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. 2020 Feb 18;59(6):818-828.
doi: 10.1021/acs.biochem.9b01008. Epub 2020 Jan 28.

Metastable HIV-1 Surface Protein Env Sensitizes Cell Membranes to Transformation and Poration by Dual-Acting Virucidal Entry Inhibitors

Charles G Ang  1   2 , Md Alamgir Hossain  1 , Marg Rajpara  1 , Harry Bach  1   2 , Kriti Acharya  1 , Alexej Dick  1 , Adel A Rashad  1 , Michele Kutzler  3 , Cameron F Abrams  4 , Irwin Chaiken  1
Affiliations

Metastable HIV-1 Surface Protein Env Sensitizes Cell Membranes to Transformation and Poration by Dual-Acting Virucidal Entry Inhibitors

Charles G Ang et al. Biochemistry. .

Abstract

Dual-acting virucidal entry inhibitors (DAVEIs) have previously been shown to cause irreversible inactivation of HIV-1 Env-presenting pseudovirus by lytic membrane transformation. This study examined whether this transformation could be generalized to include membranes of Env-presenting cells. Flow cytometry was used to analyze HEK293T cells transiently transfected with increasing amounts of DNA encoding JRFL Env, loaded with calcein dye, and treated with serial dilutions of microvirin (Q831K/M83R)-DAVEI. Comparing calcein retention against intact Env expression (via Ab 35O22) on individual cells revealed effects proportional to Env expression. "Low-Env" cells experienced transient poration and calcein leakage, while "high-Env" cells were killed. The cell-killing effect was confirmed with an independent mitochondrial activity-based cell viability assay, showing dose-dependent cytotoxicity in response to DAVEI treatment. Transfection with increasing quantities of Env DNA showed further shifts toward "High-Env" expression and cytotoxicity, further reinforcing the Env dependence of the observed effect. Controls with unlinked DAVEI components showed no effect on calcein leakage or cell viability, confirming a requirement for covalently linked DAVEI compounds to achieve Env transformation. These data demonstrate that the metastability of Env is an intrinsic property of the transmembrane protein complex and can be perturbed to cause membrane disruption in both virus and cell contexts.

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Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Structural representation of the M*DAVEI inhibitor. (A) Schematic depiction of the lectin DAVEI, starting from the N-terminus and containing hexahistidine, microvirin (Q51K/M53R), the (G4S)4 linker, and Trp3. The microvirin protein shown is Protein Data Bank entry 2Y1S visualized with BIOVIA Discovery Studio Visualizer version 19.1 (B) Sequence of the microvirin (Q51K/M53R) protein, with mutations colored red. (C) Excerpted sequence (residues 651–700) of HIV-1 Env, with MPER underlined and the Trp3 sequence (residues 664–672) colored red.
Figure 2.
Figure 2.
Flow cytometry dot plots depicting cell populations charted by calcein dye retention (Y-axis, green) and JRFL HIV-1 Env expression (X-axis, red) after MVN*DAVEI (M*D) treatment. Calcein dye on the Y-axis, 35O22 primary/PerCP secondary staining on the X-axis, on fixed cells after treatment with the indicated concentrations of M*D over 18 h. Quadrant labels show the percentage and absolute cell counts for each quadrant. Cells were gated by forward and side scatter to remove dead cells and cellular debris. Dot plots are representative for one of four independent experiments.
Figure 3.
Figure 3.
Membrane leakage and cell count effects of M*DAVEI. (A) Calcein dye retention as a percent of the median fluorescence intensity of untreated cells of the same transfection. Calcein retention values in nontreated groups were compared to those of nontransfected and nontreated populations. Fluorescence intensity is normalized for cell count and reflects the median among cells counted only. (B) Numbers of cells counted by flow cytometry for the dye retention experiment depicted in panel A. Cells were transfected with 0, 2, 4, 6, or 8 μg of HIV-1 JRFL Env gp160 DNA. Data are the average of four independent experiments. For all graphs, error bars represent the standard deviation of the mean.
Figure 4.
Figure 4.
Cell viability detection of M*DAVEI effects on HIV-1 Env-expressing cells. Bulk cell viability was measured using WST-1 mitochondrial activity reagent. An initial 10000 transfected or nontransfected cells were seeded per well and treated with the indicated concentrations of M*DAVEI for 18 h at 37 °C. Afterward, inhibitor-containing medium was removed and immediately replaced with medium containing 10% WST-1 reagent and incubated for an additional 1 h at 37 °C. Data are the average of four independent experiments. Error bars represent the standard deviation of the mean.
Figure 5.
Figure 5.
Specificity of M*DAVEI cell effects. (A) Calcein retention as the percent median fluorescence intensity relative to untreated cells at the same transfection load. No significance (p > 0.05) in calcein retention was detected between treatment with individual or unlinked components of M*DAVEI (20 μM MVN*, Trp3, or MVN*+Trp3) and untreated cells. Cells treated with linked M*DAVEI showed significant effects at multiple transfection loads; data for 0.8, 4, and 20 μM are included for comparison. (B) Cell viability as the percent absorbance at 450 nm relative to untreated, nontransfected cells. No significant loss (p > 0.05) in cell viability was detected between treatment with individual or unlinked components of M*DAVEI (20 μM MVN*, Trp3, or MVN*+Trp3) and untreated cells of the same transfection. Comparatively, cells treated with linked M*DAVEI showed significant effects at multiple transfection loads. Data for 0.8, 4, and 20 μM treatment sets are included for comparison. The significance of data was determined by ANOVA. Data are averages from three independent experiments. Error bars depict the standard deviation of the mean.
Figure 6.
Figure 6.
Schematic of the predicted interaction of M*DAVEI with Env and the membrane. (I) MVN*-L4-Trp3, with MVN* denoted “M” and Trp3 denoted “T”, approaches the surface-exposed Env trimer. (II) The MVN* component binds to gp120 via man-α(1–2)-man terminating glycans, anchoring Trp3 in the vicinity of Env and the membrane via a linker. (III) THe Trp3 component inserts into the membrane and potentially displaces one of the native Env’s MPER and TMD regions. (IV) Displacement of the native MPER/ TMD results in membrane disruption and poration.
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