Peptide PAP248-286 was synthesized using a standard solid- phase 9-fluorenylmethoxy carbonyl (Fmoc) method at GL Biochem Ltd. (Shanghai, China) and were purified by high-performance liquid chromatography (HPLC). HP, polyethyleneimine (PEI), biotin goat-anti-rabbit IgG, bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), thioflavin T (ThT), Congo Red Kits, biotinylated goat anti-rabbit IgG and FITC-goat-anti-rabbit-IgG were purchased from Sigma-Aldrich (St. Louis, MO, United States). The recombinant human CD4 protein and HIV-1_JR-FL gp120 were purchased from Sino Biological (Wayne, PA, United States).

The pNL4-3E-R-Luc plasmid, HIV-1 and VSV-G Env-encoding plasmids, the peGFP-Vpr plasmid, MT-2 cells, TZM-bl cells, CHO-WT cells, U87-CD4-CCR5 cells, U87-CD4-CXCR4 cells, HIV-1IIIB-infected H9 cells (H9/HIV-1IIIB), laboratory-adapted and primary HIV-1 strains, HL2/3 cells, HeLa cells, HeLa-CD4-LTR-β-gal cells, T20, AZT, AMD3100, Maraviroc, anti-p24 monoclonal antibody (183-12H-5C) and HIV immunoglobulin (HIV-IgG) were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. GHOST (3) Hi-5, HEK-293T cells and VK2/E6E7 cells were purchased from ATCC (Manassas, VA, United States). Plasmids encoding CXCR4-tropic HIV-1NL4-3, CCR5-tropic HIV-1SF-162, and dual-tropic HIV-181A and NL4-3 infectious clones were kindly provided by Jan Münch of Ulm University (Ulm, Badenwürttemberg, Germany). ProteoStat Amyloid Plaque Detection Kits were purchased from Enzo Life Sciences (Plymouth Meeting, PA, United States). Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors at the Nanfang Hospital by standard density gradient centrifugation by using Histopaque-1077 (Sigma, St. Louis, MO, United States).

After immunizing rabbits by PAP248-286 using conventional methods, the blood sera of rabbits containing specific polyclonal antibodies against PAP248-286 were collected by AbMax Biotechnology (Beijing, China). The rabbit anti-PAP IgG (API) was purified from the antisera by Protein A-Sepharose 4 Fast Flow column (Amersham Biosciences, Piscataway, NJ, United States). The concentration of API was detected by the BCA Protein Assay Reagent Kit (Pierce, Rockford, IN, United States). Furthermore, API was modified by HP as previously described. Briefly, saturated HP (1.19 M in DMSO) was divided into five aliquots, and then each portion was added into API solution [18.75 μM in 0.1 M phosphate buffer saline (PBS, pH 7.4)] at 12 min intervals. The final concentration of HP is 60 mM and pH value is 8.5. The mixture was kept for another 1 h at room temperature (RT), extensively dialyzed against PBS, and filtered through 0.45 μm syringe filters (Gelman Sciences, Ann Arbor, MI, United States).

The lyophilized PAP248-286 (>95% purity) was dissolved in PBS at a concentration of 3 mg/ml. SEVI fibrils were generated by incubating PAP248-286 with agitation at 1,400 rpm at 37°C for 48 h in an Eppendorf Thermomixer (Hamburg, Germany) as described previously. The binding of HP-API to PAP248-286 or SEVI fibrils were detected by ELISA and isothermal titration calorimetry (ITC).

For ELISA, wells of 96-half well polystyrene plates (Costar; Corning, Inc., Corning, NY, United States) were coated with PAP248-286 or SEVI fibrils at graded concentrations in 0.85 M carbonate-bicarbonate buffer (pH 9.6) at 4°C overnight. After blocking with 3% BSA in PBS at 37°C for 1 h, 12.5 nM HP-API was added into the wells and incubated at 37°C for another 1 h. Then, biotin-labeled goat-anti-rabbit-IgG (Sigma), SA-HRP and TMB were added into the reaction system successively. 1 N H₂SO₄ was used to terminate the reaction. The absorbance at 450 nm was measured by an ELISA reader (Ultra 384; Tecan, Research Triangle Park, NC, United States). API and normal rabbit IgG were used as controls.

