The molecular HIV-1 clone of pNL4-3 and its derivative pNL4-3 E- GFP, the latter carrying an egfp gene within the nef locus driven by the 5'-LTR, were obtained from Dr. Malcom Martin (National Institutes of Health (NIH), Bethesda, MD) and Dr. Nathaniel Landau (New York University, New York, NY) respectively, via the NIH AIDS Research and Reference Reagent Program. Pseudotyping with VSV-G, JR-FL Env and YU-2 Env was performed as reported . The molecular clones of HIV-1NL4-3 Env (V38A) and HIV-1NL4-3 Env (X23) were a kind gift from Dr. Matthias Dittmar (University of Heidelberg, Heidelberg, Germany) . The X23 env gene was derived from a T20-naive HIV-positive individual [14, 15]. Infectious stocks from proviral DNA were generated by transfection of proviral HIV plasmids into 293T cells as described . HIV-1NL4-3 virions containing β-lactamase-Vpr chimeric fusion proteins (BlaM-Vpr) were produced by triple-transfection of 293T cells with pNL4-3 proviral DNA (60 μg), pBlaM-Vpr (20 μg), and pAdVantage (8 μg) vectors (Promega, Madison, WI) per 15-cm2 dish by calcium phosphate DNA precipitation as described [17, 29]. Two days after transfection, culture supernatants were harvested and viral stocks were concentrated using Centricon® Plus-70 spin columns (Millipore, Billerica, MA). After concentration, HIV-1 virions were purified through a 20% or 30% sucrose cushion (44.000 g, 4°C, 60 min), and the virion-enriched pellet was resuspended in phosphate-buffered saline (PBS) and stored at -80°C. The p24 concentration of HIV-1 stocks was determined by antigen enzyme-linked immunosorbent assay (ELISA) as reported . The construction of the replication-competent MoMLV-GFP reporter virus has been reported .
Cell lines, monocyte-derived macrophages and primary rat T-cells
All cell lines and primary macrophages were cultivated under standard conditions in Dulbecco's modified Eagle medium (293T, TZM-bl, MDM) or RPMI 1640 (Sup-T1, C8166, CHO Tat) (both media from GIBCO, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (Invitrogen, Karlsruhe, Germany), 1% penicillin-streptomycin, and 1% L-glutamine (both from GIBCO). Cultures of monocyte-derived macrophages were prepared from Ficoll gradient-purified peripheral blood mononuclear cells isolated from individual, healthy HIV-, HCV-, HBV-seronegative blood donors (DRK Blutspendezentrale, Mannheim, Germany) by adherence and were differentiated in the presence of 10% human AB serum (Invitrogen) for 6–8 days as reported [28, 31]. Cultures of primary rat T-cells were generated as reported [28, 31].
Human Lymphoid Aggregate Culture(HLAC) from tonsil
Tonsil tissue was removed during routine tonsillectomy from HIV-, HBV-, HCV-negative patients with informed consent. To prepare HLAC, tonsil tissue was mechanically dispersed by cutting tissue in 2- to 3-mm blocks and passing them through 40-μm cell strainers (BD Falcon, Belgium). Cells were washed in PBS, and 2 × 106 cells were plated in 96-well V-bottom plates (Corning Incorporated, New York, NY) in a final volume of 200 μl. Culture medium (RPMI 1640 containing 15% fetal bovine serum, 1% L-glutamine, 1% fungizone, 1% gentamycin (all from GIBCO), 0.25% ampicillin (Roth, Karlsruhe, Germany), 1% non-essential amino acids, and 1% sodium pyruvat (both from Invitrogen)). Detailed cultivation methods have been reported [10, 11]. One day after tonsil preparation, the HLAC was inoculated with HIV-1 (5 ng p24 per 2 × 106 cells per well). Where indicated, HIV stocks were preincubated with aqueous Lamiaceae extracts or solvent alone for 1 h at 37°C prior to infection. Following overnight infection cells were washed and the culture medium was subsequently changed every two days without dispersing the pellet. At the same time intervals supernatant samples were harvested and stored at -20°C for subsequent analysis by p24 ELISA.
