Manuscript No. 16-05-2022-RA-eLife-80328  

EC50 values were determined for a subset of the selected virus cultures against a panel of protease inhibitors.  The highest level of resistance recorded was 100 µM.  Using a novel method described in the methods, these values were then pooled using several different parameters as described in the figure legends.  EC50 inhibition values are shown in Figure 1 and 5.  The Mann-Whitney rank-sum test was used to assess differences in EC50 values.    Around 10% of the curves had one point where the value was >125% infectivity of the first point.  Anomalous values were excluded from curve fitting.  4 curves did not record data.

PI dilutions were prepared by taking 10 µM stocked and performing a 5-fold serial dilution using RPMI media (final drug concentration is 100 µM). One dilution of drug was added to each well of a 24 well plate and repeated so each virus would have a full set of dilutions. Viruses for the assay were made by seeding 3 x 106 CEM cells in a 24 well plate and incubating with 250 µl of virus at 37°C for 2 to 3 h before bringing the culture to 10ml with RPMI media. After 48 h the medium was changed and repeated every 48 h after until the culture had undergone CPE. Infected CEM cells were collected and diluted so that 1ml of cells could be plated in each well containing a unique drug dilution. Then 24 hours later the virus supernatant was collected from each well followed by filtering through a 0.45 µM filter then placed in -80°C. Viruses were thawed and added to 96 well plates in triplicate. TZM-bl cells were collected and diluted to a concentration of 2x105 cells/ml, 100 µl were added on top of the pre-plated viruses. Plates were kept in 37°C, 5% CO2 in an incubator for 48 hours. After 48 hours, the cells in the plates were lysed by removing the medium, washing two times with 100 µl PBS and then lysed with 1x lysis buffer (made from 5x Promega Firefly Lysis Buffer). Plates were frozen for at least 24 hours and then thawed for 2 hours before analyzing with Promega Firefly Luciferase Kit on a luminometer.  Data was analyzed with Prism 7 to fit sigmoidal dose-response (variable slope) curves.