The epigallocatechin gallate (EGCG) is the most abundant catechin in green tea.1 Because of its unique polyphenolic structure, EGCG has been shown to have antioxidant and anti-inflammation effects.1,2 In addition, EGCG has antiviral functions on a number of viruses, including influenza A virus, hepatitis B virus, hepatitis C virus, herpes virus-1/2, and HIV.3 EGCG could block HIV entry into target cells and inhibit several steps of the viral replication, including virion destruction, suppression of viral reverse transcriptase, protease activity, and proviral DNA integration.4,5 Hauber et al6 found that EGCG could counteract semen-mediated enhancement of HIV infection in vitro.
During sexual intercourse, semen of HIV-infected men functions as a carrier of HIV, as their semen contains the virus.7 Tachet et al8 showed that a majority of HIV-infected men had lower HIV RNA levels in seminal plasma than in blood plasma. However, because some antivirals do not penetrate semen as effectively as they do blood,9 a growing number of studies have shown the persistence of HIV RNA and DNA in semen of individuals receiving highly active antiretroviral therapy.10–12 Therefore, semen might harbor replication-component virus that can be sexually transmitted.9 Men who had higher viral loads in semen relative to their plasma viral loads were shown to have higher rates of sexual transmission risk.13 Higher semen levels of HIV RNA were also noted in the semen of HIV-infected subjects with low CD4 cell counts, suggesting a high risk of HIV transmission in this population.14 In addition, semen has been reported to impair the microbicide-mediated antiviral efficacy15 and modulate the cervicovaginal microenvironment and local immunity, likely resulting in mucosal changes that enhance susceptibility to HIV infection.16 Further studies reported that in human semen, proteolytic peptide fragment of prostatic acid phosphatase (PAP248-286) can form amyloid fibrils, which could potently enhance HIV infection in vitro.17–19 Therefore, PAP248-286-formed amyloid fibrils were termed semen-derived enhancer of viral infection (SEVI).19 Because SEVI contains a high abundance of positively charged lysine and arginine residues,20 it helps the capture of negatively charged HIV particles and promotes viral attachment to the target cells.19,21–23 In addition to PAP248-286, other semen-derived peptide fragments (PAP85-120, SEM1, and SEM2) have also been identified to have the ability to form amyloid fibrils that facilitate HIV infection.17,22,24
Unlike in vitro studies, which revealed that human SEVI could enhance HIV or SIV infection,19,25 in vivo studies with animals reported conflicting data.25,26 Although human SEVI could enhance HIV infection of hCD4/hCCR5-transgenic rats by tail vein injection,19 there was little effect of human SEVI on rectal HIV transmission in humanized mice.26 Also, SEVI showed little effect on vaginal simian immunodeficiency virus (SIV) transmission in rhesus macaques.25 These conflicting in vivo observations could be due to the different animal species used in these studies. In addition, the high viral inoculum used in these studies may also have affected the magnitude of SEVI-mediated enhancement, which was inversely correlated to the virus inoculum in vitro.19 Furthermore, the use of human SEVI for the animal studies might also be a contributing factor for the discrepancy in the findings of the studies.25 Therefore, given the importance and significance of the macaque models in HIV/AIDS research, we were interested in the studying whether macaque SEVI could enhance SIV or simian/human immunodeficiency virus (SHIV) infection of macaque peripheral blood mononuclear cells (PBMCs). We also investigated the effect of EGCG on macaque SEVI-mediated enhancement of SIV or SHIV infection.
Epigallocatechin gallate (EGCG) (purity ≥95%), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Congo-red, and thioflavin T (ThT) were purchased from Sigma-Aldrich (St Louis, MO). EGCG was dissolved in water at the concentration of 20 mM and aliquots were stored at −20°C. MTT was dissolved in phosphate-buffered saline (PBS, pH 7.4) and aliquots were stored at −20°C. Congo-red and ThT were dissolved in phosphate-buffered saline (PBS, pH 7.4) and stored in a light-resistant container at ambient temperature.
