal peptide of sGFP^ZIKV.M-31/41 and involves the residues M-E33/W35/R38. Huh7 cells were transfected 24 h with plasmids expressing sGFP^ZIKV.M-31/41 or its mutants lacking a signal peptide (GFP^ZIKV.M-31/41) or bearing the Ala residues at positions M-33/35/38 (mutGFP^ZIKV.M-31/41). The sGFP plasmid construct served as a control. Caspase 3/7 enzymatic activity was measured using a caspase-3/7 assay kit, and the O.D. values were expressed as signal intensity. The results are the mean (±SEM) of three independent experiments. Statistical analysis for comparing sGFP-M constructs with sGFP was performed and noted (* p < 0.0001; n.s.: nonsignificant).

Figure 5

Schematic representation of MPF-ZAMP constructs. At the top, the organization of 69-kDa preprotein mesothelin (MSLN) that is processed into MPF (megakaryocyte-potentiating factor) and membrane-anchored MSLN by furin protease. The signal peptide of MSLN (residues 1/37) is indicated as a blue box. At the bottom, the MPF constructs with at the C-terminus, a Gly−Ser spacer followed by a FLAG epitope or ZIKV M oligopeptide ZAMP. The MPF-mutZAMP construct was made by replacing the three residues M-E31/W35/538 of ZAMP with alanine.

Figure 6

ZAMP associated to MPF has no effect on HEK-293T cells. Cells were transfected 24 h (A) or 48 h (B) with plasmids expressing MPF-FLAG, MPF-ZAMP, MPF-mutZAMP, or mock-transfected (control). In (A), FACS analysis was performed on cells expressing MPF-FLAG using anti-FLAG antibody. The percentage and the mean of fluorescence intensity (MFI) of positive cells are shown. In (B), the level of metabolic activity was measured using an MTT assay and expressed as signal intensity (O.D.). The results are the mean (±SEM) of four independent assays. Statistical analysis for comparing MPF constructs with MPF-FLAG was performed and nonstatistical differences were observed.

Figure 7

Expression of MPF-ZAMP triggers apoptosis in A549 cells. A549 cells were transfected 24 h with plasmids expressing MPF-FLAG, MPF-ZAMP, MPF-mutZAMP or GFP or transfectant alone (vehicle) or mock-transfected (control). In (A), A549 cells were transfected with plasmids expressing MPF-ZAMP or MPF-mutZAMP. Cells in apoptotic state were quantified by staining with FITC-labeled Annexin V. The percentage of Annexin V-positive cells was determined by FACS analysis. The results are the mean (±SEM) of two independent assays. In (B), caspase 3/7 enzymatic activity was measured using a caspase-3/7 assay kit, and the O.D. values were expressed as signal intensity. The results are the mean (±SEM) of four independent assays. Statistical analysis for comparing MPF constructs with GFP was performed and noted (* p < 0.001); any nonstatistically significant comparisons are omitted. In (C), the level of metabolic activity was measured using an MTT assay and expressed as signal intensity (O.D.). The results are the mean (±SEM) of three independent assays. Statistical analysis for comparing MPF constructs with MPF-FLAG was performed and noted (* p < 0.001); differences that were not statistically significant are omitted.

Figure 8

Expression of MPF-ZAMP triggers apoptosis in Huh7 cells. Huh7 cells were transfected with plasmids encoding MPF-FLAG, MPF-ZAMP, MPF-mutZAMP or transfectant alone (vehicle) or mock-transfected (control). In (A), the level of metabolic activity in Huh7 cells was determined at 18 h post-transfection using an MTT assay. The results were expressed as a percentage of cell metabolic activity in each assay relative to that in the vehicle and the results were the mean (±SEM) of four independent assays. Statistical analysis for comparing MPF constructs with MP-FLAG was performed and noted (* p < 0.001); any nonstatistically significant comparisons were omitted. In (B), caspase 3/7 enzymatic activity was measured at 18 h post-transfection using a caspase-3/7 assay kit, and the O.D. values were expressed as signal intensity. The results were the mean (±SEM) of six independent assays. Statistical analysis for comparing MPF constructs with MPF-FLAG was performed and noted (* p < 0.0001); any nonstatistically significant comparisons were omitted.