Inhibition of early steps in the lentiviral replication cycle by cathelicidin host defense peptides
© Steinstraesser et al; licensee BioMed Central Ltd. 2005
Received: 10 December 2004
Accepted: 18 January 2005
Published: 18 January 2005
The antibacterial activity of host defense peptides (HDP) is largely mediated by permeabilization of bacterial membranes. The lipid membrane of enveloped viruses might also be a target of antimicrobial peptides. Therefore, we screened a panel of naturally occurring HDPs representing different classes for inhibition of early, Env-independent steps in the HIV replication cycle. A lentiviral vector-based screening assay was used to determine the inhibitory effect of HDPs on early steps in the replication cycle and on cell metabolism.
Human LL37 and porcine Protegrin-1 specifically reduced lentiviral vector infectivity, whereas the reduction of luciferase activities observed at high concentrations of the other HDPs is primarily due to modulation of cellular activity and/ or cytotoxicity rather than antiviral activity. A retroviral vector was inhibited by LL37 and Protegrin-1 to similar extent, while no specific inhibition of adenoviral vector mediated gene transfer was observed. Specific inhibitory effects of Protegrin-1 were confirmed for wild type HIV-1.
Although Protegrin-1 apparently inhibits an early step in the HIV-replication cycle, cytotoxic effects might limit its use as an antiviral agent unless the specificity for the virus can be improved.
As a barrier and immune organ, the gastrointestinal tract, lung and skin play a key role in protecting the body from a hostile environment . The low incidence of infection at normal epithelial surfaces reflects the presence of innate, broad-spectrum antimicrobial defense mechanisms . Host defense peptides (HDPs) of the innate immune response play an important role in the protective barrier function of the epithelia . Host defense peptides have been isolated from diverse organisms, including plants, insects, bacteria and vertebrates . Several classes of mammalian peptide antibiotics have been ascribed pivotal roles in innate immunity . Among these are various cysteine-rich peptides such as defensins [6, 7] and the more structurally diverse cathelicidins . Produced as precursors, they require proteolytic processing to liberate the mature functional antimicrobial peptide. Cathelicidins contain a conserved N-terminal cathelin domain, and a structurally diverse C-terminal domain that possesses the peptide's antimicrobial activity. Rabbit CAP18 was the first cathelicidin precursor described, and its mature peptide has broad-spectrum antimicrobial activity . Cathelicidins have since been identified in many other species including hCAP18/LL37 in humans , protegrins in swine [11–13], CRAMP in mice [14, 15] and SMAP29 in sheep . Many of these peptides demonstrate extremely broad-spectrum antimicrobial activity, including Gram positive and Gram negative bacteria and fungi [4, 15]. In addition, they achieve bacterial killing much more rapidly than any commercially available antibiotic . Recently, a new family of synthetic, α-helical HDPs called "ovispirins" was described [18–20]. Although some of these modified peptides had similar antimicrobial activity of naturally occurring peptides, they manifested appreciable cytotoxicity. We have demonstrated recently that variants of Ovispirin, the so called Novispirin peptides, displayed more favorable toxic/ therapeutic ratios in vitro and broad spectrum activity in infected rat burn model [21, 22].
Some of these peptides are induced at epithelial surfaces in response to invading organisms [23–25]. Many HDPs kill microorganisms by causing membrane permeabilization, although not necessarily as their sole mode of action . Some HDPs also direct chemotaxis, promote wound healing, angiogenesis and contribute to adaptive immunity by mobilizing memory T cells and immature dendritic cells [25, 27]. Recent studies have also demonstrated antitumor activity after treatment with HDPs .
In addition, several antiviral activities were reported. Recently it has been demonstrated that rabbit neutrophil peptide alpha-defensin NP1 protects cells from infection with HSV-1 and 2 . Other studies revealed that human neutrophil peptide HNP1 to 3 and Theta-defensins also inhibit HSV infection although by different mechanisms [30–32]. The ancestral human theta-defensins retrocyclin blocked HSV attachment . The inhibition of adenovirus replication by the antimicrobial peptide awaits identification of a mechanism of action . Anti-HIV activity of defensins were first reported 1993 by Nakashima and coworkers . The alpha-defensins exhibited anti-HIV activity on at least two levels: directly inactivating virus particles; and affecting the ability of target CD4 cells to replicate the virus [35–37]. Binding to gp120 of HIV-1 and inhibition of HIV entry has also been identified as the mechanism of inhibition of HIV infection by theta defensins . Due to their inhibitory effect on HIV-1 replication and due to an association of a single-nucleotide polymorphism in a beta defensin gene human beta-defensins might also play an important role in host defense against HIV-1 .