The binding of HP-API to PAP248-286 was further detected by ITC assay using a NANO ITC (TA Instruments, New Castle, DE, United States) (Xun et al., 2015). The reference cells and the sample cells were stirred at 300 rpm at 37°C. A series of small aliquots of PAP248-286 solution (2.5 μl, 200 μM) were injected into a sample cell containing 300 μl of HP-API solution (6 μM) at intervals of 2 min for 20 injections. Blank ITC experiments were applied to correct heat-of-dilution effects. The enthalpy changes (ΔH) and the binding constant (K_d) of the interaction between HP-API and PAP248-286 in solution were calculated by a NANO ITC software. API was used as a control.

The graded concentrations of HP-API (600, 300, 150, 75, and 0 nM) were agitated with peptide PAP248-286 (3 mg/ml) at 1,400 rpm at 37°C for 48 h using an Eppendorf Thermomixer. The mixed samples were collected at different time points following agitation (0, 8, 16, 24, and 48 h) and were measured by ThT fluorescence assay, Congo red staining and transmission electron microscopy (TEM) as previously described (Chen et al., 2015). For ThT fluorescence assay, 5 μl mixed samples were stained by 195 μl ThT working solution (50 μM in PBS). Fluorescence intensity was measured immediately by an RF-5301 PC spectrofluorophotometer (Shimadzu). The excitation and emission wavelength were 440 nm (5 nm bandwidth) and 482 nm (10 nm bandwidth), respectively. For the Congo red assay, 10 μl mixed samples were added to 200 μl of Congo red solution contained in a Congo Red Kit (Sigma). And then the systems were incubated at RT for 2 min. Then the mixtures were centrifuged at 12,000 rpm for 5 min and the red-dyed fiber precipitates were dissolved in 50 μl DMSO. The absorbance at 490/650 nm was measured by an ELISA reader. The effects of HP-API on SEVI amyloid fibrils formation were further measured by TEM. In brief, SEVI fibrils (3 mg/ml) in the presence or absence of HP-API (300 nM) at different agitated time points (0, 8, 16, 24, and 48 h) were obtained and then sixfold diluted in PBS. The diluted samples were first deposited on glow-discharged, carbon-coated grids for 2 min, and then were negatively stained with 2% phosphotungstic acid for another 2 min. The morphologies of those amyloid fibrils samples were visualized at 20,000 magnifications with H-7650 transmission electron microscope at an accelerating voltage of 80 kV (Hitachi Limited, Tokyo, Japan). API was used as a control.

The secondary β-sheet structures of SEVI fibrils with or without HP-API at different agitated time points (0 and 48 h) were observed by CD spectroscopy in the far-UV spectral region between 198 and 260 nm using a Jasco 715 spectropolarimeter (Jasco Inc., Japan) equipped with thermostat-controlled cell housing and cells with a 1-mm path length. The samples containing 3 mg/ml SEVI fibrils with 300 nM HP-API were diluted 15 times in PBS. All CD spectra were collected at RT and corrected for solution baseline via the subtraction of the corresponding blank samples. The quantities of SEVI amyloid fibrils with different secondary structures were estimated from the molar residue ellipticity using Jasco software utilities as previously described (Chen et al., 2015). API was used as a control.