Macaque PAP248-286 Nucleotide Sequences Verification and Peptides Synthesis
The macaque PAP248-286 peptides (Indian macaque subspecies) are coded by the nucleotide sequences within 2 exons (26255-26387; 31256-31338; chromosome 2). Because we used PBMCs from rhesus macaque of Chinese origin, we first verified the nucleotide sequences of PAP248-286 peptides from Chinese origin rhesus macaque by polymerase chain reaction. Two sets of primers flanking the 2 exons by 76-bp upstream or 178-bp downstream were used to amplify the exons with flanking regions (Fig. S1A, http://links.lww.com/QAI/A990). Primer-1S: GAGGACAGTCATACAAATACAC, 1AS: GACAGGGTATTATGTACCAGGT. Primer-2S: GGACTTAGGATTCCTTACCTAT, 2AS: CTTAAAGGCCAGAATGATGCAT. The primers were synthesized and the amplified products were sequenced by Tianyi Huiyuan Life Science and Technology, Inc. (Wuhan, China). Genomic DNA was extracted from PBMCs or lymph nodes of the Chinese macaque. It was found that the PAP248-286 nucleotide sequences of the 2 macaque subspecies are identical (Fig. S1A, http://links.lww.com/QAI/A990). The macaque PAP248-286 peptides were then synthesized by GL Biochem (Shanghai, China) (according to GenBank accession no. AAZ82249, GIHKQKEKSRLQGGVLVNEILNHMKRATQMPSYKKLIMY) (Fig. S1B, http://links.lww.com/QAI/A990). Synthetic peptides (purity ≥98%) were characterized by high-performance liquid chromatography and mass spectrometry (data not shown). Lyophilized peptides were dissolved in PBS at a concentration of 5 mg/mL and aliquots were stored at −20°C.
Fibrils Formation and Characterization
The macaque SEVI was generated through overnight agitation of macaque PAP248-286 at 37°C and 1400 rpm by a thermomixer (Eppendorf, Germany). The macaque SEVI fibrils were characterized by Congo-red staining, ThT fluorescence assay,27 and transmission electron microscopy (TEM).19 Briefly, 5 μL of amyloid fibrils from macaque SEVI (1 mg/mL) with or without EGCG treatment was incubated with 100 μL of Congo-red solution (20 μg/mL in PBS) for 10 minutes at room temperature. The mixture was centrifuged for 5 minutes at 14,000 rpm, and the supernatant was removed. The pellets were dissolved in 100 μL dimethyl sulfoxide (DMSO), and the absorbance was read at 490–650 nm in a SpectraMax i3x microplate reader (Molecular Devices, Sunnyvale, CA).6 For ThT fluorescence assay, 5 μL of macaque SEVI (1 mg/mL) with or without EGCG treatment was mixed with 95 μL of ThT (50 μM in PBS). The mixture was then analyzed for the fluorescence intensity was measured by excitation at 440 nm and emission at 482 nm.27 For characterization of the fibrils by TEM, suspension of macaque SEVI-amyloid fibrils (100 μg/mL) in the presence or absence of EGCG (100 μM) was dropped onto the 200-mesh carbon-coated copper grids for 5 minutes. The grids were then stained with 2% aqueous uranyl acetate for 2 minutes. Fibrils were imaged by an H-8100 transmission electron microscope (Hitachi, Tokyo, Japan).19
TZM-bl cell line was obtained from the NIH AIDS Reagent Program (Bethesda, MD). The cells are highly sensitive to infections with different strains of HIV, SIV, or SHIV and enable simple and quantitative analysis of HIV, SIV, or SHIV using either β-galactosidase or luciferase as a reporter.28 The TZM-bl cells were cultured at 37°C with 5% CO2 in Dulbecco's modified Eagle's culture medium (DMEM; Gibco, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 50 units/mL penicillin, 50 μg/mL streptomycin, and 25 mM HEPES (Gibco). Before infection, cells (104) were plated in a 96-well plate for overnight. The PBMCs were isolated from blood of healthy Chinese rhesus macaques using the Ficoll-Paque PLUS (GE Healthcare Life Sciences) method as per manufacturer's instruction. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Wuhan University School of Medicine in accordance with the regulations of the National Institutes of Health “Guide for the Care and Use of Laboratory Animals.” The rhesus macaque PBMCs (macaque PBMs) in the 96-well plate (0.5 × 106 cells) were activated by phytohemagglutinin (PHA) and interleukin-2 (IL-2) in RPMI1640 medium for 72 hours before viral infections.
