Journal: Cells

Article Title: The ZIKV NS5 Protein Aberrantly Alters the Tubulin Cytoskeleton, Induces the Accumulation of Autophagic p62 and Affects IFN Production: HDAC6 Has Emerged as an Anti-NS5/ZIKV Factor

doi: 10.3390/cells13070598

Figure Lengend Snippet: Characterization of the ZIKV NS5 protein expression pattern. ( a ) Quantitative Western blot analysis of the C-terminal myc-tagged NS5 (NS5-myc) viral protein construct (1.5 µg cDNA) and total α-tubulin protein in a viral packaging HEK-293T cell model compared to those in control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA). The α-tubulin protein was used as the control for total protein loading. A representative of two experiments is shown (see associated data in ). ( b ) Left, Quantitative Western blot analysis of the cell fraction of HEK-293T cells overexpressing NS5-myc (1.5 µg cDNA) and its distribution. Histone 3 was used as the protein control for the nuclear fraction, whereas α-tubulin was used for the cytoplasmic fraction and total protein load. A representative of two experiments is shown (see associated data in ). Right, histograms quantifying the distribution of nuclear and cytoplasmic NS5-myc in lysates from ( b ) experiments, normalized to total α-tubulin. NS5 is mostly found in the nucleus. The data are presented as the means ± S.E.M. of two independent experiments. ( c ) Immunofluorescence microscopy images of HEK-293T cells overexpressing NS5-myc (1.5 µg cDNA) and fixed at 48 h post transfection. The subcellular localization of NS5 was determined using a specific α-myc mAb (green). DAPI is a blue, fluorescent probe for DNA staining. Images were captured using a 100× objective and analysed with the MetaMorph program. NS5 is found in the nucleus and forms three types of structure: uniform punctate shapes ranging from one (type i) to several (type ii) micrometres in diameter, and spheroidal and ring-shaped shapes with longer sizes (type iii). Arrowheads denote the lack of DNA and NS5 colocalization. ( d ) Line scanning of the different structures containing the NS5 viral protein (α-myc mAb (green)) (type i, black lines; type ii, green lines; and type iii, pink lines). ( e ) Quantification of the frequency of the presence of different NS5 structures (type i, black dots; type ii, green dots; and type iii, pink dots) per cell and statistical analysis ( n = 80 cells). ( f ) Average fluorescence intensity plot of NS5 spots in different structures (type i, black bar; type ii, green bar; and type iii, pink bar) per cell ( n = 80 cells). In this figure, when indicated, the p values are *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05; ns indicates not statistically significant ( p > 0.05). The p value is the comparison of the means between the two groups using the parametric Student’s t test.

Article Snippet: The pcDNA TM 3.1(+) empty vector (Life Technologies) was used as a control for cDNA transfection.

Techniques: Expressing, Western Blot, Construct, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Staining, Fluorescence, Comparison

Journal: Cells

Article Title: The ZIKV NS5 Protein Aberrantly Alters the Tubulin Cytoskeleton, Induces the Accumulation of Autophagic p62 and Affects IFN Production: HDAC6 Has Emerged as an Anti-NS5/ZIKV Factor

