The C-terminal amino acids of the OmpB orthologs and putative analogs show porin characters.

<p>Position 1 indicates the C-terminal amino acid. Hydrophobic residues are boldface. All porins shown possess the essential terminal phenylalanine and most have hydrophobic residues at positions 3, 5, 7 and 9. ORFs above the line are homologs of TM0476, those below the line are possible analogs of TM0476.</p

Characteristics of <i>T. maritima</i> OmpB orthologs and putative syntenic analogs in Thermotogales species.

<p>Characteristics are defined in the text. <b>Legend:</b> +, protein is predicted to possesses the attribute; –, protein is predicted to not possess the attribute.</p><p>ORFs are from TM, T. maritima; TRQ2, Thermotoga species strain RQ2; Tpet, Thermotoga petrophila; Tnap, Thermotoga naphthophila; CTN, Thermotoga neapolitana; Tmel, Thermotoga melanesiensis; Fnod, Fervidobacterium nodosum; Tlet, Thermotoga lettingae; THA, Thermosipho africanus; Kole, Kosmotoga olearia; Pmob, Petrotoga mobilis; and Theba, Thermotogales bacterium mesG1.Ag.4.2 (Mesotoga prima). ORFs above the line are homologs of TM0476, those below the line are possible analogs of TM0476.</p>‡<p>Values of the percent β strand content were calculated utilizing the multisequence alignment programs STRAP and SCRATCH, respectively.</p

The C-terminal amino acids of TM0476 are like those of several confirmed porins.

<p>Position 1 indicates the C-terminal amino acid. Hydrophobic residues are boldface. All porins shown possess the essential terminal phenylalanine and hydrophobic residues at positions 3, 5, 7 and 9.</p

Maximum likelihood phylogenetic tree of OmpA protein sequences from several Thermotogales species.

<p>Red labels indicate likely <i>T. maritima</i> OmpA1 (TM0477) orthologs, green labels indicate likely OmpA2 (TM1729) orthologs, and homologs whose type of homology cannot be ascertained are labeled in black. The tree was calculated as unrooted phylogeny, but is depicted as rooted between the likely OmpA1 and OmpA2 clusters. Branches with approximate Likelihood Ratio Test (aLRT) support values ≤0.75 were collapsed. aLRT, posterior probability, and bootstrap support values are given above or below the branch to which they pertain. Organism abbreviations and gene identification numbers for loci are <i>Petrotoga mobilis</i> (Pmob_0057, 160901548 and Pmob_1624, 160903060); <i>T. lettingae</i> (Tlet_1719, 157364570 and Tlet_0301, 157363168); <i>T. petrophila</i> (Tpet_1024, 148270158 and Tpet_0443, 148269583); <i>T. maritima</i> (TM0477, 15643243 and TM1729, 15644475); <i>T. napthophila</i> (Tnap_1078, 281412500 and Tnap_0259, 281411698) <i>T. neapolitana</i> (CTN_0195, 222099169 and CTN_0927, 222099901); <i>Thermotoga</i>. sp. strain RQ2 (TRQ2_0458, 170288259 and TRQ2_1096, 170288887); <i>Fervidobacterium nodosum</i> (Fnod_1724, 154250391 and Fnod_0047, 154248750); <i>Kosmotoga olearia</i> (Kole_0210, 239616617 and Kole_1500, 239617873); <i>Thermosipho africanus</i> (THA_407, 217076525 and THA_93, 217076226); <i>Thermosipho melanesiensis</i> (Tmel_0176, 150020084 and Tmel_1771, 150021641); Thermotogales bacterium mesG.Ag.4 (<i>Mesotoga prima</i>) (ThebaDRAFT_0522, 307297745; now Theba_0318).</p

Syntenic regions containing <i>T. maritima</i> OmpA1 and OmpB homologs and putative analogs mapped onto an rRNA gene reference tree.

<p>The coloring of the individual genes indicates whether the gene is a homolog, paralog, or putative analog. <i>segG</i> homologs are yellow, <i>tyrS</i> homologs are light blue, <i>ompA1</i> homologues are red, and <i>ompB</i> homologues are dark blue. <i>ompA</i> paralogs are white (see Fig. 2). <i>ompB</i> putative analogs are grey. The tree is a concatenated 23S-16S rRNA gene cladogram. Branch lengths do not reflect the extent of divergence.</p

Whole cell lipidomics reveal an altered lipid composition in DENV infected cells.

<p>Panel A and B represent an average expression (fold change) of the total number of individual lipids significantly expressed (p<0.05) per lipid class at 36 and 60 hr post-infection, respectively. The fold changes represent DENV-infected cells or UV-DENV exposed cells compared to the mock control. A lack of cones indicates that the expression level of those specific lipids were not significant (p<0.05). Panels C–H are representative lipid molecular species from specific lipid classes significantly regulated at the two different time points. The data are plotted as the integrated LC-MS peak abundance, in log 2 scale with standard deviation. PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; CER, ceramide; CER-PE, ceramide phosphoethanolamine; Lyso, lysophospholipids. See supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584.s004" target="_blank">table S1</a> for a complete list of lipid features detected in this study. Four replicates were included in the lipidomic analyses. The error bars represent standard deviation of the mean. The blue dashed line separates species that remain elevated at both time points (36 and 60 hr) from species that are only elevated at the 36 hr time point. Infections were carried out using an MOI of 20 in C6/36 cells. Significantly expressed lipids species are shown denoted with an asterisk (*).</p

The Lipid repertoire of DENV infected mosquito cells is unfavorable for replication in the presence of C75.