The enhancing activities of SEVI fibrils in the presence or absence of HP-API was further detected by a viral infection assay (Chen et al., 2015). Three kinds of HIV-1 infectious clones were produced by transfecting HEK-293T cells with proviral DNA expression plasmids (CXCR4-tropic NL4-3, CCR5-tropic SF-162, and dual-tropic 81A and NL4-3) via PEI transfection as previously described. SEVI fibrils (3 mg/ml) with or without HP-API (300 nM) were first agitated to allow fibril formation and collected at interval agitated time points (0, 8, 16, 24, and 48 h) at 1,400 rpm at 37°C. All samples were centrifuged at 12,000 rpm for 5 min and discarded the supernatant to remove the remaining HP-API. The enhancing activities of SEVI fibrils on infection by HIV-1 infectious clones (HIV-1_NL4-3, HIV-1_SF-162, and HIV-1_{81A and NL4-3}) were determined as previously described. In brief, 1 × 10⁵/ml TZM-bl cells were seeded into 96-well microtiter plates and incubated at 37°C overnight. 2 ng of p24 antigen of viruses were incubated with the diluted SEVI fibril samples (final concentration is 50 μg/ml) at RT for 10 min prior to the addition of the mixture to TZM-bl cells. After 3 h post-infection, the culture supernatants were discarded and the fresh mediums were added for avoiding cytotoxicity. At 72 h post-infection, the cells were collected and lysed with the lysing reagent included in the luciferase kit (Promega Corp., Madison, WI, United States). Aliquots of cell lysates were transferred to 96-well flat bottom luminometer plates (Costar) and added to luciferase substrate (Promega). And then the infectious activities of those fibril samples were measured in an Ultra 384 luminometer (Tecan). API was used as a control.

The effects of HP-API on the binding of HIV-1 virions to SEVI fibrils were first detected by a virus pull-down assay (Chen et al., 2015). HIV-1 Env-pseudotyped viruses (HIV-1_JR-FL), infectious clones (HIV-1_NL4-3, HIV-1_SF-162 and HIV-1_{81A and NL4-3}) and a primary HIV-1 strain (93BR020) were used. Briefly, 200 μg/ml SEVI fibrils were mixed with an equal volume of HP-API at graded concentrations (2,000, 500, 125, and 0 nM) and incubated at 37°C for 30 min. Furthermore, 10 μl viruses (about 100 ng/ml p24) were incubated with the mixed samples at 37°C for another 30 min. Then, the mixtures were centrifuged at 5,000 rpm for 3 min to pellet the fibrils and bound virions. The pellets were dissolved in PBS and mixed with equal volumes of 5% Triton X-100 at 4°C overnight. The virus lysates in the pellets were assayed for p24 antigens by ELISA as described before. Equal volumes of viruses (10 μl) were added to the ELISA reaction system and were mixed with 10 μl 5% Triton X-100 for testing the total p24 antigens. The proportion of p24 antigens in the pellets were then calculated (Pellets p24/Total p24). API was used as a control.

The inhibitory activities of HP-API against three different laboratory-adapted and primary HIV-1 strains [HIV-1 IIIB (X4), Bal (R5) and 93BR020 (X4R5)] in the presence of preformed SEVI fibrils were detected, respectively (Xun et al., 2015). Briefly, 1 × 10⁵/ml TZM-bl cells were seeded and incubated at 37°C overnight. The SEVI fibrils produced as described above were centrifuged 12,000 rpm for 5 min, and then the pellets were dissolved in fresh medium (final concentration is 50 μg/ml). The resuspended SEVI fibrils were incubated with HP-API at graded concentrations at 37°C for 30 min. Then, the mixture further co-incubated with 2 ng of p24 of viruses at RT for 10 min prior to the addition of the mixture to TZM-bl cells. After 3 h post-infection, the culture supernatants were changed with fresh medium. At 72 h post-infection, the luciferase activity was measured. The EC₅₀ values were calculated using the Calcusyn software, kindly provided by Dr. T. C. Chou at Sloan-Kettering Cancer Center (New York, NY, United States).

The antiviral activities of HP-API against infection by different laboratory-adapted HIV-1 strains (HIV-1_IIIB and HIV-1_Bal), primary HIV-1 viruses and drug-resistant HIV-1 strains (including HIV entry inhibitors and reverse transcriptase inhibitors resistant strains) without SEVI fibrils were analyzed as described elsewhere (Li et al., 2010). T20 was used as a positive control. Briefly, the graded concentrations of HP-API were incubated with 100 TCID₅₀ (50% tissue culture infective dose) of different HIV-1 strains at 37°C for 30 min. MT-2 cells were used as target cells and p24 antigen were measured by ELISA after 4 days post-infection for detecting the inhibition of infection by HIV-1_IIIB (X4 strain) and HIV-1 drug-resistant strains including T20 and A17-resistant strains. TZM-bl cells were used as target cells and the luciferase activities were measured at 72 h post-infection for detecting the inhibition of infection by HIV-1_Bal (R5 strain). PHA-stimulated PBMCs were selected as target cells and p24 antigen were measured by ELISA after 7 days post-infection for assessing the antiviral activities on HIV-1 primary strains. The EC₅₀ values were calculated using the CalcuSyn software.