Aqueous extracts from species of Lamiaceae
Dried leaves from lemon balm (Melissa officinalis L.), peppermint (Mentha × piperita L.), and sage (Salvia officinalis L.) were purchased from Caesar & Lorenz (Hilden, Germany). All plants were identified by microscopy and chromatography according to their monographs in the Pharmakopoea Europoea. Aqueous extracts were prepared as described previously . Briefly, boiling water (100 ml) was added to dried leaves (10 g) and incubated for 15 min, subsequently filtered and cooled down. The resulting extracts were sterile filtered, aliquoted, and stored at -20°C.
Enfuvirtide (Fuzeon®, (T20) Roche, Indianapolis, IN) was freshly dissolved in H2O at 9 mg/ml. Efavirenz (Sustiva®, Bristol-Myers Squibb, Jacksonville, FL) was purchased as a drinking solution at 30 mg/ml and diluted in culture medium. AMD3100 was a kind gift from Dr. José Esté (Badalona, Spain).
Treatment of virus particles and cells
First, virus stocks were incubated with the indicated concentrations (v/v) of aqueous Lamiaceae extracts or H20 as solvent control for 1 h at 37°C. Subsequently, this suspension was mixed with an equivalent volume of culture medium (1:1) and added to target cells (5 ng p24 per 4 × 104 cells). Following overnight exposure, cells were washed and cultivated for 4 more days. Productive HIV-1 infection was assessed by the p24 concentration in culture supernatants.
Exclusive treatment of cells
Cells were exposed to the indicated concentrations of aqueous extracts in culture medium for 1 h at 37°C. Subsequently, the supernatant was discarded, cells were washed once with PBS and challenged overnight with HIV-1 (5 ng p24 per 4 × 104 cells).
Cell viability assay
In parallel to all infection assays, uninfected cells were cultivated in the presence of aqueous Lamiaceae extracts at the identical concentrations used in the infection assays, or in the presence of solvent alone (reference control). Following overnight exposure, cells were washed and cultivated for 3 more days, at which time the cytotoxicity was determined by quantifying the amount of a formazan product metabolized by viable cells from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma) as reported .
HIV-1 virion-fusion assay
The flow cytometry-based HIV-1 virion-fusion assay was performed essentially as described [12, 17, 29]. Briefly, C8166 T-cells were pretreated with enfuvirtide (2 μM), AMD3100 (10 μM), or efavirenz (10 μM) for 15 min. Alternatively, HIV-1NL4-3 BlaM-Vpr virions were pretreated for 1 h at 37°C with the indicated concentrations of aqueous lemon balm extract or solvent. Subsequently, C8166 T-cells were exposed to HIV-1NL4-3 BlaM-Vpr virions (40 ng p24 per 2 × 106 cells) for 6 h, washed and then loaded with CCF2/AM dye overnight. Fusion was monitored with a three-laser BD FACSAria Cell Sorting System (Becton Dickinson, San Jose, CA).
Luciferase reporter virus assay
HIV-1NL4-3 wt virions, or T20-insensitive HIV-1NL4-3 Env (V38A) or HIV-1NL4-3 Env (X23) virions were exposed to lemon balm extract at concentrations from 0.00001 to 1% or solvent for 1 h at 37°C and subsequently added to TZM-bl cells, carrying an LTR-driven firefly luciferase gene. In parallel, TZM-bl cells were pretreated with enfuvirtide at concentrations from 0.0032 to 10 μM for 15 min and then challenged with virus. TZM-bl cells were washed the following day. 48 h after infection a luciferase reporter assay system (Promega) was used to monitor the enzymatic activity.
