LAI HIV-1 Env mutant virus library (replicate 2) passaged in SupT1 cells treated with 2 g/mL doxycycline and 10 M trimethoprim (+XBP1s/+ATF6) for 96 h
SupT1DAX cells were seeded in T175 vented tissue culture flasks (Corning) at a density of 100,000,000 cells/flask in RPMI media. The cells were pre-treated with 0.01% DMSO, 2 g/mL dox, 10 M TMP, or 2 g/mL dox and 10 M TMP for 18 h. Pre-treated cells were infected with the mutant viral libraries at an MOI of 0.005 based on the infectious (TZM-bl) titers. In addition, one flask was either mock-infected (negative control) or infected with wild-type virus (to enable error correction for DMS data analysis). To remove unbound virions from culture, 6 h post-infection the cells were pelleted at 2,000 rpm for 5 min, washed twice with 25 mL PBS, and then resuspended in 50 mL of RPMI media treated with 0.01 % DMSO, 2 g/mL dox, 10 M TMP, or 2 g/mL dox and 10 M TMP. Viral supernatant was harvested 96 h post-infection by pelleting cell debris at 2,000 rpm for 5 min. Cell pellets were washed twice with PBS and then resuspended in 1 mL of PBS. Aliquots (100 L) were added to Eppendorf tubes and stored at 80 C for subsequent DNA extraction.  To generate samples for Illumina sequencing, non-integrated viral DNA was purified from aliquots of frozen SupT1DAX cells using a mini-prep kit (Qiagen) and ~10,000,000 cells per prep. PCR amplicons of Env were prepared from plasmid or mini-prepped non-integrated viral DNA by PCR following a previously described protocol [Haddox Plos Pathogens 2016]. The amplicons were sequenced using barcoded-subamplicon sequencing, dividing Env into six sub-amplicons. As previously described, at least 1,000,000 Env molecules were PCR-amplified for preparation of sub-amplicon sequencing libraries to ensure sufficient sampling of viral library diversity [Doud Viruses 2016].