Time course of DENV NS5 glutathionylation in the virus infected cell.

<p>HEK293T/17 cells were mock infected and infected with DENV 2 for 2 h. The infected cells were harvested at 0, 2 and 6 hour post infection (hpi). The immunoprecipitates with DENV NS5 protein antibody were run on non-reduced SDS gel and western blot was performed using anti-GSH antibody.</p

Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities

<div><p>It has been estimated for dengue infection that the global population at risk is 3.5 billion people, which makes dengue an important public health problem. The causative agents of dengue are dengue viruses. For dengue virus replication, the dengue virus NS5 protein is of special importance as it has several enzyme activities important for viral replication. Previous reports of phosphorylation and SUMOylation of dengue NS5 have shown these protein modifications have important consequences for NS5 functions. In this report we identify glutathionylation, another reversible post translation modification that impacts on NS5 enzyme activity. Using dengue virus infected cells we employed specific antibodies and mass spectrometry to identify 3 cysteine residues of NS5 protein as being glutathionylated. Glutathionylation is a post translational protein modification where glutathione is covalently attached to a cysteine residue. We showed glutathionylation occurs on 3 conserved cysteine residues of dengue NS5. Then we generated two flavivirus recombinant full length proteins, dengue NS5 and Zika NS5, to characterize two of the NS5 enzyme activities, namely, guanylyltransferase and RNA-dependent RNA polymerase activities. We show glutathionylation of dengue and Zika NS5 affects enzyme activities of the two flavivirus proteins. The data suggests that glutathionylation is a general feature of the flavivirus NS5 protein and the modification has the potential to modulate several of the NS5 enzyme functions.</p></div

Amino acid alignment of flavivirus NS5 proteins.

<p>The sequences are nonstructural protein NS5 from Dengue virus 2 NP_739590.2, Dengue virus 1 NP_722465.1, Dengue virus 3 YP_001531176.2, Dengue virus 4 NP_740325.1, Zika virus AMR39834.1, Japanese encephalitis virus NP_775674.1, West Nile virus YP_001527887.1, Tick-borne encephalitis virus NP_775511.1, Yellow fever virus NP_776009.1, Murray Valley encephalitis virus NP_722539.1, Langat virus NP_740302.1. Cysteines are highlighted in yellow with the positions of the 3 glutathionylated cysteines identified in this study shown in green highlight in the alignment. Blue bars illustrate the residues of the finger subdomains, the red bars show the residues of the palm domain and the purple bars show the residues of the thumb domain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193133#pone.0193133.ref030" target="_blank">30</a>]. Conservation of amino acid sequence is shown by 100 percent conserved as white on black, between 80 to 100 percent conserved as white on dark grey, between 60 to 80 percent conserved as black on light grey, and less than 60 percent conserved as black on white.</p

Immunoprecipitation result of the second method.

<p>The second method was to immunoprecipitate the dengue protein in cell lysate with specific dengue protein antibody and protein A/G beads and determine that the dengue protein was glutathionylated as shown by western blot detection with an anti-GSH antibody. The quantity of NS5 is not detectable in whole lysate. Lane 1 is DENV-infected cell lysate. Lane 2 is IP sample of NS5.</p

Glutathionylation effects on dengue and Zika NS5 guanylyltransferase activity.

<p>A) DENV and Zika NS5 protein were not treated and pre-treated with 5 mM GSSG for 10 min at RT. These proteins then were used to perform the guanylyltransferase activity assay. The samples were resolved on 10% SDS-PAGE. The extent of enzyme guanylylation activity was measured by GTP-Cy5 signal tracking using Azure^™ cSeries. Gels were then stained with Coomassie Blue to normalize for protein loading. The bottom panel shows equivalent aliquots of the same samples used in a parallel gel that was transferred for Western blot and detection by anti-GSH antibody. B) The ratio of quantified guanylyltransferase bands to protein load of panel A is shown as a bar graph.</p

Plots of V versus S for dengue and Zika NS5 RdRp activity before and after glutathionylation with the kinetic parameter values generated by non-linear regression of this secondary plot.

<p>On the dengue plot the 95% confidence limits for the curves are shown by dotted lines. The plots were generated by GraphPad Prism version 5.04. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193133#pone.0193133.s004" target="_blank">S4 Fig</a> for an example of the primary plot.</p

Correlation between the percentages of lectin-positive cells and the viral titers.

<p>Dot plots of percentages of lectin-positive cells versus maximum viral titers produced from the same tissue samples show linear correlation with Pearson correlation coefficient of 0.889 for SNA (p = 0.003) and 0.859 for MAA I (p = 0.006). The data were derived from the same experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012973#pone-0012973-g001" target="_blank">Figure 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012973#pone-0012973-g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012973#pone-0012973-g004" target="_blank">4</a>.</p

Distribution of receptor and infection of nasal polyps and adjacent normal mucosa.

<p>Distribution of sialic acid receptors (a) and outputs of viral infection (b) of tissue samples from nasal polyps and adjacent normal mucosa from the same patients. The data were derived from experiments using nasal polyps and adjacent normal nasal mucosal tissue samples from two patients.</p

Representative micrographs of nasal polyp and nasal turbinate mucosa showing distribution of α2,3- and α2,6-sialic acid.

<p>Tissue sections were stained with FITC conjugated lectin MAA I (left panel) or SNA (right panel), specific toward α2,3- or α2,6-linked sialic acid, respectively (a). To confirm the specificity and the presence of α2,3-linked sialic acid on nasal polyp, tissues were digested with 1U/ml of sialidase before MAA I staining. The sialidase-treated tissue (right) lost the staining signal on the apical surface, while non-treated section was positive (left) (b).</p

In situ hybridization of H5N1 AIV-infected (upper) and non-infected (lower) polyp tissues.

<p>The hybridization signal is shown in red-brown color in the epithelial cells on the mucosal surface of infected tissue.</p