The antibacterial activity of HDPs is largely mediated by pore formation leading to permeablization of the bacterial membrane. Although some selectivity for bacterial membranes has been described, the lipid membrane of enveloped viruses might also be a target of antimicrobial peptides [32, 40]. This might allow development of antiviral effector molecules for topical application against a broad spectrum of enveloped viruses. Targeting host cell-derived membrane components might be a particularly interesting approach to inhibit viruses that rapidly develop resistance to compounds directed against viral proteins such as HIV. Therefore, we screened a panel of different natural occurring and designer HDPs for Env-independent inhibition of HIV infection at an early step in the viral replication cycle.
Summary of the radial diffusion assay results comparing host defense peptides with a clinically used antibiotic (Ampicillin)
M E C (μg/ ml)
0,91 ± 0,04
4,74 ± 0,1
9,13 ± 0,5
2,42 ± 0,4
4,2 ± 0,4
10,50 ± 0,2
4,48 ± 0,2
13,09 ± 0,6
8,96 ± 0,2
8,20 ± 0,5
2,8 ± 0,02
4,46 ± 0,3
13,61 ± 0,7
10,60 ± 0,7
4,3 ± 0,3
28,85 ± 0,6
3,3 ± 1,1
12, 28 ± 0,5
6,87 ± 0,8
1,56 ± 0,4
2 ± 0,1
11,66 ± 1,5
3,66 ± 0,3
79,10 ± 0,3
1,63 ± 0,3
18,9 ± 0,9
5,01 ± 0,2
13,13 ± 0,2
3,19 ± 0,5
4,5 ± 0,02
From the panel of five HDP studied, the cathelicidin LL37 and PG-1 were found to specifically inhibit lentiviral and retroviral vector, but not adenoviral vector infectivity. The strongest inhibition was seen if the lentiviral vectors were preincubated with LL37 and PG-1. This suggests that these HDPs directly interacted with the vector particles, which is consistent with our observation that inhibition was dependent on the HDP concentration during preincubation of the vectors with HDPs, but not on the HDP concentration during infection of the cells. Since lentiviral vectors and retroviral vectors were inhibited to a similar degree although they do not share any viral protein, the target for the HDP on the particles is probably cell-derived. This could either be the lipid membrane derived from the cell, which surrounds the vector particles or cellular membrane proteins that are frequently incorporated in lentiviral and retroviral particles during budding . A permeabilizing effect of LL37 and PG-1 on the viral particles would be consistent with our data, but other mechanisms of inhibition cannot be excluded. While the inhibitory effect of PG-1 was also detected with wild type HIV-1 on P4CCR5 cells, LL37 inhibited HIV-1 to lesser degree then the lentiviral vectors. Due an IC50 of 88 μg/ml against wild type HIV-1, it is questionable whether LL37 concentrations are sufficiently high at mucosal membranes to play a role in host defense against HIV-1.
Modulation of cell metabolism was generally seen at concentrations of HDPs exceeding 50 μg/ml, while the MEC of the antibacterial activity ranged from 1 to 10 μg/ml. This might leave a sufficient window for therapeutic intervention of bacterial infection. However, for the treatment of HIV-1, the therapeutic window of LL37 and PG-1 is rather narrow. It should also be noted that the HDP-induced modulation of cell metabolism and cytotoxicity can be cell type dependent. Therefore, increasing the selectivity of HDPs for early steps in the viral replication cycle seems to be necessary for further development of the human cathelicidin LL37 and the porcine Protegrin-1 as antiviral agents for systemic or topical applications.
Preparation of vectors transferring the luciferase or GFP genes
To generate lentiviral vector particles transferring the luciferase gene, a codon-optimized (Geneart GmbH, Regensburg, Germany) HIV-1 gag-pol expression plasmid (Hgpsyn)  and a VSV-G expression plasmid (pHIT-G)  were used to package the SIV-based vector VLΔBH. This vector contains the luciferase gene replacing the GFP gene of VGΔBH .5 μg of Hgpsyn, 2 μg of pHIT-G and 5 μg of VLΔBH were transiently cotransfected by the CaPO4coprecipitation method into 293T cells as previously described . An HIV vector construct containing the GFP reporter gene (HIV-CSCG)  was also used to prepare lentiviral vector particles by cotransfection with Hgpsyn, pcTat , pcRev  and pHIT-G or pSVIIIenv3-2, an HIV-1 envelope expression plasmid . The MLV vectors were prepared by cotransfection of pHIT-456, pHIT-60 and pRV-172 . Two days after transfection, the supernatants were cleared from cellular debris by low speed centrifugation (10 minutes, 1000 × g) and filtration through 0.2 μm filters from Roth (Karlsruhe, Germany). Aliquots were stored at -80°C.