Cytotoxicity Assay of Macaque SEVI and EGCG
TZM-bl cells (104 cells per well) or PHA/IL-2-activated macaque PBMCs (0.5 × 106 cells per well) were cultured in a 96-well plate overnight and then treated with various concentrations of macaque SEVI or EGCG for 3 hours. After washed once with PBS, the cultures were further incubated for another 48 hours (for TZM-bl) or 6 days (for macaque PBMCs). Subsequently, MTT (5 mg/mL) was added to the cell cultures (1:10 vol/vol) and incubated for 3 hours. Finally, the culture media were discarded and 100 μL of DMSO was added to dissolve the formazan crystals. The plate was then read on a SpectraMax i3 microplate reader (Molecular Devices) at OD490-650 nm.19
The SIVmac239, SIVmac251, and SHIVSF162P3N were obtained from the NIH AIDS Reagent Program (Bethesda, MD). SHIVSF162P3N derived from the HIVSF162 primary isolate was29–31 generously provided by Dr. Cecilia Cheng-Mayer (The Aaron Diamond AIDS Research Center, New York, NY). Equal amount p27 of the viruses was mixed with different concentrations (0, 5, 10, 25, or 50 μg/mL) of macaque SEVI and incubated at 37°C for 1 hour. For EGCG treatment, the mixture of the virus and macaque SEVI was incubated with different concentrations (10, 25, or 50 μM) of EGCG or PBS for 30 minutes. The mixture was then added to infect TZM-bl cells or macaque PBMCs, respectively. Three hours after the viral infection, the cells were washed once by PBS and replenished with fresh medium. For TZM-bl cells, the culture media were discarded at 48 hours after infection and the cells were lysed in 40 μL of cell culture lysis buffer (Promega). Luminescence was measured by a SpectraMax i3x reader (Molecular Devices), and luciferase activity was expressed as relative light units per second (RLU/s) according to the manufacturer's protocol (Promega). For macaque PBMCs, 50% of the culture media was collected every 48 hours after infection and the cultures were replenished with fresh media. Finally, cellular RNA was harvested at postinfection day 6 for determination of the intercellular copies of the SIV gag gene by the quantitative real-time polymerase chain reaction.
Nucleotide Sequences of Macaque PAP248-286
The macaque PAP248-286 is coded by the nucleotide sequences within 2 exons (26255-26387; 31256-31338) on chromosome 2 (Fig. S1A, http://links.lww.com/QAI/A990). To verify whether the coding sequences of Chinese macaque PAP248-286 are the same as those of the Indian macaque, we designed 2 sets of primers flanking the 2 exons, which amplify the corresponding nucleotide sequences of the Chinese macaque genome (Fig. S1A, http://links.lww.com/QAI/A990). As shown in Figure S1A, http://links.lww.com/QAI/A990, the amplified Chinese macaque coding sequences of PAP248-286 by the primers are identical to those of the Indian macaque genome. Similarly, the amino sequences of PAP248-286 are identical in both Indian macaque and Chinese macaque. We thus synthesized the peptides based on the amino acid sequences (GenBank accession no. AAZ82249) in Protein Database of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov). The size and purity of the synthesized macaque PAP248-286 peptides were characterized by mass spectrometry and high-performance liquid chromatography, respectively (data not shown).
