Figure 1.
HIV-1 gp41 extra-cellular domain.
A scheme showing the functional regions within the extra-cellular portion of gp41 (aa 512–684).
Table 1.
Designations and Sequences of the Peptides Investigated in This Study.
Figure 2.
Bioinformatics database analysis.
A. The sequence alignments obtained from the EMBOSS package: Needle global alignment analysis. I) TCRα CP / SIV gp41 TMD sequences alignment II) TCRα CP / HIV-1 gp41 TMD sequences alignments. B. Z-Score alignment histogram. TMD Entries scores were clustered according to taxonomic species division. The mean of each cluster was Z-normalized. The histogram describes the distribution of alignment scores over the 265 clusters dataset C. Wilcoxon Rank Test. The HIV-1 gp41 cluster (n = 47) was compared against all the other 264 clusters using Wilcoxon Rank Test. p-value results of comparisons are presented in a log scale. Significance of ranking was determined according to Benjamini-Hochberg method (E (FDR)<0.05). Clusters which are significantly lowered ranked over HIV-1 gp41 are marked (black dot) and presented left to the arrow. Cluster which were not significantly distinct in ranking from HIV-1 gp41 are marked (diamond).
Figure 3.
Gp41 TMD inhibits T-cell proliferation in vitro.
T-cells were activated with the MOG 35–55 peptide and APC in the presence of gp41 TMD (black) or FP5–13 AAAA as control peptide (grey) in three different concentrations (50, 25 and 5 µg/ml) and the proliferative responses were assayed. The data are presented as mean inhibitions+SD (n = 4 or more, ** p<0.01). The uninhibited T-cell proliferative responses were 9382±891 cpm. The background proliferation in the absence of antigen was 232±36 cpm.
Figure 4.
Gp41 TMD inhibit T-cell activation induced by mitogenic antibodies to CD3.
T-cells were activated with 1µg/ml anti-CD3 and APC in the presence of 50µg/ml gp41 TMD or TCRα CP and the proliferative responses were assayed. The uninhibited T-cell proliferative responses were 9103±208 cpm. The background proliferation in the absence of antigen was 257±41 cpm. The data are presented as normalized mean proliferation percentage (control, no peptide = 100%) + SD (n = 5 or more, ** p<0.01). There was no significant statistical difference between the TCRα CP and the control.
Figure 5.
Gp41 TMD does not inhibit T-cell activation induced by PMA/ionomycin.
T cells were stimulated with PMA/ionomycin or in the presence of gp41 TMD or TCRα CP, and T cell proliferation was studied. The uninhibited T-cell proliferative responses were 12220±871 cpm. The background proliferation in the absence of antigen was 246±38 cpm. The data are presented as normalized mean proliferation percentage (control, no peptide = 100%)+SD (n = 5 or more). There was no significant statistical difference between all groups.
Figure 6.
Gp41 TMD co-localizes with CD3 within the TCR complex.
Gp41 TMD-NBD and AMP-NBD were used to study peptide binding to the membranes of resting and activated T cells in combination with Sy5-labeled antibodies to CD3. The T cells had been activated by incubation with APC and the MOG 35–55 peptide. (A) Distribution of gp41 TMD-NBD, AMP-NBD and CD3 molecules in resting and activated T cells. (B) Co-localization of gp41 TMD-NBD with the CD3 molecules. (C) Co-localization of AMP-NBD with the CD3 molecules.
Figure 7.
Fluorescence Energy Transfer (FRET) measurements reveal a specific interaction of the gp41 TMD and the TCRα CP.
Fluorescence spectra were obtained at room temperature, with excitation set at 467 nm (5-nm slit) and emission scan at 500–600 nm (10-nm slits). NBD-labeled CP peptide was added first from a stock solution in DMSO (final concentration 0.1 µM and a maximum of 0.25% (v/v) DMSO) to a dispersion of PC LUV (100 µM) in PBS. This was followed by the addition of: A. Rho labeled gp41 TMD peptide B. Rho labeled TCRα CP C. Rho labeled TAR/PS TM control peptide. Fluorescence spectra were obtained in four different ratios of Rho-peptide∶NBD-peptide: 0∶4 (black line), 1∶4 (black triangle), 2∶4 (black circle) and 3∶4 (dashed line). The fluorescence values were corrected by subtracting the corresponding blank (buffer with the same vesicles concentration). Statistical analysis was performed at the pick measurements (∼537 nm, n = 5, * p<0.05).