doi: 10.3390/cells13070598

Figure Lengend Snippet: Characterization of the effect of the ZIKV NS5 protein on microtubule stabilization and reorganization and on the accumulation of the autophagy marker p62. ( a ) Immunofluorescence microscopy analysis of the effect of NS5 on microtubules (MTs) in HEK-293T cells overexpressing NS5-myc (1.5 µg cDNA) compared to that in control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA). In this representative experiment, cells were processed for immunostaining with antibodies against the acetylated Lys 40 residue in α-tubulin (green) and/or NS5 (using an anti-myc mAb) (red). DAPI is a blue, fluorescent probe for DNA staining. Images were captured using a 40× objective and analysed with the MetaMorph program. ( b ) Immunofluorescence microscopy images showing the exacerbated nested structure of acetylated MTs promoted in cells overexpressing NS5 (1.5 µg cDNA) (images captured using a 100× objective). As a control for the acetylation of MTs, cells were treated with tubacin (1 μM), a specific inhibitor of the deacetylase activity of HDAC6. This image (captured using a 40× objective) also serves as a control for the NS5-mediated acetylation of MTs images presented in panel ( a ). In these representative experiments, cells were processed for immunostaining with antibodies against the acetylated Lys 40 residue in α-tubulin (green) and/or NS5 (using an anti-myc mAb) (red). DAPI is a blue, fluorescent probe for DNA staining. Images were captured using a 100× or 40× objective, as indicated, and analysed with the MetaMorph program. ( c ) Quantitative Western blot analysis of the dose–response effect of NS5 overexpression on MT stabilization, as monitored by the acetylation of MTs at Lys 40 α-tubulin residues. The α-tubulin protein was used as the loading control protein. A representative of nine independent experiments is shown (see associated data in ). Histograms quantifying the amount of acetylated α-tubulin induced by NS5 in stabilized MTs from nine replicates of the experiment shown in panel ( c ). The data were normalized to the total α-tubulin protein. ( d ) Western blot analysis of the effect of the NS5 viral protein (1.5 µg cDNA) on the accumulation of the autophagic protein p62 in HEK-293T cells. NS5, p62 and total α-tubulin are shown. Histograms quantifying the amount of p62 stabilized by NS5 from the experiments are shown in panel ( d ). The data were normalized to the total α-tubulin protein. The data are presented as the means ± S.E.M. of three independent experiments (see associated data in ). ( e ) Quantitative Western blot analysis of the dose–response effect of NS5 overexpression on the accumulation of the autophagic protein p62 in HEK-293T cells overexpressing NS5. NS5, p62 and total α-tubulin are shown. The data are presented as the means ± S.E.M. of four independent experiments (see associated data in ). In panels ( c – e ), when indicated, the p values are **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.1; ns indicates not statistically significant ( p > 0.05). The p value is the comparison of the means between the two groups using the parametric Student’s t test.

Article Snippet: The pcDNA TM 3.1(+) empty vector (Life Technologies) was used as a control for cDNA transfection.

Techniques: Marker, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation, Immunostaining, Residue, Staining, Histone Deacetylase Assay, Activity Assay, Western Blot, Over Expression, Comparison

Journal: Cells

Article Title: The ZIKV NS5 Protein Aberrantly Alters the Tubulin Cytoskeleton, Induces the Accumulation of Autophagic p62 and Affects IFN Production: HDAC6 Has Emerged as an Anti-NS5/ZIKV Factor

doi: 10.3390/cells13070598

Figure Lengend Snippet: HDAC6 targets the ZIKV NS5 protein in a deacetylase-dependent manner, regulating the expression of NS5 in the cytoplasm. ( a ) Quantitative Western blot analysis of HEK-293T cells overexpressing NS5-myc (lane 2), N-terminal HA-tagged wt-HDAC6 (HA-wt-HDAC6) (lane 3) or both constructs (lane 4) (1.5 µg cDNA) and comparison to control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA) (lane 1). The negative effects of HA-wt-HDAC6 on NS5-myc expression (lane 4), the stabilization of acetylated MTs by NS5 (lane 2) and the deacetylase activity of HDAC6 on acetylated MTs (lanes 3 and 4) are shown. The α-tubulin protein was used as the loading control protein. A representative experiment of four is shown (see associated data in ). Histograms quantifying the amount of NS5-myc expressed in the absence (corresponding to lane 2 in the left panel) or presence of overexpressed HDAC6 (corresponding to lane 4 in the left panel) normalized to total α-tubulin. The data are presented as the means ± S.E.M. of four independent experiments. ( b ) Immunofluorescence images showing NS5 labelling 48 h post transfection in cells cotransfected with HDAC6-DsRed (red), fixed and labelled with an α-myc antibody for NS5 detection (green). NS5 was detected only in cells not overexpressing wt-HDAC6-DsRed (white asterisks). The white dotted lanes indicate the cell perimeters of three cells (1–3) that expressed wt-HDAC6-DsRed in the field. DAPI is a blue, fluorescent probe for DNA staining. Images were captured using a 100× objective and analysed with the MetaMorph program. ( c ) Immunofluorescence microscopy analysis of NS5 cell distribution in cells treated or not treated (control) with tubacin (1 μM), a deacetylase inhibitor of HDAC6, in cells overexpressing NS5-myc (1.5 µg cDNA). The cells were processed for immunostaining with an anti-myc mAb (green). DAPI is a blue, fluorescent probe for DNA staining. Images were captured using a 100× objective and analysed with the MetaMorph program. ( d ) Quantitative Western blot analysis of the effect of functional (HA-wt-HDAC6) or deacetylase inactive (HA-dm-HDAC6) HDAC6 on the stability of the ZIKV NS5 protein (NS5-myc) in HEK-293T cells overexpressing the different constructs (1.5 µg cDNA per construct) and comparison with cells overexpressing either only NS5 or only HA-wt-HDAC6 or HA-dm-HDAC6. The levels of acetylated α-tubulin and total α-tubulin under the different experimental conditions are shown. A representative experiment of two is shown (see associated data in ). Histograms quantifying the amount of NS5 detected in cells from the experiment ( d ). The data are presented as the means ± S.E.M. of two independent experiments. For the quantitative analysis of the Western blots, when indicated, the p values are *** p ≤ 0.001 and ** p ≤ 0.01. The p value is the comparison of the means between the two groups using the parametric Student’s t test.