<p>The specific lipid classes differentially regulated by C75 treatment of cells are shown. Panel A represents the fold changes of lipid classes expressed in DENV infected cells (MOI 3) compared to C75 treated DENV infected cells. Panels B–D show fold changes for individual molecular species within each lipid class that are regulated. PI, phosphatidylinositol; PG, phosphatidylglycerol; MG, monoacylglycerol; DG, diacylglycerol; PA, phosphatidic acid. See supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584.s006" target="_blank">table S3</a> for a complete list of lipid features detected in this analysis and supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat-1002584-g002" target="_blank">figure 2</a> for a heat map representation of the data. Three replicates were included in the lipidomic analyses.</p

Bioactive sphingolipid species are differentially regulated in replication complex membranes isolated from DENV-infected mosquito cells.

<p>Multiple Reaction Monitoring (MRM) analysis of sphingolipids species differentially regulated in DENV-infected cells (MOI 20) or UV-DENV exposed cells compared to the mock control (see also supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584.s006" target="_blank">table S3</a>). The data represent fold changes observed in three subcellular fractions that were analyzed in this study; 16K, replication complex membranes; CE, cytoplasmic extracts following removal of replication complex membranes and nuclei; N, nuclear fraction. Panels A–C represent ceramide, sphingomyelin and monohexosylceramide species respectively. The dashed line highlights values equal to the mock. The data represent three independent experiments. The error bars represent standard deviation of the mean.</p

Newly synthesized lipids and viral RNA in subcellular fractions show a dynamic distribution with time of infection.

<p><b>A.</b> A pulse-chase analysis of ¹⁴C-acetate incorporation into newly synthesized lipids. The results show total labeled lipid in post-nuclear supernatants of C6/36 cells infected with DENV for 36 and 60 hr at an MOI of 20. <b>B.</b> The same pulse-chase analysis showing ¹⁴C-acetate incorporation into newly synthesized lipids in subcellular fractions (16K and CE) at 36 and 60 hr post-infection. <b>C.</b> The ratio of viral RNA genome copies per labeled lipid in subcellular fractions (described in B) at 36 and 60 hr post-infection. 16K, membrane fraction (pellet) following centrifugation of post-nuclear supernatants at 16, 000× g. CE, cytoplasmic extract following centrifugation of post-nuclear supernatants at 16,000× g. cpm, counts per minute.</p

Dengue virus infection perturbs lipid homestasis in infected mosquito cells.

<p>The lipidomic analyses of dengue virus infected C6/36 mosquito cells suggest several metabolic pathways that may be significantly up regulated during infection. The grey dashed line highlights specific pathways of interest. Black arrows highlight reactions suggested by the lipidomic data and grey arrows represent reactions not observed in the data. Metabolites highlighted in boxes (solid line) are up regulated (white) or down regulated (grey) in DENV infected mosquito cells. <b>1.</b> Through the recruitment and activation of FAS, DENV stimulates <i>de novo</i> phospholipid biosynthesis in the replication complex <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584-Heaton1" target="_blank">[20]</a>. <b>2.</b> Inhibition of this process with C75 disrupts the cellular lipid repertoire in mosquito cells to be unfavorable for virus replication. <b>3.</b> The lipidomic analyses reveal an up regulation of fatty acids such as palmitic (C16) and stearic (C18) acid. These fatty acids are intermediates in the biosynthesis of phospholipids, which is up regulated during DENV infection. Interestingly, in DENV infected cells the prevalent phospholipids primarily consist of C16 and C18 unsaturated acyl chains. Very long chain fatty acids are not significantly up regulated during infection. <b>4.</b> FAS activity also stimulates <i>de novo</i> sphingolipid biosynthesis. In the lipidomic analyses, the up regulation of intermediates such as N-palmitoylesphingosine suggests sphingolipid biosynthesis is activated during DENV infection. Specifically, SM and CER are enriched in DENV infected cells. Alternately, the up regulation in CER (and DG) during infection could result from the degradation of SM through the activity of sphingomyelinases (Smase). The resulting CER and DG could be redirected into several signaling pathways or be utilized for <i>de novo</i> phospholipid biosynthesis. The glycopshingolipids, GlcCER and GalCER are down regulated during DENV infection, which suggest that they are catabolized to produce CER. <b>5.</b> Lipidomic analyses also suggest the up regulation of triacylglycerol catabolism (Lipolysis) in DENV infected cells. This pathway results in the generation of MG, DG and palmitic acid. These intermediates are all up regulated in DENV infected cells and could be utilized for downstream signaling or <i>de novo</i> phospholipid biosynthesis. It has also been shown that TG catabolism is necessary for mitochondrial β-oxidation during DENV infection <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584-Zaitseva1" target="_blank">[50]</a>. <b>6.</b> Elevated levels of LPC in DENV infected cells also suggest activation of PC hydrolysis by PLA₂. This enzyme is activated during DENV infection. The elevated levels of other phospholipids such as PA, PI, PE, PG as well as PC suggest that the CDG-DG pathway for phospholipid biosynthesis could also be activated. FAS, fatty acid synthase; DENV, dengue virus; C75, inhibitor of FAS; SM, sphingomyelin; CER, ceramide; MG, monoacylglycerol; DG, diacylglycerol; TG, triacylglycerol; LPC, lysophosphatidylcholine; PLA₂, phospholipase A₂; PA, phosphatidic acid; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PC, phosphatidylcholine.</p