Introduction

Lactoferrin is a mammalian single-chain iron-binding glycoprotein in whey with a molecular weight in the vicinity of 80 kDa. Its diversity of activities includes bone regenerating, anti-obesity, anti-viral, anti-bacterial, and immunomodulatory activities [1–6]. Oral administration of lactoferrin exerted an immunomodulatory action in HIV-infected, anti-retroviral therapy-naïve children [7]. Many protein anti-HIV activities, including trypsin inhibitor, lectins, and ribonuclease, have been reported [8–10]. The lactoferrin-derived peptides lactoferricin and lactoferrampin display anti-bacterial, anti-viral, anti-fungal, anti-inflammatory, immunomodulatory, anti-angiogenic, and anti-metastatic activities [11].

Bovine lactoferrin exerted pronounced HIV-1 reverse transcriptase inhibitory activity but only meager suppressive activity on HIV-1 protease and integrase. In the presence of bovine and human lactoferrin fragments, the activities of reverse transcriptase, protease, and integrase of the HIV-1 were attenuated [12]. It was found that human lactoferricin attenuated the activity of HIV-1 reverse transcriptase with the highest potency (IC50 = 2 μM), followed by bovine lactoferricin (IC50 = 10 μM) which had a dissimilar sequence, and then bovine lactoferrampin (IC50 = 150 μM). Bovine lactoferrin [1–11], human lactoferrin [1–11], and human lactoferrampin failed to affect HIV-1 reverse transcriptase activity at 1 mM concentration. Bovine but not human lactoferriampin inhibited HIV-1 RT, although they were structurally similar. The peptides exhibited only a weak (from slightly below 2 to 6 % inhibition) HIV-1 protease inhibitory activity [13].

Integrase is not present in humans, and this explains why HIV-1 integrase is a target for combating HIV [14]. In recent years, the research on HIV-1 integrase inhibitors has been intensive [15]. A Triumeq® tablet which contains dolutegravir (an integrase strand transfer inhibitor) and abacavir and lamivudine nucleoside/nucleotide reverse transcriptase inhibitors is deployed against HIV-1 [16]. Dolutegravir appears to be the best HIV-1 integrase inhibitor [17]. Nevertheless, resistance may arise under certain circumstances [18–20].

Research efforts have been directed to inhibitors of nuclear translocation of HIV-1 integrase [21]. We have previously identified some lactoferrin-derived peptides as inhibitors of HIV-1 integrase activity. In view of the fact that inhibition of HIV-1 integrase activity and inhibition of nuclear translocation of HIV-1 are two different processes, we tested the lactoferrin-derived peptides for the ability to inhibit nuclear translocation of the retroviral enzyme in the present investigation.

Materials and Methods

Peptide Synthesis

All peptides were synthesized following manual solid phase synthesis protocols and were synthesized by Shanghai Biotech Bioscience and Technology Company, China. Purity was confirmed by high-performance liquid chromatography (HPLC) analyses. Mass spectrum was also used for confirmation of product quality. Fmoc solid-phase peptide synthesis was employed. In the case of bovine lactoferricin (peptide with free C-terminal carboxyl group), the C-terminal amino acid residue (Fmoc-Ser(tBu)) was attached to the 2-chlorotrityl chloride resin (substitution of Cl 1.46 meq/g; Calbiochem-Novabiochem AG, Switzerland) in the presence of an equimolar amount of DIPEA in relation to the amino acid in anhydrous DCM solution. Peptide chains were elongated in the consecutive cycles of deprotection and coupling. Deprotection was performed with 20 % piperidine in the mixture of DMF/NMP (1:1, v/v) with addition of 1 % Triton X-100, whereas the chain elongation was achieved with standard DIC/HOBt chemistry; three equivalents of protected amino acid derivatives were used. After completing the syntheses, the peptides were cleaved from the resin simultaneously with the side chain deprotection in a one-step procedure, using a mixture of TFA/phenol/triisopropylsilane/H2O (88:5:2:5, v/v/v/v). The crude peptides were purified by HPLC on a Beckman Gold System (Beckman, USA) using an RP Kromasil-100, C8, 5-μm column (8 × 250 mm; Knauer, Germany). The solvent system was 0.1 % TFA (A) and 80 % acetonitrile in A (B). Either isocratic conditions or a linear gradient were applied (flow rate 3.0 ml/min, monitored at 226 nm). The purity of the synthesized peptides was checked on another RP Kromasil 100, C8, 5-μm column (4.6 × 250 mm) (Knauer, Germany). The solvent system was 0.1 % TFA (A) and 80 % acetonitrile in A (B). Linear gradient from 10 to 90 % B in 40 min with a flow rate 1 ml/min was employed and monitored at 226 nm. The mass spectrometry analysis was carried out on a MALDI MS (a Biflex III MALDI-TOF spectrometer, Bruker Daltonics, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix. The analysis confirmed the correctness of molecular weight values of all six synthesized peptides [22].