Article Snippet: The pcDNA TM 3.1(+) empty vector (Life Technologies) was used as a control for cDNA transfection.

Techniques: Histone Deacetylase Assay, Expressing, Western Blot, Construct, Comparison, Transfection, Plasmid Preparation, Activity Assay, Immunofluorescence, Staining, Microscopy, Immunostaining, Functional Assay

Journal: Cells

Article Title: The ZIKV NS5 Protein Aberrantly Alters the Tubulin Cytoskeleton, Induces the Accumulation of Autophagic p62 and Affects IFN Production: HDAC6 Has Emerged as an Anti-NS5/ZIKV Factor

doi: 10.3390/cells13070598

Figure Lengend Snippet: HDAC6 promotes the autophagic clearance of the ZIKV NS5 protein. ( a ) Quantitative Western blot analysis of HEK-293T cells overexpressing NS5-myc (lane 2) or the N-terminal HA-tagged HDAC6 mutant lacking the C-terminal BUZ domain (HA-HDAC6-DBUZ) (lane 3) or both constructs (lane 4) (1.5 µg cDNA) and comparison to control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA) (lane 1). The absence of a degradative effect of HA-HDAC6-DBUZ on NS5-myc expression (lane 4) and the deacetylation of MTs by HA-HDAC6-DBUZ, which is deacetylase active (lanes 3 and 4) are shown. NS5 could not stabilize acetylated MTs under these experimental conditions (lane 4). The α-tubulin protein was used as the loading control protein. A representative experiment of three is shown (see associated data in ). Left histograms quantifying the amount of NS5-myc expressed in the absence (corresponding to lane 2 in the western blot panel) or presence of overexpressed HA-HDAC6-DBUZ (corresponding to lane 4 in the western blot panel), normalized to total α-tubulin. Right histograms quantifying the amount of p62 protein expressed in cells overexpressing NS5-myc, HA-HDAC6-DBUZ or both constructs (corresponding to lanes 2–4 in the western blot panel, respectively) and compared with that in control cells (corresponding to lane 1 in the western blot panel). These data are presented as the means ± S.E.M. of three independent experiments. ( b ) Immunofluorescence microscopy analysis of NS5 cell distribution in cells treated or not treated (control) with 3-MA (5 mM), an inhibitor of HDAC6-mediated aggresome formation and further autophagy clearance of targeted proteins, in cells overexpressing NS5-myc (1.5 µg cDNA). The cells were processed for immunostaining with an anti-myc mAb (green). DAPI is a blue, fluorescent probe for DNA staining. Images were captured using a 100x objective and analysed with the MetaMorph program. ( c ) Quantitative Western blot analysis of HEK-293T cells overexpressing NS5-myc (lane 2), the functional HA-HDAC6 construct (lane 3) or both constructs (lane 4) (1.5 µg cDNA), compared to control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA) (lane 1) under control conditions (PBS-treated cells, vehicle for 3-MA) or cells treated with 3-MA (5 mM). The absence of a degradative effect exerted by HA-HDAC6 on NS5 in the presence of 3-MA (lane 4 in 3-MA-treated cells compared to lane 4 in PBS-treated cells) and the deacetylation of MTs by HA-HDAC6 are shown. The α-tubulin protein was used as the loading control protein. A representative experiment of 2 is shown (see associated data in ). Histograms quantifying the amount of NS5-myc expressed in the absence (lane 2) or presence of overexpressed HA-HDAC6 (lane 4), normalized to total α-tubulin, in both PBS- and 3-MA-treated cells are shown. The data are presented as the means ± S.E.M. of 2 independent experiments. ( d ) Quantitative Western blot analysis of the effect of siRNA-mediated knockdown of endogenous HDAC6 (siRNA-HDAC6, 25 pmol/well) on the level of NS5 protein expression in HEK-293T cells overexpressing NS5-myc and compared to that in control and scrambled (1 µM)-treated cells. A representative experiment of four is shown (see associated data in ). Histograms quantifying the amount of endogenous HDAC6 and NS5 detected in cells treated with siRNA-HDAC6 or scrambled oligos, normalized to total α-tubulin. The data are presented as the means ± S.E.M. of four independent experiments. For quantitative analysis of Western blots, when indicated, the p values are **** p ≤ 0.0001 and * p ≤ 0.1; ns indicates not statistically significant ( p > 0.05). The p value is the comparison of the means between the two groups using the parametric Student’s t test.