To further verify the possible target(s) of HP-API, the antiviral activity of HP-API against HIV-1_IIIB infection on being pre-incubated either cells or virus particles prior to infection were analyzed. Briefly, the graded concentrations of HP-API were pre-incubated with 100 TCID₅₀ of HIV-1_IIIB or MT-2 cells at 37°C for 30 min, respectively. After 4 days infection, TZM-bl cells were used as target cells and the luciferase activities were measured for detecting the inhibition of infection. The EC₅₀ values were calculated using the CalcuSyn software.

The in vitro cytotoxicities of HP-API on HIV target cells (MT-2, U87-CD4-CXCR4, and U87-CD4-CCR5) and the human vaginal epithelial cells (VK2/E6E7) were measured by MTT assay. Briefly, equal amounts of HP-API at graded concentrations with cells described above were co-cultured at 37°C for 3 days. Then, 10 μl of 5 mg/ml MTT solution was added to each well. After incubating at 37°C for 4 h, the supernatants were discarded gently and 100 μl of DMSO was added for 10 min. The absorbance at 570 nm was measured by an ELISA reader. API was used as a negative control. The 50% cytotoxicity concentrations (CC₅₀) were calculated using the CalcuSyn software.

A time-of-addition assay was performed as previously described (Li et al., 2010). HP-API, API and the corresponding control anti-HIV compounds were added to the infection systems at different post-infection (0, 0.5, 1, 2, 4, 6, and 8 h). The antiviral activities of those compounds on the infection by HIV-1_IIIB and HIV-1_Bal were determined as described above. A nucleoside reverse transcriptase inhibitor (NRTI), AZT, was used as a positive control for inhibiting both HIV-1_IIIB and HIV-1_Bal infection. A CXCR4 co-receptor inhibitor, AMD3100, was chose as a negative control for inhibiting HIV-1_IIIB infection. In addition, a CCR5 co-receptor inhibitor, Maraviroc, was selected as a negative control for inhibiting HIV-1_Bal infection.

The HIV-1-mediated cell–cell fusion assay was measured by two different methods as previously described (Li et al., 2010). MT-2 cells expressing CD4 receptor and CXCR4 co-receptor were used as the target cells. HIV-1_IIIB infected H9 (H9/HIV-1_IIIB) cells or CHO-WT cells expressing gp120/gp41 were used as the effector cells. Briefly, MT-2 cells were incubated with the effector cells in the absence or presence of HP-API at graded concentrations at 37°C for 2 h (Calcein-AM labeled H9/HIV-1_IIIB cells) or 48 h (CHO-WT cells). The fused and unfused Calcein-labeled cells or the syncytia were counted under an inverted fluorescence microscope (Zeiss, Germany) or an inverted microscope, respectively. Four fields per well were examined randomly. T20 was used as a positive control.

In order to characterize the antiviral activity of HP-API on a single-cycle HIV-1 infection, HIV-1 viruses pseudotyped with different HIV-1 envelope proteins in comparison to VSV-G pseudovirus and H5N1/TG pseudovirus were generated as described above (Li et al., 2010). Briefly, HEK-293T cells were cotransfected with pNL4-3E^-R^-Luc plasmid and HIV-1 Env-encoding plasmids derived from HIV-1_HXB2 (X4 strain), HIV-1_JR-FL (R5 strain), VSV-G Env-encoding plasmid or H5N1/TG Env plasmids by PEI transfection reagent. The inhibitions of HP-API against HIV-1, VSV-G and H5N1/TG pseudotyped virus infection were detected as described previously. Briefly, 1 × 10⁵/ml U87-CD4-CXCR4 cells, U87-CD4-CCR5 cells or MDCK cells were seeded in a 96-well plate at 37°C overnight. Then, HP-API at graded concentrations was preincubated with 100 TCID₅₀ HIV-1, VSV-G and H5N1/TG pseudotyped virus for 30 min at 37°C. And then, the mixtures were transferred to the wells with the target cells. At 48 h post-infection, the luciferase activity was detected as described above. T20 was used as a positive control.

