Hydrolytic activity of PTE<i>_Ar</i>.

<p>A) Schematic showing the PTE<i>_Ar</i>-mediated hydrolysis of malathion. B) Structure of the OP insecticides demeton, chlorpyrifos, parathion and diazinon.</p

Brønsted plot of leaving group pKa values <i>vs</i> log(<i>k</i>_cat/<i>K</i>_M) for a range of substates.

<p>The p<i>K</i>_a values of the leaving groups 2,6-difluoro-4-nitrophenol; quinoxalin-2-ol; 2-fluoro-4-nitrophenol; 2-isopropyl-6-methylpyrimidin-4-ol; 3-fluoro-4-nitrophenol; 4-nitrophenol; 4-hydroxybenzaldehyde; 2,2-dichloroethylenol; 4-hydroxybenzonitrile; 1-(4-hydroxyphenol)ethanone; methyl 4-hydroxybenzoate; 4-hydroxybezamide; 3-chloro-7-hydroxy-4-methyl-2H-chromen-2-one; 2-(ethylthio)ethanethiol; 2-(diethylamino)ethanethiol; 2-(diisopropylamino)ethanethiol; and 4-(methoxymethyl)phenol plotted (left to right) against their log(<i>k</i>_cat/<i>K</i>_M) values. p<i>K</i>_a values were as published elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Caldwell1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Jackson4" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Jackson7" target="_blank">[20]</a> or as calculated using the SPARC online p<i>K</i>_a calculator (<a href="http://ibmlc2.chem.uga.edu/sparc/" target="_blank">http://ibmlc2.chem.uga.edu/sparc/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Hilal1" target="_blank">[38]</a>. The biphasic dependence of the enzyme on p<i>K</i>_a as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Caldwell1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094177#pone.0094177-Jackson7" target="_blank">[20]</a> is shown: the curve flattens below a p<i>K</i>_a of ∼8.0 and there is a linear dependence on p<i>K</i>_a at values below <i>ca</i>. 8.0.</p

Kinetic parameters of purified PTE<i>_Ar</i> and most active variant against malathion for a range of OP insecticides.

<p>Standard deviations for the <i>k</i>_cat and <i>K</i>_M values are given in parentheses below the mean values obtained for triplicate experiments.</p

Data collection and refinement statistics for structures reported in this work.

<p>^*Values in parenthesis are for the highest-resolution shell.</p>†<p><i>R</i>_merge(I)  =  (Σ<i>_hkl</i> Σ<i>_j</i> |<i>I_hkl,j−</i>〈<i>I_hkl</i>〉|)/(Σ<i>_hkl</i> Σ<i>_j I_hkl,j</i>) where 〈<i>I_hkl</i>〉 is the average intensity of <i>j</i> symmetry-related observations of reflections with Miller indices <i>hkl</i>.</p>#<p>CC_1/2  =  percentage of correlation between intensities from random half-datasets.</p>‡<p><i>R</i>_work  =  Σ<i>_hkl</i>|F_(obs)−F_(calc)|/Σ<i>_hkl</i>|F_(obs)|; 5% of the data that were excluded from the refinement were used to calculate <i>R</i>_free.</p

Amino acid frequencies at sites that exhibit amino acid changes between sequenced TF/CC pairs are shown and compared to HxB2 (reference sequence).

(PDF)</p

Transmitted HIV-1 of the CH040 virus pair is more resistant to IFN and AZT than the matched chronic virus.

(A-B) TMZR5 cells were treated with indicated doses of IFNα14 for 24 hours before being challenged with the CH040 TF and CC viruses. Cells were sampled daily to monitor virus spread and GFP-positive cells were enumerated via flow cytometry. (C-D) TMZR5 cells were pre-treated with a range of azidothymidine (AZT) doses for 2 hours prior to infection with the CH040 TF or CC viruses. Cells were sampled daily to monitor virus spread and GFP-positive cells were quantified via flow cytometry.</p

Statistical modelling of virus growth curves indicates that CH058 CC has a lower growth rate than its TF progenitor but is not otherwise more sensitive to IFN.