Article Snippet: The pcDNA TM 3.1(+) empty vector (Life Technologies) was used as a control for cDNA transfection.

Techniques: Western Blot, Mutagenesis, Construct, Comparison, Transfection, Plasmid Preparation, Expressing, Histone Deacetylase Assay, Immunofluorescence, Microscopy, Immunostaining, Staining, Functional Assay

Journal: Cells

Article Title: The ZIKV NS5 Protein Aberrantly Alters the Tubulin Cytoskeleton, Induces the Accumulation of Autophagic p62 and Affects IFN Production: HDAC6 Has Emerged as an Anti-NS5/ZIKV Factor

doi: 10.3390/cells13070598

Figure Lengend Snippet: The ZIKV NS5 protein inhibits RNA-triggered RIG-I-mediated IFN production, and this effect is enhanced by autophagy inhibition. ( a ) Quantitative assay of the inhibitory effect of NS5 on RNA-triggered RIG-I-mediated IFN production in HEK-Lucia TM RIG-I cells expressing NS5 (1.5 µg cDNA) and treated with 3p-hpRNA (100 nM), an RNA-specific RIG-I agonist, for 24 h before the IFN-I response was quantified by QUANTI-Luc. The data obtained from control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA) and treated with 3p-hpRNA (100 nM) are shown. The effect of 3-MA (5 mM) on the NS5-mediated inhibition of 3p-hpRNA-triggered RIG-I-mediated IFN production is shown. The data are presented as the means ± S.E.M. of two independent experiments ( n = 6). ( b ) Quantitative Western blot analysis of the effect of 3-MA treatment on HEK-Lucia cells overexpressing NS5-myc (1.5 µg cDNA). This biochemical analysis corresponds to the cells shown in panel ( a ) for RNA-mediated RIG-I IFN production. Lanes 1 and 2 represent control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA) and treated with PBS (vehicle for 3-MA) or 3-MA (5 mM), respectively. Lanes 3 and 4 represent cells overexpressing NS5 (NS5-HA plasmid, 1.5 µg cDNA) and treated with PBS or 3-MA (5 mM), respectively. The stabilization effect of 3-MA on NS5 (lane 4 in 3-MA-treated cells compared to lane 3 in PBS-treated cells) and the NS5-mediated acetylation of MTs are shown. The α-tubulin protein was used as the loading control protein. A representative experiment of two is shown (see associated data in ). Histograms quantifying the amount of NS5-HA expressed, normalized to total α-tubulin, in both control (PBS)- and 3-MA-treated cells from the top Western blot experiments. The data are presented as the means ± S.E.M. of two independent experiments. In ( a ), the p values are *** p ≤ 0.001 and * p ≤ 0.1, and in ( b ), the p value is 0.15. The p value is the comparison of the means between the two groups using the parametric Student’s t test.

Article Snippet: The pcDNA TM 3.1(+) empty vector (Life Technologies) was used as a control for cDNA transfection.

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Western Blot, Comparison