The binding of HP-API to cells expressing HIV-1 Env or CD4 receptor was detected by ELISA, flow cytometric analysis and surface plasmon resonance (SPR) analysis.

For ELISA and flow cytometric analysis, Hela-CD4-LTR-β-gal cells that express CD4 receptor were used to analyze the binding of HP-API to CD4 receptor. HL2/3 cells expressing high levels of HIV-1 Env proteins were used to evaluate the binding of HP-API to HIV-1 Env. HeLa cells bearing neither HIV-1 Env nor CD4 were chose as a negative cell control. API was also used as a negative drug control (Li L. et al., 2013).

For ELISA, 2 × 10⁵/well Hela-CD4-LTR-β-gal or HL2/3 cells were seeded at 37°C overnight, followed by fixing with 4% formaldehyde at RT for 15 min. After three washes, wells were blocked by 1% dry fat-free milk at 37°C for 1 h. HP-API at graded concentration was added and incubated for another 1 h at 37°C. Biotin-labeled goat-anti-rabbit-IgG, SA-HRP and TMB were added successively. 1 N H₂SO₄ was used to terminate the reaction. The absorbance at OD₄₅₀ was measured by an ELISA reader.

For flow cytometric analysis, 1 × 10⁶/well Hela-CD4-LTR-β-gal or HL2/3 cells were suspended in PBS containing 10% goat serum (PBS-GS) and incubated at 4°C for 1 h. 10 nM HP-API was added to cells and incubated at 4°C for 1 h. After three washes, FITC-goat-anti-rabbit-IgG was added and incubated at 4°C for another 1 h. Then, the cells were washed six times and resuspended in 500 μl of wash buffer, followed by flow cytometry analysis.

The affinity of binding of HP-API to HIV-1 gp120 or CD4 was measured using BIAcore T100 system (GE Healthcare, Sweden) as previously described (Avramis et al., 2009). Briefly, gp120 derived from HIV-1_JR-FL (20 μg/ml) or CD4 (6 μg/ml) was immobilized onto a CM5 sensor chip, HP-API or API (5.00, 2.50, 1.25, 0.63, 0.31, and 0.16 nM) was injected at a flow rate of 20 μl/min with a contact time of 2 min and a dissociation time of 2.5 min. The running buffer was water. The chip platform was regenerated with 10 mM HEPES, 150 mM NaCl, and 0.01% vol/vol Tween 20 (pH 7.4) and washed with the running buffer. A binding affinity (K_D) value was calculated using BIAcore software (GE Healthcare).

A specific antibody of PAP248-286 or SEVI fibrils might be a good choice for blocking the SEVI-mediated enhancement of HIV infection due to its antigen-antibody specific effects. Here, we first prepared 3-hydroxyphthalic anhydride-modified rabbit anti-PAP IgG (HP-API) by chemical modification and further detected the binding of HP-API to the peptidic fragment of PAP248-286 or SEVI fibrils. Rabbit anti-PAP IgG (API) was used as a positive control antibody. Results showed that both HP-API and API could bind to the peptide PAP248-286 (Figure 1A) or SEVI fibrils (Figure 1B) in a dose-dependent manner by ELISA. Neither PAP248-286 nor SEVI fibrils could bind to the negative control antibody, a normal rabbit IgG (Figures 1A,B). Furthermore, ITC was conducted to obtain the thermodynamic parameters of the interaction between HP-API and PAP248-286 in solution. HP-API or API bound to PAP248-286 with a binding constant (K_d) of 7.92 × 10^-7 M (Figure 1C) or 7.72 × 10^-7 M (Figure 1D), respectively, which indicated that both HP-API and API can bind to PAP248-286 with high affinity.

