Individual data points are also shown. are successfully and differentially induced. Representative immunoblot image showing specific upregulation of XBP1s (Sec24D) and ATF6 (BiP) protein targets in SupT1DAX cells upon vehicle treatment (basal), dox treatment (+XBP1s), TMP treatment (+ATF6), and co-treatment of dox and TMP (+XBP1s/+ATF6).(TIF) pbio.3001569.s012.tif (138K) GUID:?4A08C695-691A-43F5-A653-22B6F0E3A2F1 S2 Fig: ER proteostasis perturbation has no deleterious effects on cell viability and does not restrict HIV replication. (A) Induction of XBP1s, induction of ATF6, or co-induction of XBP1s and ATF6 did not alter the metabolic activity of SupT1 cells, as measured by a resazurin assay. The average of biological quadruplicates is plotted, with error bars representing the standard deviation. Individual data points are also shown. (B) Induction of XBP1s and co-induction of XBP1s and ATF6 did not restrict, and actually slightly increased, HIV infectious titers, while induction of ATF6 did not influence HIV replication in SupT1 cells, as measured by TZM-bl infectious units. The average of biological triplicates is plotted, with error bars representing the standard deviation. Individual data points are also shown. For (A) and (B), replicate data are provided in S3 Data.(TIF) LRP2 pbio.3001569.s013.tif (141K) GUID:?3C166BE8-66CD-4C20-9EED-AA2204FA966F S3 Fig: Library coverage was generally consistent throughout the Env sequence. The number of codons observed fewer than 3 times after summing the codon counts over the 3 biological replicate libraries is plotted against the amino acid site number. Sites with lower coverage were not localized to any specific domain of structural or functional importance. Data values for library coverage are provided in S4 Data.(TIF) pbio.3001569.s014.tif (185K) GUID:?10941C54-4D25-4AB6-AE2F-8E136C3F797F S4 Fig: Subamplicon sequencing strategy ensures greater accuracy of reads during deep sequencing. The full-length Env gene was divided into Prazosin HCl 9 subamplicons. In the first round of PCR, unique, random barcodes and part of the Illumina adapter were appended to the Env subamplicon molecules. In the second round of PCR, the complexity of the uniquely barcoded subamplicons was controlled to be less than the sequencing depth, and the remainder of the Illumina adapter was appended. The resulting libraries were sequenced on an Illumina HiSeq 2500 in rapid run mode with 2 250-bp paired-end reads.(TIF) pbio.3001569.s015.tif (275K) GUID:?F0FE5B8C-1F2D-492E-895A-1D47A815F48E Prazosin HCl S5 Fig: Env variants with negative diffsel exhibit processing defects. Immunoblots in biological triplicates showing gp160 and gp41 bands for selected variants with (A) negative diffsel and (B) positive diffsel upon XBP1s induction.(TIF) pbio.3001569.s016.tif (873K) GUID:?3D950CD5-D30F-493B-894C-C895CA5B53DE S6 Fig: Transient transfection of Env variants with highly negative diffsel does not induce UPR. RT-PCR analysis of in HEK293T cells expressing GFP (negative control), wild-type Env, and 3 Env variants that were strongly negatively selected in +XBP1s versus basal (C54W, L111P, and L556R). As a positive control for UPR induction, HEK293T cells expressing GFP were treated with thapsigargin (Tg; 2 M) for 6 h (GFP + Tg). RT-PCR data are presented as fold increase relative to GFP-transfected negative control. RT-PCR data values are provided in S7 Data.(TIF) pbio.3001569.s017.tif (88K) GUID:?1EAD0D83-D8D7-467A-9340-0E16D9E4A238 S7 Fig: Sequence logo plots reveal diffsel across Env upon co-induction of XBP1s and ATF6. Logo plot displaying averaged diffsel for +XBP1s/+ATF6 normalized to the basal proteostasis environment. The height of the amino acid abbreviation corresponds to the magnitude of diffsel. The amino acid abbreviations are colored based on their side-chain properties: negatively charged (D, E; red), positively charged (H, K R; blue), polar uncharged (C, S, T; orange/N, Q; purple), small non-polar (A, G; red), aliphatic (I, L, M, P, V; green), and aromatic (F, Prazosin HCl W, Y; dark brown). The quantities and words below the logos suggest the Env site in HXB2 numbering as well as the identity from the wild-type amino acidity for this site, Prazosin HCl respectively. The colour club below the logos signifies the function (F) that.