Datasets for Selection of HIV-1 for Resistance to Fifth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

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Datasets collected for manuscript: Selection of HIV-1 for Resistance to Fifth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance.

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1. Km and Ki Dataset for Selection of HIV-1 for Resistance to Fifth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

Title Tesim:
Km and Ki Dataset for Selection of HIV-1 for Resistance to Fifth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance
Creator:
Swanstrom, Ron and Spielvogel, Ean
Date of publication:
July 8, 2022
Abstract Tesim:
Ki values were determined against all UMASS inhibitors (Table 1). Brackets above the bars represent significant p-values between the two groups using the unpaired t-test. Data were pooled in different ways for the following analyses: A) The Ki values against the inhibitors with the larger R1-2 moiety (n=34) were more potent against resistant proteases compared to the inhibitors with the smaller R1-1 moiety (n=35) B) Ki values for the inhibitors with the R2-2 group (n=10) showed a trend toward being more potent against the highly mutated proteases compared to the other R2 groups (n=40). C) Ki values for enzymes with the I50V mutation (n=33) showed greater resistance to the inhibitors compared to enzymes with the I84V mutation (n=22). D) The R1-2 moiety provided increased potency to enzymes with the I84V mutation (n=20) but not the I50V mutation (n=20). E) The R2-2 moiety was more potent against the enzymes with I84V mutation (n=10) compared to the enzymes with the I50V mutation (n=10). The unpaired t-test was used to assess differences in Ki values.
Resource type:
Dataset
Affiliation Label Tesim:
Department of Microbiology and Immunology
Type:
http://purl.org/dc/dcmitype/Dataset
DOI:
https://doi.org/10.17615/rb7r-tx62
Kind of Data:
Other
Language Label:
English
Last Modified Date:
2022-07-08
License Label:
CC0 1.0 Universal
Methodology:
Km Assay. Km values were determined as previously described (Henes et al., 2019; Lockbaum et al., 2019; Matayoshi et al., 1990; Windsor and Raines, 2015). Briefly, a 10-amino acid substrate containing the natural MA/CA cleavage site with an EDANS/DABCYL FRET pair was dissolved in 8% DMSO at 40 nM and 6% DMSO at 30 nM. The 30 nM substrate was 4/5 serially diluted from 30 nM to 6 nM. HIV-1 protease was diluted to 120 nM and, and 5 µl were added to the 96-well plate to obtain a final concentration of 10 nM. Fluorescence was observed using a PerkinElmer Envision plate reader with an excitation at 340 nm and emission at 492 nm, and monitored for 200 counts. A FRET inner filter effect correction was applied as previously described (Liu et al., 1999). Data corrected for the inner filter effect was analyzed with Prism7. Datasets were uploaded to the Carolina Digital Repository. Ki Assay. Enzyme inhibition constants (Ki values) were determined as previously described (Henes et al., 2019; Lockbaum et al., 2019; Matayoshi et al., 1990; Windsor and Raines, 2015). Briefly, in a 96-well plate, inhibitors were serially diluted down from 2000-10,000 nM depending on protease resistance. All samples were incubated with 5 nM protein for 1 hour. A 10-amino acid substrate containing an optimized protease cleavage site (Windsor and Raines, 2015), purchased from Bachem, with an EDANS/DABCYL FRET pair was dissolved in 4% DMSO at 120 µM. Using a PerkinElmer Envision plate reader, 5 µl of the 120 µM substrate were added to the 96-well plate to a final concentration of 10 µM. Fluorescence was observed with an excitation at 340 nm and emission at 492 nm and monitored for 200 counts. Data was analyzed with Prism7. Datasets were uploaded to the Carolina Digital Repository.
ORCID:
0000-0001-7777-0773 and
Other Affiliation:
Person:
Swanstrom, Ron and Spielvogel, Ean
Rights Statement Label:
In Copyright

2. EC50 Dataset for Selection of HIV-1 for Resistance to Fifth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

Title Tesim:
EC50 Dataset for Selection of HIV-1 for Resistance to Fifth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance
Creator:
Swanstrom, Ron and Spielvogel, Ean
Date of publication:
July 8, 2022
Abstract Tesim:
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.
Resource type:
Dataset
Affiliation Label Tesim:
Department of Microbiology and Immunology
Type:
http://purl.org/dc/dcmitype/Dataset
DOI:
https://doi.org/10.17615/3697-3v58
Kind of Data:
Numeric
Language Label:
English
Last Modified Date:
2022-07-08
License Label:
CC0 1.0 Universal
Methodology:
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.
ORCID:
0000-0001-7777-0773 and
Other Affiliation:
Person:
Swanstrom, Ron and Spielvogel, Ean