Construction and Production of Ad.OW126 Vector
Beginning with a first generation E1- and E3-deleted adenoviral vector, we generated a replication-competent adenoviral vector Ad.OW126, which harbors in the E1 region the firefly luciferase cDNA (subcloned from pGEM-Luc (Promega, Madison, WI)), a IRES element [50, 51], and an Ad5 E1A ΔE1B-55K gene. The entire expression cassette is driven by the human CMV-IE promoter in parallel to the transcriptional orientation of the adenovirus E1 gene products and terminated by the bovine growth hormone polyadenylation site. The expression cassette was flanked upstream by the Ad5 packaging sequence and downstream by the Ad5 pIX. The Ad.OW126 vector was generated by in vitro ligation  to H5dl327 (kindly provided by T. Shenk, Princeton University, Princeton, NJ), utilizing the unique Bst1107 I restriction site. The vector was propagated in 293 cells and purified by two rounds of CsCl density centrifugation , dialyzed (Slide-A-Lyzer, Pierce, Rockford, IL) against 1500 ml of PBS with 1 mM MgCl2 and 10% glycerol four-times (1 hour each) at 4°C, and stored at -80°C until use. The concentration of the vector was determined by measuring absorbency at 260 nm , and the infectious titer was determined by plaque assay on 293 cells . The ratio of infectious to non-infectious virus particles was approximately 1:80.
Generation of 293A cells stably transduced with a luciferase gene
To stably transduce the luciferase gene into 293A cells, a self-inactivating version of VLΔBH, VLΔBH-SIN similar to VGΔBH-SIN  was packaged by cotransfection with Hgpsyn and pHIT-G. 293A cells were plated in 24 well plates at a density of 50.000 cells / well and transduced with 200 μl of VLΔBH-SIN vector for two hours. Two days after plating cells were transferred to one well of a six well plate and transduced again with 1 ml of VLΔBH-SIN vector. Cells were subsequently expanded resulting in 293-Luc cells.
The supernatant of infected 293A or 293-Luc cells, cultured in 96 well plates was removed and cells were lysed in 50 μl of cell lysis buffer (Promega, Pittsburgh, PA). 20 μl of the cell lysates were used in the firefly luciferase assay system of Promega as described by the manufacturer. Each single value of the triplicates was expressed as percent of the mean of triplicates of control cultures infected with the same vector in the absence of HDPs and the mean and the standard deviation of the percent values was calculated for each triplicate.
Host defense peptides
The antimicrobial peptides (human LL37, porcine PG1-1, mutants from the ovine SAP29: Novispirin G10 and fungal Plectasin) used in this study were prepared by solid phase synthesis and purified by RP-HPLC. Recombinant human β-Defensin-2 was produced by a molecular farming approach in transgenic potato tubers and purified by perfusion chromatography (data not shown). The peptides (≥ 98% pure) were dissolved in 0.01% acetic acid and used for all in vitro and in vivo studies. Potential endotoxin contamination was monitored with the chromogenic Limulus amoebocyte lysate assay (BioWhittaker, Walkersville, MD) using Escherichia coli endotoxin (supplied with the kit) as the standard. Endotoxin levels for the peptides were not detectable.
The following strains were used in this study: Gram-negative strains: Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) Gram-positive strains: Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228) and Enterococcus faecalis (ATCC 29212).
All bacterial strains were analyzed with API test strips (BioMerieux, Hazelwood, MO) to confirm identity and aliquots were stored frozen in 50 % skim milk at -80°C. Bacteria were grown overnight in trypticase soy broth (Becton Dickinson, Franklin Lakes, NJ) at 275 rpm and 37°C. An aliquot of the resulting stationary phase cultures was then transferred to 20 ml of trypticase soy broth and incubated at 37°C for 2.5 hours to reach log phase. This subculture was transferred to a 50 ml conical polystyrene tube and centrifuged for 10 min at 4°C at 880 g. The bacterial pellet was washed once with chilled phosphate buffered saline, pH 7.4, and resuspended in 5 ml of the same cold buffer. One milliliter was removed to measure its optical density at 620 nm. The bacterial concentration was calculated from the following formula: CFU/ml = OD620 × 2.5 × 108.