The effects of HP-API on the kinetics of SEVI fibril formation formed by peptide PAP248-286 were further detected by ThT and Congo red assay. Both HP-API and API interfered with fibrillogenesis of PAP248-286 in a dose-dependent manner, as assessed by ThT (Figure 2A) and Congo red assay (Figure 2B). At the high concentrations (600 and 300 nM), HP-API and API could completely abrogate the formation of SEVI fibrils in vitro even at 48 h after agitation, while PAP248-286 alone showed typical fibril growth with a lag time of approximately 8–16 h after shaking. Those results were further observed by TEM directly (Figure 2C). At 8 h after agitation, branching-needle-like long fibrils were revealed in the solution of PAP248-286 alone (3 mg/ml), suggesting that PAP248-286 slowly forms SEVI fibrils under agitation. However, no clear amyloid fibrils were imaged in the solution of PAP248-286 in the presence of HP-API or API (300 nM) even after shaking 48 h. All above results suggested that both HP-API and API could antagonize SEVI fibrils formation obviously.

The high-level association β-sheet secondary structure in proteins has been confirmed to be a distinguishing feature in the formation of the protein aggregates and amyloid fibrils in many diseases (Nabers et al., 2016). Here, we further detected the conformational changes associated with the aggregation of PAP248-286 in the presence or absence of HP-API shown by CD spectroscopy distinctive of random coils. As illustrated in Figure 2D, the spectra of PAP248-286 with or without HP-API or API at 0 h after agitation exposed a characteristic random coiled structure. After shaking at 37°C for 48 h, the spectrum of PAP248-286 alone revealed a typical β-sheet component, containing a single minimum at approximately 218 nm (Figure 2E). Of note, the spectra of PAP248-286 in the presence of HP-API or API at 48 h after agitation did not manifest any stable conformation, indicating that β-sheet aggregation had not occurred, which demonstrated that the addition of HP-API or API failed to induce the structural transition of PAP248-286 from random coil to a cross-β-sheet structure (Figure 2E).

We next focused on the effects of HP-API on SEVI-mediated enhancing activities of different infectious HIV-1 clones using a viral infection assay. For avoiding the effects of free HP-API on viral infectivity, all collected samples at different agitated time points (0, 8, 16, 24, and 48 h) as described above were centrifuged and the supernatants were discarded. The samples of PAP248-286 alone collected after shaking from 16 to 48 h could increase the infectivity of three infectious HIV-1 clones significantly, including CCR5-tropic HIV-1_SF-162 (Figure 3A), CXCR4-tropic HIV-1_NL4-3 (Figure 3B) and dual-tropic HIV-1_{81A and NL4-3} (Figure 3C). The enhancing temporal trends by a viral infection assay are coincided with the formation of SEVI amyloid fibrils by ThT, Congo red, and TEM assay (Figure 2). In contrast, the samples of PAP248-286 with HP-API or API (300 nM) were not contributed to the HIV-1 infection enhancement of three infectious HIV-1 clones even at agitated 48 h after agitation (Figures 3A–C). Those results further certified that HP-API could induce the loss of SEVI-mediated HIV-1 infection enhancing activity due to inhibiting amyloid fibrils formation by PAP248-286.

All above results demonstrated that HP-API could abrogate the formation of SEVI amyloid fibrils and block SEVI-mediated HIV infection enhancing activity because of the special antigen-antibody interaction. Here, we further detected the inhibitory activities of HP-API against infection by laboratory-adapted HIV-1 strains and primary HIV-1 isolates in the presence of preformed 50 μg/ml SEVI fibrils using a viral infection assay. As shown in Figure 4, SEVI fibrils could obviously enhance HIV-1 infection by different laboratory-adapted and primary strains up to 54.60 times. HP-API showed strongly inhibitory activity against infection by all tested strains, including HIV-1_IIIB (Figure 4A), HIV-1_Bal (Figure 4B), and HIV-1_93BR020 strains (Figure 4C) in the presence preformed SEVI fibrils. The effective concentration for 50% inhibition (EC₅₀) values of HP-API with preformed SEVI fibrils were 17.61–45.01 nM. Even though unmodified API could slightly decrease the enhanced times of HIV-1 infection by SEVI fibrils, it is regrettable that API did not exhibited anti-HIV-1 activities in the presence of SEVI fibrils even at the concentration of 300 nM (Figure 4).