We next determined whether there are differences in the coding nucleotide sequences between the human PAP248-286 and macaque PAP248-286. There are differences in 2 nucleotides within the coding sequence 2 between human PAP248-286 and macaque PAP248-286 (Fig. S1A, http://links.lww.com/QAI/A990). Given the reading frame of PAP248-286 starts within the coding sequence 1, the first transition of A to G (in red) in coding sequence 2 results in the amino acid change from Ile277 to Met277. This change of Ile277 to Met277 had little effect on the overall positive charges of the peptides, as these amino acids are neutral and nonpolar. The isoelectric points (pI) of both macaque and human PAP248-286 are 10.21. In contrast, the second transition of C to T (in green) results in no change of the amino acid as both codons (CTC and CTT) that code for Leu283 (Fig. S1A, B, http://links.lww.com/QAI/A990). We then compared the 3-D structure of the macaque PAP248-286 with the human PAP248-286 using the Swiss-Model structure prediction server.32,33 Based on the conformational structure, the amino acid Ile277 in the human PAP248-286 has the 2 methyl groups oriented toward the hydroxyl phenol group of the Tyr280 (Fig. S1C, http://links.lww.com/QAI/A990). In contrast, the amino acid Met277 in the macaque PAP248-286 has a conformational structure with the methylthio group toward the reverse side of the hydroxyl phenol group of Tyr280 (Fig. S1C, http://links.lww.com/QAI/A990).
Cytotoxicity of Macaque SEVI and EGCG
Before we examined the impact of SEVI and/or EGCG on SIV or SHIV infection, we examined the cytotoxicity effect of SEVI or EGCG on TZM-bl cells and macaque PBMCs. Macaque SEVI up to 50 μg/mL had no significant cytotoxicity on macaque PBMCs (Fig. S2, http://links.lww.com/QAI/A990) although slightly reduced cell viability of TZM-bl cells. Furthermore, EGCG (up to 50 μM) had no cytotoxicity on both TZM-bl cells and macaque PBMCs (Fig. S2, http://links.lww.com/QAI/A990). Therefore, we used EGCG at the concentrations of 50 μM or less and macaque SEVI at dose of 50 μg/mL or less in the following studies.
Macaque SEVI Enhances SIV or SHIV Infection
To determine the effect of macaque SEVI on SIV or SHIV infection/replication in vitro, we preincubated SIVmac239, SIVmac251, or SHIVSF162P3N with macaque SEVI at different concentrations for 1 hour and then added the mixture to the TZM-bl cells or macaque PBMCs. As shown in Figure 1, macaque SEVI dose-dependently enhanced the viral infection of TZM-bl cells. The maximal enhancing effect of macaque SEVI on the viruses was a 32.9-fold for SIVmac239, 94.0-fold for SIVmac251, and 68.9-fold for SHIVSF162P3N, respectively (Fig. 1). We next examined whether macaque SEVI enhances SIV or SHIV infection of macaque PBMCs. As shown in Figure 2, macaque SEVI dose-dependently enhanced SIV (mac239 and mac251) or SHIV (SF162p3N) infection of macaque PBMCs. The cell-associated SIV or SHIV gag expression at postinfection day 6 was significantly increased by macaque SEVI at the doses of 25 and 50 μg/mL. In addition, viral RNA in the culture supernatant (extracellular) at postinfection days 2, 4, and 6 was also higher in the presence of macaque SEVI.
