EMBO/SourceData huggingface dataset:
Figure 2A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A, B)A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, metabolic activity was assessed by a WST-1 assay (C, D).Data information: Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value 100% (C, D). The selected doses were indicated by "an arrow". Plasmatic concentrations after usual dosage (Cmax) of each drug in human therapy are indicated below the LRA names and by a box in the graph.
Figure 3A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. Viral production was measured by quantifying p24 antigen production in culture supernatants (A, B)A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. metabolic activity was assessed by a WST-1 assay (C, D). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 100% (C, D).E From data of ex vivo cultures of CD8+-depleted PBMCs isolated from 24 HIV+ patients presented in Appendix Table S2, the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented. One night after cell purification, cells were mock-treated or simultaneously treated with 5-AzadC (1µM) and/or SAHA (1µM). Six days after treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.
Figure 4A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: viral p24 production in cell supernatant was measured (A and B)A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows:FACS analyses were performed and the percentages of GFP+ cells are presented as histograms (C and D)A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR and results were normalized using the β-actin gene primers and are presented as histograms representing fold-inductions compared to the mock-treated condition (E and F). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 1 (E, F).
Figure 5One night after cell purification, CD8+-depleted PBMCs from 12 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added to the cultures. Six days after 5-AzadC treatment, WST-1 assay was performed. Mock value was arbitrary fixed at 100% for each individual.
Figure 6Figure 6: Representation of reactivation status of ex vivo cultures of CD8+-depleted PBMCs isolated from HIV+ patients.A-C From data of ex vivo cultures of CD8+-depleted PBMCs isolated from HIV+ patients (Table 2), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.
Figure 7A Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART-treated HIV+ patients (shown in Appendix Table S2).B Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART-treated HIV+ patients (shown in Table 2).Data information: The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented by a line.
Figure 8Figure 8: Representation of reactivation status of ex vivo cultures of resting CD4+ T cells.A-C From data of ex vivo cultures of resting CD4+ T cells isolated from HIV+ patients (Table 3), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.
Figure 9A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.
Figure 10One night after cell purification, HLA DR-CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The median fluorescence intensity of CD4 receptor of viable CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.
Figure 1(A) RFP-ATG8g-labeled autophagosomes were quantified from plants syringe infected with mock or Xcv ΔxopQ (oD600=0.2) at 2 dpi in the presence or absence of ConA (bars = 20 μm). Puncta were calculated from z-stacks (15) of n=6 individuals using ImageJ. Central band of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically different groups (P < 0.05) as determined by one-way ANOVA. The experiment was repeated twice with similar results.(B) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ or mock infected N. benthamiana plants at 1 and 2dpi. Agrobacterium-mediated transient expression of AIMp-RFP serves as a control for autophagy suppression. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.(C) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ was co-infiltrated with Agrobacteria containing the luciferase reporter constructs. Coexpression of RFP-AIMp serves as a control for autophagy inhibition. Expression of the latter was confirmed with western blot (inset). Renilla (RLUC) and Firefly (FLUC) luciferase activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P<0.001) was revealed by Student's t-test. The experiment was repeated more than 3 times with similar results.(D) RT-qPCR analysis of NbATG8-1.1/1.2, NbATG8-2.1/2.2 and NbJoka2 transcript levels upon challenge of N. benthamiana plants with Xcv ΔxopQ for 1 and 2 dpi compared to mock infected plants. Values represent expression relative to mock control of respective time point and were normalized to actin. Values are biological replicates (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (*P < 0.05, **P < 0.01, *** P < 0.001) was revealed by Student's t-test.(E) Bacterial density in leaves of N. benthamiana infected with Xcv in the presence or absence autophagy suppressor AIMp-RFP. Leaves were syringe-infiltrated with OD600 = 0.0004, and colony-forming units were counted at 6 dpi. Compared to empty vector control (EV), AIMp expressing plants (n=6) harbour significantly more bacteria. Bacterial growth was repeated with the same result in 12 plants over two independent experiments. Red and yellow data points indicate independent repeats of the experiment. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P < 0.001) was revealed by Student's t-test. Expression of RFP-AIMp was verified at 6 dpi with an anti-RFP blot (inset).