Since the infectivity-enhancing activity of SEVI fibrils is associated with the electrostatic attraction between HIV-1 particles and SEVI fibrils (Roan et al., 2009, 2011), we observed the effects of HP-API and API on the binding of HIV-1 virions to SEVI fibrils by a virus pull-down assay. As demonstrated in Figures 5A,B, SEVI fibrils alone bound to more than 90% of the input HIV-1 virions including HIV-1 pseudoviruses, HIV-1 infectious clones and primary HIV-1 strain. It is gratifying that HP-API could significantly inhibit the binding of all of the HIV-1 virions to SEVI fibrils in a dose-dependent manner (Figure 5A). However, only the highest concentration of API showed slight inhibition on the binding of HIV-1 virions to SEVI fibrils (Figure 5B). Above results indicated that HP-API abrogated SEVI-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes.

In addition to abrogating the ability of SEVI amyloid fibrils and weakening HIV infection indirectly, HP-API also displayed a strong antiviral activity directly in vitro. We first evaluated the anti-HIV-1 activities of HP-API on infection by different laboratory-adapted and primary HIV-1 isolates. The unmodified API was used as a negative control, an HIV entry/fusion inhibitor T20 was chosen as a positive control. As shown in Table 1, HP-API showed extremely high inhibitory activities on all tested primary HIV-1 isolates with EC₅₀ values in the low nM range. For the representative primary HIV-1 isolates of clades A to F and group O (X4, R5, or X4R5), the EC₅₀ values of HP-API were ranged from 1.48 to 72.96 nM. However, the control unmodified API had no obviously inhibitory activity against any of the above viruses even at the highest tested concentration (3,000 nM). The anti-HIV-1 activities of the positive control T20 were also showed in Table 1. On the other hand, HP-API-mediated anti-HIV-1 activity was not significantly difference regardless of being pre-incubated either target cells or virus particles. HP-API was pre-incubated cells prior to infection with an EC₅₀ at 2.34 ± 0.41 nM (Supplementary Figure 1B), which was similar to being pre-incubated with virus particles with an EC₅₀ at 1.48 ± 0.39 nM (Supplementary Figure 1A).

We further detected the inhibition of HP-API on infection by the A17 strain, a non-nucleoside reverse transcriptase inhibitor (NNRTI) resistant strain, and by HIV entry/fusion inhibitor T20-resistant strains. Results showed that the drug-resistant HIV-1 strains were highly resistant to such drugs as UC781 (a NNRTI) and T20, as expected; therefore, the EC₅₀ values could not be determined, even at concentrations up to 10 μM or 2 μM, respectively (Table 2). Notably, HP-API exhibited similar antiviral activity on wild-type HIV-1 strains and two types of drug-resistant HIV-1 strains, with EC₅₀ values at the same approximate nM range.

The cytotoxicity of HP-API on target cells (MT-2, U87-CD4-CXCR4, and U87-CD4-CCR5 cells) and vaginal epithelial cells (VK2/E6E7) was detected by MTT assay. In Table 3, both HP-API and API at concentrations up to 3 μM had no cytotoxicity against any tested cells, which was more than 700 times its EC₉₀ against HIV-1_Bal infection. The low cytotoxicity indicated that HP-API might be a safe microbicide when administered to the human vagina or anus.

Our previous studies have reported that some anhydride modified proteins might be HIV entry/fusion inhibitors by blocking viral entry (Li et al., 2010; Li L. et al., 2013; Li M. et al., 2013). In order to illuminate the role of HP-API on the stages of HIV-1 infection, we first carried out a time-of-addition assay for evaluating the antiviral activities when HP-API was added to cells at different intervals post-infection. As shown in Figures 6A,B, a fixed concentration of the nucleoside reverse transcriptase inhibitor, AZT, showed similar antiviral activity against both HIV-1_IIIB and HIV-1_Bal when it was added to the target cells before and after viral infection. However, a fixed concentration of HIV entry inhibitors, including AMD-3100 (against HIV-1_IIIB) and Maraviroc (against HIV-1_Bal), exhibited significantly decreased inhibitory activity when they were added into cells at 0.5 to 2 h post-infection. Of note, HP-API showed inhibitory profiles similar to those of HIV entry inhibitors (Figures 6A,B), suggesting that HP-API only exerts its antiviral activities at the early stage of HIV-1 infection.

