Fig 1.
LEN potency is influenced by p24 levels.
(A–D) HIV-1 virions were produced by transfecting HEK293T cells (producer cells) with indicated concentrations of full-length WT pNL4.3. Indicated concentrations of LEN or DMSO control were added to HEK293T cells, and viruses isolated by ultracentrifugation through 20% sucrose cushions were used to infect HeLa TZM-bl cells (Target cells). After 48 h of infection, luciferase activity was measured to determine EC50 values of LEN. The averaged data (+ / − SD) from three independent experiments are shown.
Fig 2.
LEN treatments yield virions with malformed capsids.
(A) Representative micrographs of virions used during morphological classification. For each experimental condition, > 200 virions were counted. Across cumulative experiments (n = 3 for DMSO treatment; n = 2 for LEN treatments), > 520 virions were counted in total. Virions with visibly electron-dense outer membranes were classified as: mature, containing a single circular or conical electron-dense region; immature, containing outer, comparatively thick toroidal or semi-hemispheric electron density; the following were grouped as atypical or aberrant: empty, comparative lack of luminal contents; rod, electron-dense or electron-lucent core lacking obvious conical shape; multiple cores, > 1 conical, circular, or rod-like capsid shape that was electron-dense or lucent; multiple densities; > 1 distinct electron-dense region wherein at least one of the densities could not be readily attributed to a core structure; empty core, roughly conical shapes that lacked associated electron density. (B) Quantitation of virions belonging to indicated morphological categories (average +/ − SD for n = 2−3 independent experiments). (C) p24 concentrations were determined by p24 ELISA assay after DMSO or LEN treatments (average +/− SD for n = 2−4 independent experiments).
Fig 3.
Sub-stoichiometric LEN:CA ratios inhibit late steps of HIV-1 replication.
(A) The experimental design. The same virus preparation was used for three different assays: 1) LC-MS/MS to determine LEN amounts bound to virions, 2) p24 ELISA to determine CA amounts; and 3) infectivity of the viruses produced in the presence of LEN or DMSO control in HeLa TZM-bl cells. (B) Representative LC-MS/MS results of viruses produced in the presence of LEN (the top panel), the identical concentration of LEN added to HEK293T cells in the absence of pNL4.3 (the middle panel), and the control of the medium without LEN or the virus (the bottom panel). (C) Infectivity of the viruses at indicated [LEN]:[CA] ratios or DMSO control. The [LEN]:[CA] ratios were measured using p24 ELISA and LC-MS/MS results for the same virus preparations.
Fig 4.
Sub-stoichiometric LEN:CA ratios impair formation of mCLPs assemblies in vitro.
Representative micrographs of WT CA (117 µM) in the presence of (A) 500 µM IP6; (B) 117 µM LEN; and (C) 500 µM IP6 + 117 µM LEN. In vitro assembly reactions were performed in 50 mM MES (pH 6.0) and 40 mM NaCl. (D) Quantification of in vitro assembly products using 117 µM WT CA, 500 µM IP6 and varying amounts of LEN. The [LEN]:[CA] ratios for each reaction is indicated. The averaged results (± SD) from three independent experiments are shown.
Fig 5.
LEN specifically blocks formation of cross-linked pentamers but not hexamers.
Representative SDS-PAGE images for formation of cross-linked pentamers (A) and cross-linked hexamers (B). 1 µM CA was used in all reactions. Lanes 1 (A and B): molecular weight markers; Lanes 2 (A and B): the control assembly reactions in the presence of 40 mM βME, which inhibits the cross-linking reactions. Lanes 3 (A and B): DMSO was added to monomeric CA in the absence of the reducing agent and then cross-linked products were monitored for pentamers (A) and hexamers (B). Lanes 4 (A and B) 2 µM LEN was added to monomeric CA in the absence of the reducing agent and then cross-linked products were monitored for pentamers (A) and hexamers (B).
Fig 6.
Mass photometry analysis of WT CA assembly intermediates in the presence of IP6 or LEN.