(A) TMZR5 cells were pre-stimulated with different doses of IFNα14 for 24 hours prior to infection with CH058 TF or CC virus and sampled daily to monitor virus spread. GFP-positive cells were enumerated using flow cytometry. Blue and orange points show means (+/- standard error) across 4 experimental replicates, while translucent points show individual observations. Viral spreading replication experiments took place on two occasions, a typical result is shown. (B) Model fit for the differential sensitivity model, in which viruses are allowed to vary in both baseline growth rate (i.e., growth rate in the absence of interferon) and in their sensitivity to interferon. (C) Model fit for the constant sensitivity model, in which viruses differ only in their baseline growth rate. In both B and C, opaque points show predictions from the fitted model, along with 95% confidence intervals. Translucent points and lines underneath show the observed (experimental) replicate growth curves also present in A. For each treatment, only the initial timepoints until at least one replicate curve decreased by >30% relative to its preceding observation were used in model fitting (i.e. points after HIV-1 had overwhelmed the culture were not considered). (D) Effect sizes for both differential and constant sensitivity models (maximum likelihood estimates and 95% confidence intervals). Model fit was compared by likelihood ratio test and AIC, with results shown in the legend. (E) Illustration of effects making up the fitted growth rates in the differential sensitivity model. Effects were modelled as additive, allowing us to separate discrete contributions to the growth rates needed to recapitulate the in vitro data as illustrated in B. Points show maximum likelihood estimates, while error bars show 95% confidence intervals. An inset in the final panel (right) illustrates expected patterns under different hypothesized scenarios; in particular, if the CC virus was more sensitive to IFN, its achieved growth rate would have diverged from that of the TF virus at increasing IFN doses.</p

S2 Fig -

Western blot gels assessing protein expression levels and IFN induction of expression of (A) CD80, (B) FNDC3B, (C) MICB (D) TMEM140, (E) CD38, (F) SCARB2. (G) TMZR5 cells (modified to express CD38 and SCARB2) were challenged with NHG and sampled daily to monitor virus spread. GFP-positive cells were enumerated via flow cytometry. (H) Western blots of the seven CRISPR guides and non-targeting control guide cell lines for CD38 in PM1 cells. (I) PM1 cell lines were pre-treated for 24 hours with the IFNα14 doses indicated and were subsequently challenged with NHG and sampled daily to monitor virus spread. (J) Western blots of the seven CRISPR guides and non-targeting control guide cell lines for SCARB2 in TMZR5 cells. (K) TMZR5 cell lines were pre-treated for 24 hours with the IFNα14 doses indicated and were subsequently challenged with NHG and sampled daily to monitor virus spread. Viral spreading replication experiments took place on two occasions, a typical result is shown. The white line in A indicates that the CD80 image was flipped for this blot to correct sample order, but the blot is the same for both portions of this image. Raw western blot images can be viewed in S7 Fig. (PDF)</p

Anti-HIV-1 ISGs inhibit the CC virus more potently than transmitted HIV-1.

(A) Candidate anti-HIV-1 effectors that were more inhibitory than the known anti-HIV-1 effector IFITM3 were tested against CH058 GIN GFP-IRES-Nef) TF and CC viruses on MT4-R5 cells, as in 2F-G, normalised to the level of infection observed in the presence of an empty vector control. (B-C) ISGs in Fig 3A were tested for ability to induce cell death or ISRE (IFN-stimulated response element). In B, MT4 and TMZR5 cells were tested for cell death when expressing ISGs by flow cytometry using the LIVE/DEAD fixable dead cell stain kit (Invitrogen) and MT4 cell viability was additionally tested using the luminescence based CytoTox-Glo Cytotoxicity assay (Promega). In C, IFN induction by candidate ISGs was measured by flow cytometry using MT4 cells expressing an ISRE-GFP construct. (D) Candidate ISGs from Fig 3A were checked for their changes in expression upon type I IFN stimulation using the Interferome database to determine their ‘ISG-ness’. (E-F) Validation of the 8 most potent anti-HIV-1 effector ISGs, along with IFITM1 and IFITM2 controls, against CH058 TF and CC viruses, In E, TMZR5 cells transduced with pLV constructs containing the indicated ISGs or RFP as a control, were challenged with CH058 TF or CC and sampled daily to monitor virus spread. GFP-positive cells were enumerated using flow cytometry. Viral spreading replication experiments took place on two occasions, a typical result with contemporaneous controls is shown. In F, data from panel E represented as area under the curve (AUC).</p

Sequencing coverage statistics.

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