Cell-Cell fusion assay
This assay was performed in principle as described [16, 17]. Briefly, CHO Tat cells were transiently transfected with expression plasmids encoding for HIV-1 JRFL Env or HIV-1 YU-2 Env together with pCMV-Rev. One day later, transfected CHO Tat cells and TZM-bl cells, the latter stably expressing CD4, CCR5, and an LTR-driven β-galactosidase gene, were harvested and cocultured in a 1:1 ratio in 96-well plates (2 × 104 cells per well in 200 μl of a 1:1 mixture of complete RPMI and DMEM culture medium). Where indicated, transfected CHO Tat cells were exposed to aqueous lemon balm extract or solvent for 1 h at 37°C prior to mixing. The following day, cells were washed once with PBS, and the β-galactosidase enzyme activity and protein concentration in cell lysates were determined with the Galacto-Star™ System (Applied Biosystems) and the BCA™ Protein Assay Kit (Pierce), respectively. The luminometric activity was analyzed with a Luminoskan Ascent (Thermo Labsystems) luminometer and Ascent Software 2.0.
HIV-1 single-round infections
TZM-bl cells were seeded at a density of 1 × 105 cells per well and challenged with single-round HIV-1NL4-3 E- GFP reporter viruses (HIV-1 GFP) pseudotyped with JR-FL Env, YU-2 Env, or VSV-G (all 20 ng p24 per well). Prior to infection, virions were exposed to peppermint extract at concentrations ranging from 0.001 to 1% or solvent for 1 h at 37°C. TZM-bl cells were washed the following day. Three days after infection, the percentage of GFP-positive cells was determined on a FACSCalibur using BD CellQuest Pro 4.0.2 Software (BD Pharmingen).
For the dialysis experiment, lemon balm extract was dialyzed for 24 h at room temperature against water at molecular weight cut-offs of 3.5 or 12 kDa (Spectra/Por® Regenerated Cellulose (RC) Dialysis Membranes). Dialysis-induced changes in extract volume were accounted for prior to virus exposure.
Exploring the activity of lemon balm extract on surface-bound viral particles, TZM-bl cells were inoculated with HIV-1 GFP (200 ng p24 per well) and incubated for 2 h at 4°C. Cells were washed and then solvent or lemon balm extract was added at the indicated concentrations and cells were cultivated at 37°C until analysis.
Western blot analysis
Virions were exposed to lemon balm extract (1%), solvent (H2O; 1%), or Triton X-100 (0.5%) for 1 h at 37°C, pelleted through a 20% sucrose cushion by ultracentrifugation (44.000 g, 4°C, 60 min) and resuspended in PBS (4°C, 30 min) for subsequent analysis by Western blotting. Briefly, samples were boiled in sodium dodecyl sulphate (SDS) sample buffer, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. After incubation with primary antisera (rabbit anti-gp120 Env, 1:5000 (kind gift from Dr. Valerie Bosch, DKFZ, Heidelberg); rabbit anti-24 Gag, 1:4000 (kind gift from Dr. Hans-Georg Kräusslich)) and secondary antibodies (goat anti-rabbit-horseradish peroxidase, 1:10.000; Dianova, Hamburg, Germany), viral structural proteins were detected with an ECL staining solution as reported .
Sucrose-density equilibrium gradient analysis
HIV-1NL4-3 virions (~500 ng p24), which had been purified and enriched by ultracentrifugation through a 30% sucrose cushion, were incubated with either aqueous Lemon balm extract (1%), solvent (H2O; 1%), Triton X-100 (0.5%), or left untreated for 1 h at 37°C. Subsequently, the virion suspensions were loaded onto a 20 to 60% linear sucrose gradient (total volume: 3.5 ml). After ultracentrifugation at 44.000 rpm for 16 h at 4°C in a SW60 rotor, 24 fractions of each 150 μl were carefully collected from top to bottom and the p24 concentration was analyzed. The sucrose density of fractions from an additional tube (without virus) run in parallel was determined by refractometry.