Figure 2(A) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. XopL or GFP constructs were co-infiltrated. RLUC and FLUC signals were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities to the mean of control (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (P < 0.01) was shown by Student's t-test. The experiment was repeated more than 3 times by with similar results.(B) Immunoblot analysis of NBR1 and ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL or GFP control at 2dpi verified with an anti-GFP antibody (same blot was split for visualization purpose). Ponceau Staining (PS) served as a loading control. The experiment was repeated at least three times with similar results.(C) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing XopL or GUS control at 2dpi after ConA or DMSO treatment. Expression of GFP-XopL was verified with an anti-GFP antibody, while expression of GUS-HA was confirmed with an anti-HA antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.(D) GFP-ATG8g-labeled autophagosomes were quantified from plants infected with Xcv or Xcv ΔxopL at 2 dpi in the presence or absence of ConA. Puncta were calculated from z-stacks (15) of n=12 individuals using ImageJ. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (** P < 0.01, *** P < 0.001) was determined by one way ANOVA. The experiment was repeated twice with similar results.(E) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ, Xcv ΔxopQ ΔxopL or mock infected N. benthamiana plants at 2dpi. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.(F) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ and Xcv ΔxopQ ΔxopL were co-infiltrated with Agrobacteria containing the respective constructs. RLUC and FLUC activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance comparing Xcv ΔxopQ and Xcv ΔxopQ ΔxopL values (***P < 0.001) was revealed by Student's t-test. The experiment was repeated 3 times with similar results.
Figure 4(A) SH3P2-GFP was transiently coexpressed together with GUS-HA and GFP-XopL in N. benthamiana using agroinfiltration. At 42 hpi, 200 μM MG132 was infiltrated into A. tumefaciens-inoculated leaves, and leaf material was collected 48 hpi. Expression of SH3P2-GFP (lower band) and GFP-XopL (upper band) was detected using an anti-GFP antibody. GUS-HA expression was confirmed with an anti-HA antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.(B) In vitro ubiquitination assay reveals ubiquitination of SH3P2 by XopL. GST-XopL, and MBP-SH3P2 were tested using the Arabidopsis His-AtUBA1 and His-AtUBC8. Lanes 2 to 4 are negative controls. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. Asterisks indicate bands that are not ubiquitinated an present in controls. The experiment was repeated twice with similar results.(C) SH3P2-GFP was transiently coexpressed together with GFP, GFP-XopL and GFP-XopL ΔE3 in N. benthamiana using agroinfiltration. GFP protein levels were detected with an anti-GFP antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.(D) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL, GFP-XopL ΔE3 or GFP control at 2dpi. Expression of binary constructs was verified with an anti-GFP antibody. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.(E) RLUC-ATG8a constructs were coexpressed with internal control FLUC in N. benthamiana. GFP-XopL, GFP-XopL ΔE3 or GFP control were co-infiltrated together with RLUC/FLUC mixture. Renilla and Firefly luciferase activities were simultaneously measured in leaf extracts at 48 hpi using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (* P < 0.5, ** P < 0.01) was revealed by Student's t-test. The experiment was repeated 3 times.
Figure 1C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind epitopes of antigenic site II (Mota and 47F) or antigenic site IV (101F). mAs specific of hMPV F bind epitopes of antigenic site II (MF9, MF12, MF14 and MF15) or antigenic site IV (MF16).