HIV Env-mediated membrane fusion is an early and a critical step of HIV entry into a target cell. We further tested the inhibition of HP-API on HIV-1 Env-mediated cell–cell fusion. Here, we chose non-infectious CHO-WT cells or infectious H9/HIV-1_IIIB cells as the effector cells and MT-2 cells as the target cells for simulating the early steps of the HIV-1 replication cycle. The numbers of syncytia or fluorescence-labeled fused cells were counted under an inverted microscope. As shown in Figures 7A,B, HP-API inhibited both the syncytium formation and the dye transfer between non-infectious or infectious effector cells and the target MT-2 cells in a dose-dependent manner. The EC₅₀ values of HP-API were 167.85 nM within a non-infectious system and 64.18 nM within an infectious system, respectively. The positive control drug, T20, inhibited cell–cell fusion, with EC₅₀ about 3.33 and 15.33 nM, respectively. These results indicated that HP-API might inhibit viral entry by blocking HIV-1 Env-mediated cell–cell fusion.

A single-round entry assay is another commonly used method to identify a HIV entry/fusion inhibitor by assessing the effects on HIV-1 virus–cell fusion directly. Therefore, we further tested the inhibitory activity of HP-API on virus–cell fusion using pseudotyped viruses expressing HIV-1_JR-FL (R5 strain) and HIV-1_HXB2 (X4 strain) Env. For analyzing the specificity of HP-API on HIV-1 Env, VSV-G pseudovirus expressing VSV Env G-protein and H5N1/TG pseudovirus expressing NA and HA from influenza were used as negative controls. T20 was used as a positive control drug. Similar to cell–cell fusion, HP-API inhibited the infection by both pseudotyped HIV-1_JR-FL (Figure 8A) and HIV-1_HXB2 (Figure 8B) viruses significantly, with the EC₅₀ values were 1.90 nM and 2.25 nM, respectively. Here, we further verified that HP-API could not block a single round entry of both VSV-G pseudovirus and H5N1/TG pseudovirus even at the concentration up to 200 nM (Figures 8C,D), indicating the specific role of HP-API in HIV-1 membrane fusion.

The binding of HIV-1 Env surface subunit gp120 to CD4 receptor on a CD4⁺ target cell is the first step leading to HIV-1 viral entry. To further elucidate the binding target(s) of HP-API, the interaction of HP-API with gp120 or CD4 was examined using ELISA and flow cytometry assay. HL2/3 cells expressing high levels of HIV-1 Env proteins and HeLa-CD4-LTR-β-gal cells expressing CD4 receptor were used here. HeLa cells expressing neither HIV-1 Env nor CD4 receptor were used as a negative control. The dose-dependent binding effects of HP-API on both HL2/3 and HeLa-CD4-LTR-β-gal cells were displayed by ELISA (Figure 9A), suggesting its binding to both HIV-1 Env and CD4 receptor. However, the negative control, API, could not bind to above cells (Figure 9B). Similar results showed by flow cytometry assay. HP-API could significantly bind to both HL2/3 and HeLa-CD4-LTR-β-gal cells, while it had only background binding to HeLa cells (Figure 9C).

For further determining if HP-API indeed binds specifically to both CD4 and gp120, the interaction of HP-API with gp120 from the HIV-1_JR-FL or recombinant human CD4 was examined using a SPR assay. As shown in Figures 10A,C, HP-API could bind to both gp120 and CD4 with a binding affinity (K_D) of 5.27 × 10^-7 M and 4.43 × 10^-8 M, respectively. However, API could neither bind to gp120 nor CD4 even at the concentration of 5 μM (Figures 10B,D). These results confirmed that HP-API could bind to both gp120 and CD4 specifically and then interfere with their interaction, resulting in the inhibition of HIV-1 entry.