Assay of Hemolytic Activity

Rabbit erythrocytes were washed four times with 10 mM phosphate-buffered saline (PBS; pH 7.5) and adjusted to a final concentration of 2 % (v/v) in PBS. A sample solution (50 μl) was mixed with rabbit erythrocytes (50 μl) and incubated at room temperature for 1 h before centrifugation at 400×g for 5 min. The amount of hemoglobin released from disrupted erythrocytes was determined spectrophotometrically. One-hundred-percent hemolysis was defined as OD540 of hemoglobin released from erythrocytes treated with 0.1 % Triton X-100.

Assay of Inhibitory Activity on Translocation of HIV-1 Integrase into the Nucleus

The translocation of HIV-1 integrase assay was conducted by transfected pcDNA3.1/HIV IN into HeLa cells, and the HIV-IN nuclear distribution was probed by using both nuclear protein western blot and localization image analysis.

First of all, HeLa Tet-Off Advanced Cells (Clontech) were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 μg/ml G418, and 1 % of streptomycin–penicillin (Invitrogen) at 37 °C in a 5 % CO2 incubator. Twenty-four hours before transfection, 5× 106 HeLa Tet-Off Advanced Cells were seeded onto a 12-well plate (BD) in DMEM containing 10 % FBS. IN expression vector was transfected into HeLa Tet-Off Advanced Cells using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer’s instruction. Then, the medium was removed 4 h after transfection. Fresh medium containing test peptide was added. Compound Y, a newly synthesized compound from our collaborator, was used as a positive control. At the end of another 24 h, for western blot analysis, the cells were rinsed with PBS, dislodged, pelleted by centrifugation, and resuspended in the cell lysis buffer [10 mM HEPES; pH 7.5, containing 0.5 mM PMSF, 0.5 % Nonidet-40, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 10 mM KCl, together with the protease inhibitor cocktail (Sigma)] and left to swell on ice for 15–20 min with intermittent mixing. Tubes were vortexed to disrupt cell membranes followed by centrifugation for 10 min at 12,000g and 4 °C. The supernatant was discarded. The nuclei in the pellet were rinsed thrice with the cell lysis buffer and resuspended in the nuclear extraction buffer containing 20 mM HEPES (pH 7.5), 1 mM PMSF, 1 mM DTT, 1 mM EDTA, and 400 mM NaCl, with protease inhibitor cocktail, followed by incubation in ice for 30 min. After centrifugation at 12,000g for 15 min at 4 °C, the nuclear extract was collected. Determination of protein concentration of the nuclear extract was carried out using Bradford’s reagent (Bio-Rad, USA). The extract was immediately used for western blot.

For localization image analysis, after 24-h transfection, cells were fixed in 4 % paraformaldehyde in PBS for 15 min at room temperature. The cells were rinsed in PBS, postfixed, and made permeable in 0.5 % Triton X-100 at room temperature for 15 min. After rinsing with PBS, the cells were incubated in 3 % BSA for 1 h, washed in PBS, and incubated in anti-integrase-1 antibody (Santa Cruz Biotechnology) diluted with PBS containing 1 % BSA for 1 h at 37 °C in a humid chamber. Then, cells were washed for 30 min in three changes of PBS at room temperature and then incubated in FITC-conjugated antibody (Santa Cruz Biotechnology) diluted with PBS containing 1 % bovine serum albumin for 1 h at 37 °C in a humid chamber. Then, cells were washed as before. Cells were stained with PI (1 μg/ml) for 20 min, and pictures were taken under a confocal fluorescence microscope.

Results

Peptide Synthesis

The amino acid sequences of the peptides used in this study are presented in Table 1, and their molecular masses are presented in Fig. 1.

Table 1 Amino acid sequences of human and bovine lactoferricins, lactoferrins 1-11, and lactoferrampins
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Fig. 1
figure 1figure 1figure 1

Mass spectrometric analysis of peptides

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Assay of Nuclear Translocation of HIV-1 Integrase

Western blot results for HIV-1 integrase nuclear translocation assay indicated that human lactoferricin (peptide 1), human lactoferrin 1-11 (peptide 2), bovine lactoferrampin (peptide 6), and compound X (positive control) could significantly reduce HIV-1 integrase nuclear distribution. Reduced protein concentration of HIV-1 integrase/lamin B1 was observed as indicated by the attenuated HIV-1 integrase after treatment with band intensity human lactoferricin (peptide 1), human lactoferrin 1-11 (peptide 2), and bovine lactoferrampin (peptide 6). However, human lactoferrampin (peptide 3), bovine lactoferricin (peptide 4), and bovine lactoferrin 1-11 (peptide 5) and raltegravir did not inhibit HIV-1 integrase nuclear distribution (Fig. 2). A comparison with previously published data on HIV-1 integrase inhibitory activity revealed that only bovine lactoferrampin was capable of inhibiting nuclear translocation as well as activity of HIV-1 integrase.