(A) Unliganded WT CA; The observed peak of 29 ± 9 kDa corresponds to the theoretical MW of a monomer (25.4 kDa). (B) cross-linked CA(N21C/A22C/W184A/M185A) pentamer; (C) cross-linked CA(A14C/E45C/W184A/M185A) hexamer; (D) WT CA + 10 mM IP6 after 10 min incubation. The main peak of 30 ± 4 kDa is close to the theoretical MW of a monomer (25.4 kDa) with a shoulder at 47 ± 7 kDa indicating the formation of a dimer (the theoretical MW of ~ 51 kDa). (E) WT CA + 10 mM IP6 after 1 h incubation. The major peak of 119 ± 48 kDa correlates to the theoretical MW of the pentamer (~ 127 kDa) with a shoulder at 156 ± 14 kDa indicating the presence of a hexamer (the theoretical MW of ~ 152 kDa). (F) WT CA + 10 mM IP6 after 16 h incubation. The major peak of 488 ± 82 kDa indicates higher-order CA assembly products. (G) WT CA + LEN after 10 min incubation. The single symmetrical peak of 55 ± 10 kDa corresponds to the theoretical MW of the CA dimer + LEN is ~ 52 kDa; (H) WT CA + LEN after 1 h incubation. The single symmetrical peak of 62 ± 11 kDa indicates that LEN stabilizes CA dimers; (I) CA + LEN after 16 h incubation. The single symmetrical peak with MW of ~ 172 ± 31 kDa is in the range of the theoretical MW of CA hexamer + LEN (~ 158 kDa).
Fig 7.
CA NTD adopts an opened conformation in the presence of LEN.
(A) The X-ray crystal structure of NTD + LEN (NTDLEN, PDB: 8V23). Side chains of Gly60 and Met66 are indicated to delineate the opened and closed conformations. (B) Our X-ray crystal structure of NTDLEN (steel blue) superimposed onto the apo NTD (NTDApo, magenta, PDB: 5HGK). (C) The X-ray crystal structure of NTDLEN (steel blue) superimposed onto the native CA hexamer (CAHex, orange, PDB: 7URN). (D) X-ray crystal structure of NTDLEN (steel blue) superimposed onto the native CA pentamer (CAPnt, scarlet, PDB: 7URN).
Fig 8.
LEN specifically induces formation of CA(M66A) hexameric lattices.
Representative micrographs of 117 µM CA(M66A) in the presence of (A) DMSO; (B) 10 mM IP6; (C) 1.5M NaCl; or (D) 117 µM LEN. In vitro assembly reactions were performed in 50 mM MES (pH 6.0). (E–G) Quantification of the assembly products shown in B-D. The averaged data (+/− SD) from three independent experiments are shown. CLP, capsid-like particles. Pnt, pentameric spheres. Hex, hexameric lattices/nanotubes. Atp, atypical assemblies. Clt, clusters. Scale bars: 200 nm.
Fig 9.
HIV-1 capsids produced in the presence of LEN show increased stability in vitro.
(A and B) Quantification of the CDR/capsid marker loss from INmNG labeled WT HIV-1 capsids produced in the presence of DMSO (vehicle control) or indicated concentrations of LEN. Kinetics of CDR loss (A), and endpoint (25 min) measurements of stable HIV-1 capsid (B) are shown. (C and D) Quantification of the CDR/capsid marker loss from INmNG labeled HIV-1(M66A CA) capsids produced in the presence of DMSO (vehicle control) or indicated concentrations of LEN. Kinetics of CDR loss (C), and endpoint (25 min) measurements of stable HIV-1 capsid (D) are shown. Student ‘t’-test was used to determine statistical significance in (B and D).
Fig 10.
A schematic for inhibition of mature capsid assembly by LEN.
Addition of LEN to CA stabilizes dimeric and hexameric intermediates, and specifically impairs formation of pentamers. More complex interplay in the presence of both IP6 and LEN with CA is expected as discussed in the text. CAMon, CADim + LEN, CAHex + LEN (PDB id: 6VKV); CAPnt (PDB id: 7URN); mature capsid (PDB id: 3J3Q).