EGCG Counteracts Macaque SEVI-Mediated Enhancement of SIV or SHIV Infectivity
We next examined the effect of EGCG on macaque SEVI-mediated enhancement SIV or SHIV infection of TZM-bl cells and macaque PBMCs. As shown in Figure 3, exposure of macaque SEVI and SIV or SHIV mixture to EGCG resulted in the suppression of macaque SEVI-mediated enhancement of viral infectivity in TZM-bl cells. This inhibitory effect of EGCG on macaque SEVI-mediated viral enhancement was dose-dependent (10–50 μM) and 50 μM EGCG could completely block the enhanced viral infection. Similarly, EGCG counteracted macaque SEVI-mediated enhancement of SIV or SHIV infection of macaque PBMCs (Fig. 4). EGCG pretreatment of the virus/macaque SEVI mixture dose-dependently inhibited the expression of SIV gag at both cell-associated and extracellular levels. The inhibitory effects of EGCG at doses of 10 and 25 μM were more significant at postinfection days 2 and 4 than at day 6 (Fig. 4). This would be attributed to the wash-off of EGCG after 3 hours of infection and no EGCG presence during the postinfection period, which will not affect the produced viruses for new infection of initially uninfected cells. However, 50 μM of EGCG very significantly inhibited the infection at all time points, suggesting almost complete abolishment of initial viral infection by pre-exposure of EGCG to the virus/macaque SEVI mixture.
EGCG Degrades Macaque SEVI Fibrils
The SEVI fibrils provide a cationic bridge between negatively charged virions and cells by human SEVI is implicated as a mechanism for human SEVI-mediated enhancement of HIV infection.19,21–23 We thus examined whether EGCG could affect the formation of macaque SEVI fibrils. As shown by Congo-red staining (Fig. 5A) and ThT fluorescence assay (Fig. 5B), the macaque SEVI was stable in PBS over the 48-hour incubation in the absence of EGCG. In contrast, in the presence of EGCG, the macaque SEVI was completely degraded in 24 hours (Fig. 5A). Formation of the macaque SEVI-derived amyloid fibrils could be visualized by TEM (Fig. 5C). Incubation with EGCG demolished the structures of macaque SEVI amyloid fibrils (Fig. 5C).
Sexual transmission contributes to more than 80% of new HIV infections,34 and semen plays a critical role in HIV sexual transmission.7 Semen provides a protective environment for HIV virions, and some of the seminal fluid peptides can enhance HIV infection in vitro.16,17,19,21–23 Among the HIV-enhancing elements in human semen, the PAP248-286-derived amyloid fibril was the first and the most extensively studied peptide fragment from prostatic acid phosphatase of semen.19 Thus, the PAP248-286 amyloid fibril was termed as semen-derived enhancer of viral infection (SEVI).19 Although in vitro studies have clearly shown that SEVI could enhance HIV infection/replication, in vivo investigations on the role of SEVI in enhancing intravaginal or intrarectal HIV infection in the animal models have reported conflicting results.25,26 The difference in epithelial integrities of rectal or reproductive mucosa of different animal species35 could contribute the discrepancy. In addition, the high viral inoculum used in the animal studies may also affect the magnitude of SEVI-mediated HIV infectivity enhancement.19 Another possible defect in these animal studies could be the use of human SEVI or semen. It is likely that the exposure of macaque vaginal mucosa to human semen/SEVI can induce local inflammation and immune reactions to the foreign proteins. Therefore, it is appropriate to use macaque SEVI for studies of SIV or SHIV sexual transmission with macaque models.
In this study, we showed that macaque SEVI could significantly enhance the infectivity of the SIV (mac239 and mac251) and the SHIV (SF162P3N). These findings support the early study that human SEVI could facilitate HIV virion attachment to target cells and enhance the infection.20 Comparison of the SEVI precursor peptides PAP248-286 of human and macaques showed only one amino acid difference at the site of 277 (Fig. S1A, http://links.lww.com/QAI/A990). The residue 277 in human PAP248-286 is isoleucine, whereas that in macaque PAP248-286 is methionine, both of which are neutral and nonpolar amino acids. Therefore, the substitution of Ile277 by Met277 in macaque PAP248-286 should not affect the overall positive charge of the peptides (pI = 10.21) (Fig. S1B, http://links.lww.com/QAI/A990), indicating that macaque SEVI has the ability to promote SIV or SHIV virions to attach/fuse with target cells, a key mechanism for macaque SEVI-induced enhancement of SIV or SHIV infection.