Fig. 2
figure 2

Western blot results for HIV-IN translocation assay (upper panel) and graphical presentation of western blot results in Fig. 1 after scanning of band intensities (lower panel). At lower panel, left-hand-side graph is showing the concentration of HIV IN in nucleus; right-hand-side graph is showing the concentration of HIV IN in whole cells. Numbers 1 to 6 present results for peptides 1 to 6, respectively. Data represent mean ± SD (n = 3). Asterisk indicates statistically significant difference (p < 0.01) compared with the other treatments (control, peptide 3, peptide 4, and peptide 5) when analyzed by ANOVA

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Figure 3 shows green HIV integrase staining with FITC, red nucleus staining with PI, and merged picture obtained after treatment with the various peptides. Accumulation of HIV integrase in the cytoplasm signifying inhibition of nuclear translocation of HIV integrase was observed following treatment with human lactoferricin (peptide 1), human lactoferrin 1-11 (peptide 2), bovine lactoferrampin (peptide 6), and compound X (positive control) but not after treatment with human lactoferrampin (peptide 3), bovine lactoferricin (peptide 4), and bovine lactoferrin 1-11 (peptide 5). The merged picture shows merging of the green and red colors after treatment with human lactoferrampin (peptide 3), bovine lactoferricin (peptide 4), and bovine lactoferrin 1-11 (peptide 5), indicating absence of an inhibitory action on nuclear translocation of HIV integrase. The green fluorescence due to HIV integrase did not merge with the red nucleus staining with PI after treatment with human lactoferricin (peptide 1), human lactoferrin 1-11 (peptide 2), bovine lactoferrampin (peptide 6), and compound X (positive control) indicating an inhibitory action on nuclear translocation of HIV integrase.

Fig. 3
figure 3

Observation under a confocal fluorescence microscope for HIV-IN nuclear translocation assay. Human lactoferricin (peptide 1), human lactoferrin 1-11 (peptide 2), and bovine lactoferrampin (peptide 6) could reduce HIV-1 IN transport into cell nucleus. The white arrows pointed at the accumulation of HIV IN in the cytoplasm. Compound Y employed as a positive control in the present study was the next generation of compound X which was used as a positive control for the HIV-IN translocation assay

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Discussion

A great deal of effort has been dedicated to ascertain compounds with ability to inhibit nuclear translocation of HIV-1 integrase which is a focus of anti-HIV research. 3-(1,3-benzothiazol-2-yl)-8-{[bis(2-hydroxyethyl)amino]methyl}-7-hydroxy-2H-chrom en-2-one (D719) demonstrated HIV-1 integrase nuclear translocation inhibitory activity in cell imaging [23]. 1,4-bis(5-(naphthalen-1-yl)thiophen-2-yl) naphthalene [24] and wikstroelide obstructed nuclear translocation of HIV-1 integrase by attenuating the interaction between LEDGF/p75 and the enzyme. Importin α3 (Impα3) interacts with HIV-1 integrase via the (211) KELQKQITK and (262) RRKAK regions of the C-terminal domain of retroviral enzyme and plays a role in viral cDNA nuclear import [25]. Ivermectin hindered importin α/β-mediated transport of HIV-1 integrase and potently inhibited HIV-1 [26]. Mifepristone impeded nuclear translocation of HIV-1 integrase [27]. LEDGINs are HIV inhibitors targeting interaction between HIV integrase and LEDGF/p75 which is a coactivator of cellular transcription employed by HIV to tie the preintegration complex to the chromosome. Transportin-SR2 is utilized by HIV for nuclear import [28].

HIV-1 preintegration complex-associated integrase binds to the C-terminal domain of nucleoporin NUP153 for nuclear import of the preintegration complex [29]. The HIV-1 Rev protein facilitates nuclear export and prevents nuclear import of HIV-1 integrase [30]. Peptides that disrupt interaction of HIV-1 integrase with either importin α or TNPO3 impede nuclear import of the integrase-DNA complex and HIV-1 infection [31]. Amino acid residues in the C-terminal domain of HIV integrase play a role in HIV-1-cellular protein interaction. Importin alpha3, an HIV integrase-interacting cofactor, expedites nuclear import and replication of HIV-1 in cells undergoing cell division and also in nondividing cells [32].

Hence, the mechanism of inhibitory action of human lactoferricin, human lactoferrin 1-11, and bovine lactoferrampin on nuclear translocation of HIV-1 integrase awaits elucidation. We believe that the peptides inhibit IN nuclear translocation through blockade of p75/HIV-IN interaction. It is likely that these lactoferrin-derived peptides interact with some of the aforementioned entities. The efficacy of bovine lactoferrampin in attenuating HIV-1 integrase activity and preventing HIV-1 integrase nuclear translocation makes it a promising candidate for development into an anti-HIV-1 drug. Attempts have been made to search for natural products and synthetic compounds with an inhibitory action on activity of HIV enzymes including HIV-1 integrase and on HIV-1 integrase nuclear translocation [33–36]. The finding of bovine lactoferrampin with suppressive activity on both HIV-1 integrase activity and on HIV-1 integrase nuclear translocation may facilitate anti-HIV therapy.