Growth Inhibition Assay
To monitor bacterial growth inhibition in vitro a radial diffusion assay was performed as previously described . Briefly, the underlay agar consisted of 1% agarose (A-6013, Sigma Chemical, St. Louis, MO) and 0.3 mg/ml trypticase soy broth (TSB) powder in 10 mM sodium phosphate with 100 mM NaCl (normal salt medium), pH 7.4. Bacteria (approximately 5 × 106 CFU) were mixed with 10 ml of underlay gel (43°C) and immediately poured into square 9 × 9 cm petri dishes. A series of 3 mm wells was punched after the agarose solidified. After appropriate serial dilutions were done, 5 μl of HDP, vancomycin (Abbott Labs, Chicago, IL), gentamicin, ciprofloxacin, or fluconazole (Sigma-Aldrich, St. Louis, MO) were added to the designated wells. Plates were incubated at 37°C for 3 hours. The bacteria-containing layer was covered with a 10 ml overlay of the nutrient rich agar. The overlay agar consisted of 6% (w/v) TSB and 1% agarose in PBS for all assays. After 18 h of incubation at 37°C, the plates were stained with 0.001% Coomassie blue for 10 h. The clear zones (bacterial growth inhibition) around the punched wells indicated antibacterial activity. The diameters of the clear zones were converted into units by subtracting the well diameter and multiplying the difference by 10. Results were plotted using a semi log scale and correlation coefficients and X-intercepts obtained from linear regression analysis. The minimal effective concentration (MEC) corresponded to the X-intercept value. All assays were performed in triplicates and repeated at least once.
Cytotoxicity and proliferation Assay
293-Luc cells were plated in 96 well plates at a density of 2 × 103 cells / well. After 48 hours, 50 μl of MTT-solution (3 mg/ml) was added and incubated at 37° C under 5% CO2 for 1 hour. After this time medium was removed and 100 μl 0.04 N HCL + 10% SDS was used to dissolve the resulting blue formazan crystals in living cells. The optical density was determined at 550 nm. Each single value of the triplicates was expressed as percent of the mean of triplicates of control cultures infected with the same vector in the absence of HDPs and the mean and the standard deviation of the percent values was calculated for each triplicate. In addition the BrdU Cell proliferation ELISA with chemiluminescence detection (Roche Diagnostics GmbH, Mannheim, Germany) was performed. After 293-Luc cells or P4CCR5 cells were plated in 96 well plates at a density of 2 × 103 cells/well BrdU (5-bromo-2'-deoxyuridine) was added to the cells with a resulting concentration of 10 μM for the last 22 h of the incubation period. After removing the culture medium, the cells were fixed and DNA was denatured in one step with Fixdenat. Thereafter the cells were incubated with Anti-BrdU-POD for 1 h at room temperature. The chemiluminescence detection was measured after automatic injection of substrate solution with a microplate-luminometer (Orion, Berthold detection systems, Pforzheim, Germany).
Inhibition of vector infectivity
To determine the effect of HDPs on vector infectivity and cell metabolism, 293A target cells were plated in triplicates into 96-well plates at 2 × 103 cells / well. After overnight incubation, the supernatant of the wells were removed and replaced by 25 μl vector preparation and 25 μl HDP at twice the final concentration indicated. 50 μl fresh medium with HDP at the final concentration indicated was added after two hours. One (adenoviral vector) or two (lentiviral and retroviral vector) days after infection, the supernatant was removed and 50 μl of cell lysis buffer (Promega) was added. Lysates were stored at -80°C until determination of the luciferase activity of the extracts. The affect of HDPs on cell metabolism was determined in parallel by plating 293-Luc cells exactly the same way as 293A cells and the luciferase activity was determined after one (as a control to inhibition of adenoviral vector infectivity) or two (as a control of lentiviral infectivity) days of incubation with HDPs. For GFP-expressing vectors, vector titers were calculated from the number of GFP positive cells per well as previously described  and the BrdU incorporation assay was used to monitor cytotoxic effects.
Inhibition of HIV-1
Stocks of HIV-1 were generated by transient transfection of 293T cells with the molecular clone pNL4-3. 25 μl of the virus stock were incubated for 30 minutes at room temperature with 25 μl of LL37 or PG-1 adjusted to twice the final concentration indicated. The mixture was added to P4CCR5 cells plated the day before at a density of 2 × 103 cells / well of 96 well plate. 50 μl fresh medium with HDP at the final concentration indicated was added after two hours. Two days after infection, the supernatant was removed and cells were stained by X-Gal. The number of infected cells per well were counted in the microscope.
Recombinant human β-Defensin-2 was kindly provided by Michael Kleine at Planton GmbH (Kiel, Germany). Novispirin G-10 and Plectasin (WO 03/044049) were kindly provided by Hans-Henrick Christensen and the AMP team at Novozymes A/S (Copenhagen, Denmark). We would like to thank Ralf Wagner for providing Hgpsyn, Ulrike Blömer for HIV-CS-CG, Joachim Hauber for pcTat and pcRev, Michael Malim for pHIT-G, and Paula Cannon for pHIT456, pHIT60 and pRV172. This work was supported by a grant from the FORUM program of the Ruhr-University Bochum.
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