Selection of HIV-1 for resistance to fifth-generation protease inhibitors reveals two independent pathways to high-level resistance
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, United States
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, United States
- Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, United States
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, United States
Figures
Figure 1
Darunavir (DRV) structure as a PR substrate analog.
The chemical structure of DRV is shown. Protease substrates are labeled following a convention where the scissile bond is flanked upstream by amino acids labeled P1, P2, etc., with the amino acids …
Figure 2 with 4 supplements
Features of viruses selected to high-level resistance against darunavir (DRV) and the UMASS-1 through -10 inhibitor series.
Infected cultures were put under drug selection starting with either wild-type virus or a pool of mutant viruses. (A) Most abundant variants present at final timepoint. Resistance mutations in the …
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Figure 2—source data 1
Source sequence data for top abundant variant in panel A.
- https://cdn.elifesciences.org/articles/80328/elife-80328-fig2-data1-v2.xlsx
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Figure 2—source data 2
Source sequence data for top 3 most abundant variants in panel A.
- https://cdn.elifesciences.org/articles/80328/elife-80328-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
EC50 inhibition curves for 2nd, 3rd, 4th, and 5th generation protease inhibitors.
EC50 inhibition curves for (A) old and new protease inhibitors and (B) fifth-generation analogs of darunavir (DRV) (UMASS-1–10).
Figure 2—figure supplement 2
Enzymatic inhibition constant (Ki) values for UMASS-1–10 in the presence of pro with I84V or I50V/A71V mutations.
UMASS-1, -3, -6, and -8 show less potency toward these variants and are shaded in a red tint matching R2 groups. UMASS-2, -5, -7, and -10 show increased potency toward these variants and are shaded …
Figure 2—figure supplement 3
Increasing inhibitor concentrations of viral selection passages.
(A, B) Viral selections with darunavir (DRV) and UMASS-1–10 have increasing inhibitor concentrations during passaging. Passages are increased when extensive cytopathic effect (CPE) is observed …
Figure 2—figure supplement 4
Shannon’s entropy in the protease region through five of five darunavir (DRV) selections.
(A) Shannon entropy was calculated using sequence diversity in the protease region through five darunavir (DRV) selections. Abundant mutations are shown and fixed mutations are underlined. (B) The …
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Figure 2—figure supplement 4—source data 1
Source sequence data for top abundant variant DRV passages in panel A.
- https://cdn.elifesciences.org/articles/80328/elife-80328-fig2-figsupp4-data1-v2.xlsx
Figure 3 with 2 supplements
Evaluation of sequence diversity in the viral population during increasing selective pressure with protease inhibitor (PI) inhibitors.
(A) Shannon entropy was calculated using sequence diversity in the protease region. Abundant mutations are shown and fixed mutations are underlined. Selections from wild type are in orange (n = 6) …
Figure 3—figure supplement 1
Shannon’s entropy of viral cultures undergoing difference resistance pathways.
(A, B) Shannon’s entropy of viral cultures that utilize either (A) I84V or (B) I50V as their resistance pathway. When I84V or I50V arise in the selection cultures above the Poisson distribution, …
Figure 3—figure supplement 2
Abundance of I50V and I84V mutations at each drug concentration of all UMASS inhibitors that reached at least 1000 nM concentrations in culture.
Abundance limit of detection cutoffs are shown as black lines on each timepoint to resemble our template consensus sequences (TCS) Poisson distribution calculation as shown in Zhou et al., 2015.
Figure 4 with 1 supplement
Patterns of accumulation of amino acid substitutions associated with resistance during selection.
(A) Abundance data from selections for each indicated amino acid substitution were pooled and examined sequentially at different levels of drug concentration. Selections that reached the maximum …
Figure 4—figure supplement 1
Introduction of mutations at increasing drug concentrations.
Phylogenetic trees of wild-type selection cultures against UMASS-1 and UMASS-8, showing the addition of mutations found in I84V and I50V pathways. Each mutation’s position and color denote the drug …
Figure 5
Analysis of Ki values for mutant enzymes.
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 …
Figure 6
Analysis of EC50 values for mutant virus cultures.
EC50 values were determined for a subset of the selected virus cultures against a panel of inhibitors (n = 50). For (A–E), the EC50 data were pooled using the same methods and sample numbers as in Fi…
Figure 7
Protease cleavage site mutations observed after selection for high-level resistance.
(A) Culture names, final inhibitor concentration, and resistance pathway are shown. Changes in amino acid sequence are shown for the NC/SP2 and SP2/p6 cleavage sites. (B) P2 substitution in the …
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Figure 7—source data 1
Sequenceing data for protease cleavage sites.
- https://cdn.elifesciences.org/articles/80328/elife-80328-fig7-data1-v2.xlsx
Figure 8
Structural interpretation of protease inhibitor resistance and of residual inhibitor potency.
(A–C) Hydrophobic packing in the S1’ subsite in complex with R1 structural groups; darunavir is shown in cyan, UMASS-1 in magenta, and UMASS-6 in salmon. (A) The two forms of R1 and darunavir (DRV) …
Tables
Table 1
Structures, Ki constants, and EC50 values of the UMASS series of HIV-1 protease inhibitors (PIs).
| Inhibitor | Structure | Ki (pM) | EC50 (nM) | |||
|---|---|---|---|---|---|---|
| WT | I84V | I50V/A71V | WT | Viral culture (5000 nM) | ||
| DRV |  | <5.0* | 25.6 ± 5.6* | 74.5 ± 5.6* | 7.7 ± 1.6 | >100,000 |
| UMASS-1 |  | <5.0* | 26.1 ± 3.7* | 110.3 ± 8.8* | 5.9 ± 1.0 | |
| UMASS-2 |  | <5.0 | <5.0 | 15.0 ± 2.7 | 2.4 ± 0.3 | |
| UMASS-3 |  | <5.0 | 9.9 ± 2.7 | 79.9 ± 5.9 | 9.1 ± 1.0 | 14,800 ± 6800 |
| UMASS-4 |  | <5.0 | 10.5 ± 1.8 | 32.9 ± 3.0 | 3.2 ± 0.4 | 13,700 ± 6600 |
| UMASS-5 |  | <5.0 | 7.0 ± 1.7 | 7.8 ± 0.9 | 4.0 ± 0.5 | >100,000 |
| UMASS-6 |  | <5.0* | 12.8 ± 3.1* | 100.0 ± 9.9* | 5.2 ± 0.8 | |
| UMASS-7 |  | <5.0 | 12.1 ± 4.5 | 18.2 ± 3.0 | 3.1 ± 0.5 | |
| UMASS-8 |  | <5.0 | <5.0 | 55.4 ± 4.0 | 4.2 ± 0.9 | |
| UMASS-9 |  | <5.0 | 7.6 ± 1.6 | 42.3 ± 2.6 | 6.4 ± 1.2 | >100,000 |
| UMASS-10 |  | <5.0 | 14.3 ± 9.3 | 5.8 ± 1.1 | 4.1 ± 0.9 | |
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*
Previously reported in Lockbaum et al. (ACS Infect Dis. 2019 Feb 8; 5 (2): 316–325).