Figure 3Groups of BALB/c female mice (n = 5) were inoculated twice (four weeks apart) i.m. with 10 µg/dose of the proteins indicated at the top of each panel, folded in their respective postfusion conformations. One week after the last dose, mice were sacrificed and their blood collected. A) Each mouse serum (identified by a number) was tested in ELISA for antibody binding to the postfusion F proteins indicated at the bottom. The results represented in the y axis for each individual mouse serum correspond to the inverse dilution that gave 50% of maximal binding, expressed in log10 units (EC50 (log10)). Each number represents an individual mouse. Short horizontal bars indicate mean values for each group. P values were calculated as indicated under Statistical Analysis. Only relevant P values are shown. *P = 0.00045. Differences were considered significant when P < 0.05.Groups of BALB/c female mice (n = 5) were inoculated twice (four weeks apart) i.m. with 10 µg/dose of the proteins indicated at the top of each panel, folded in their respective postfusion conformations. One week after the last dose, mice were sacrificed and their blood collected. B) Mouse sera were also tested in microneutralization assays with hRSV and hMPV, as indicated at the bottom. The results represented in the y axis correspond to the inverse dilution that inhibited 50% of the viral infectivity (IC50 (log10)). P values: *P = 0.01001 and **P = 0.00271. Long horizontal lines in A and B indicate detection limits.
Figure 4Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. A) Mouse sera, collected just before challenging with hRSV (A2 strain), were used in microneutralization assays against the viruses indicated at bottom. Statistical results are shown as in Fig. 3B. *P = 0.00085 and **P = 0.01986.Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. B) Five days after virus inoculation, mice were sacrificed and the amount of virus in lung extracts was measured in a plaque assay. Mean virus titer for each group is indicated by short horizontal bar.
Figure 5C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind to epitopes of antigenic site Ø (D25), antigenic site II (Mz and 47F) or antigenic site IV (101F and 107F). mAs specific of hMPV F bind to epitopes of antigenic site II (MF14), antigenic site IV (MF11, MF16 and MF29) or an epitope shared by hRSV F and hMPV F (MPE8).
Figure 6F. Groups of BALB/c female mice (n = 5) were inoculated twice with 10 µg of the proteins indicated at the top of each panel, folded in either the prefusion conformation for hRSV F, F-410 and F-411 or in the postfusion conformation for hMPV F. One week after the last dose, mice were sacrificed and their blood collected. A) Each mouse serum was tested in ELISA for antibody binding to the F proteins indicated at the bottom of each panel. *P = 0.00046 and **P = 0.02459.Groups of BALB/c female mice (n = 5) were inoculated twice with 10 µg of the proteins indicated at the top of each panel, folded in either the prefusion conformation for hRSV F, F-410 and F-411 or in the postfusion conformation for hMPV F. One week after the last dose, mice were sacrificed and their blood collected. B) Mouse sera were also tested in microneutralization assays with hRSV and hMPV, indicated at the bottom. *P = 0.04138, **P = 0.04831 and ***P = 0.00339. The results in A and B are presented as in Fig. 3.
Figure 1A Northern blots comparing GlmY and GlmZ levels in wild type strain Z8 and the ∆rapZ mutant Z28 under normal growth and GlcN6P starvation conditions. Both strains, which also carried a chromosomal glmS'-lacZ fusion, were treated with 60 μg/ml Nva-FMDP or H2O ("mock"). Samples were harvested hourly for Northern analysis and determination of β-galactosidase activity. Growth curves are shown in Appendix Fig S1. Blots were re-probed using a 5S rRNA specific probe to provide loading controls.B The purification profile of Strep-RapZ from the ∆glmS strain Z904 under GlcN6P replete and depletion conditions is shown (top). The cleared lysate (CL), flow through (FT), washing steps (W) and the elution fractions (E1-3) from StrepTactin affinity chromatography were separated on 12.5 % SDS-PAA gels and stained with Coomassie blue. Metabolites were extracted from E2 and analyzed by HILIC-MS/MS. The extracted ion chromatograms of the LC-MS analysis targeting GlcN6P (retention time 16.6 minutes) are shown below. The samples derived from purification of Strep-RapZ (panels i and ii) or Strep-KdpE (panels iii and iv) were analyzed with the SRM transition m/z 258.1 to m/z 97 in the negative ion mode. The identity of the metabolite detected in panel i was confirmed by adding chemically pure GlcN6P to a final concentration of 100 pg/μl (panel v).C SPR analysis addressing interaction of RapZ variants with GlcN6P and similar metabolites. The Strep-tagged proteins were captured onto a sensor chip and various concentrations of the respective metabolite (i.e. 100, 500, 1000, 2500 and 5000 nM) were injected using a single cycle kinetics approach.