The importance of drug–semen interaction has been emphasized in evaluating the anti-HIV efficacy of microbicides.36,37 Zirafi et al15 reported that semen could impair the antiviral efficacy of multiple classes of microbicides, including anionic polymers (eg, polystyrene acid, polynaphthalene sulfonate, and cellulose sulfate), neutralizing antibodies (nAbs; eg, gp41-specific 2F5 nAb), or antiviral drugs that act on intracellular targets at different steps of the viral replication cycle (eg, reverse transcription, integration). In the case of polyanion-containing microbicides, the reduced anti-HIV activity may be due to the fact that polyanions have the ability to interact with SEVI and form SEVI amyloid fibrils, which counteract with polyanionic microbicides in preventing HIV infection.20 These findings indicate that the in vitro efficacy of candidate microbicides should be evaluated in the presence of semen to identify the best candidates for the prevention of HIV sexual transmission.
The polyphenolic compounds have been found to be one of the most effective antiaggregative agents that can effectively inhibit aggregation of a broad spectrum of amyloidogenic proteins.38 As a polyphenolic compound found in green tea, EGCG has the ability to degrade the amyloid fibrils of human SEVI and block human SEVI-mediated HIV enhancement.6,39–41 In this study, we showed that EGCG could also degrade macaque SEVI-formed amyloid fibrils (Fig. 5). Direct imaging of amyloid fibrils in human semen demonstrated the amyloid aggregates that partially consist of prostatic acid phosphatase fragments.21 On the contrary, nonamyloidogenic peptides were demonstrated to be defective in boosting HIV infection.42 Thus, the capability of SEVI to form orderly amyloidogenic aggregation is critical for its enhancing effect on HIV.43 EGCG has also been reported to interact with human monomeric PAP248-286 peptide. The sites for the specific interaction lie within the side chains of 2 regions (K251-R257 and N269-I277) of human SEVI, primarily charged residues, particularly lysine.41 Our finding that EGCG could potently degrade the amyloid fibrils of macaque SEVI (Fig. 5) indicates that the substitution of I277 by M277 in macaque SEVI does not affect the ability of EGCG to block HIV-enhancing effect of the macaque SEVI. In addition to its effect on semen-derived SEVI, EGCG possesses direct anti-HIV ability, as EGCG could inhibit the integration, reverse transcription of HIV, and even directly degrade the virions.5,44 EGCG was shown to bind to the same molecular pocket on CD4 as does HIV gp120.45,46 These further in vivo studies using macaque SEVI and SHIV in macaque models should be of great interest.
In summary, our study for the first time identified the nucleotide sequences that code the macaque PAP248-286 peptides. Importantly, we demonstrated that the macaque PAP248-286-derived SEVI fibrils could enhance SIV or SHIV infection of macaque PBMCs. These findings in conjunction with the studies by others25,26,35 argue for future investigations, particularly in vivo studies, to determine whether macaque SEVI fibrils can facilitate SIV or SHIV mucosal transmission in the macaques. In addition, given the findings that EGCG could remodel seminal amyloid fibrils and effectively suppress SEVI-mediated HIV enhancement in vitro, it is necessary to examine whether EGCG can protect macaques from SIV or SHIV infection through rectal or reproductive tract mucosa.
The PBMCs were isolated from blood of healthy Chinese rhesus macaques using the Ficoll-Paque PLUS (GE Healthcare Life Sciences) method as per manufacturer's instruction. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Wuhan University School of Medicine in accordance with the regulations of the National Institutes of Health “Guide for the Care and Use of Laboratory Animals.”
The authors thank Dr. Shun-Yi Li (Hubei University, Wuhan, China) for assistance with the 3-D structure analysis of the human PAP248-286 and macaque PAP248-286.
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