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• W2032706681 abstract "SEVI and SEVI bind by transmission electron microscopy (View interaction) Early in our exploration of the mechanism of anti-HIV activity of T-20, we found that peptides 6312 and 6313 derived from the HIV-1 gp120 co-receptor binding region could significantly abrogate T-20-mediated inhibition of HIV-1 infection [1]. Thus, we speculated that T-20 might interact with the HIV-1 co-receptor binding region of gp120 as its potential mechanism of action. However, no direct interaction between T-20 and either peptide 6312 or 6313 was detected in vitro. Interestingly, these two peptides individually could form amyloid fibrils that drastically enhanced HIV-1 infection. These amyloid-forming peptides were defined as enhancing peptides (EPs). Amyloid fibrils are critical aetiologies of several neurodegenerative diseases and systemic amyloidoses because they induce abnormal accumulation of protein or peptide aggregates [2]. In addition, amyloid fibrils play an important role in microbial infection [3, 4] . For example, some bacteria and fungi can employ amyloid fibrils to enhance their virulence and to invade the host [5-7]. Recent studies have shown that HIV-1 might also utilise amyloid fibrils to promote viral entry. Beta-amyloid fibrils, the pathogenic agent of Alzheimer's disease, could enhance HIV-1 infection at the stage of virus attachment [8]. Peptides derived from prostatic acidic phosphatase (PAP, predominant component PAP248–286) in semen, designated as semen-derived enhancer of viral infection (SEVI), significantly enhanced HIV-1 infection via the formation of amyloid fibrils. Aside from PAP, peptides derived from physiologically cleaved coagulum proteins in semen could also form amyloid fibrils to enhance HIV-1 infection [9]. Most of the amyloid fibrils reported thus far originate from the host environment. Limited investigation has been performed to examine whether some viral protein-derived amyloid fibrils might exert an effect on HIV-1 infection or pathogenesis. In this study, we investigated the ability of EPs to form amyloid fibrils and, in turn, to enhance HIV-1 infectivity. Interestingly, EPs exhibit a strong tendency to form amyloid fibrils in vitro, which can remarkably enhance HIV-1 infection by promoting the interaction between viruses and cells. Significantly, the antiviral efficacies of a panel of antiretroviral drugs (ARVs) are impaired in the presence of EPs. The addition of epigallocatechin gallate (EGCG), which has been shown to inhibit amyloid fibrillogenesis and to disrupt amyloid fibrils derived from several proteins [10, 11], also displayed similar effects on EP-derived fibrils. Based on our results, the potential roles of EPs in viral pathogenesis and their biochemical applications are discussed. MT-2 cells, TZM-bl cells, primary HIV-1 strains, the anti-p24 monoclonal antibody (183-12H-5C), antiviral compounds and HIV-1 clinical isolates were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. CEMx 174 5.25M7 cells were kindly provided by C. Cheng–Mayer. Thioflavin T (ThT), Congo red and EGCG were purchased from Sigma (St. Louis, MO). The plasmids of CXCR4-tropic NL4-3, CCR5-tropic 92TH014.12 and dual-tropic 81A and NL4-3 infectious clones were gifts from Jan Münch of Ulm University, Germany. Various EPs, including EP1, EP2, EP3 and scrEP1, were synthesised with purities above 90% by Huada Biotech Company (Shanghai, China). Lyophilised peptides were dissolved in DMSO at a concentration of 10 mg/ml as stock solutions. Aliquots were stored at 20 °C. The peptides were diluted in PBS at various concentrations before use. PAP248–286 (SEVI) [12] was synthesised by GL Biochem (Shanghai, China). This lyophilised peptide (>95% purity) was dissolved in PBS at a concentration of 10 mg/ml. Formation of SEVI fibrils was promoted via agitation at 37 °C for 2–10 days at 1200 rpm using an Eppendorf Mixmate. Amyloid fibril formation was monitored using ThT and Congo red [13]. Briefly, peptides at 25 μM were added to 50 μM ThT. The fluorescence intensity was then measured. For the time-resolved ThT assay, 500 μM EP2 was incubated with different concentrations of EGCG at 37 °C. At the indicated time points, 5 μl aliquots were removed and mixed with 95 μl of 50 μM ThT solution, and the fluorescence intensity was measured. The structure of fibrils were visualised using a Tecnai 12 (Philips/FEI, USA) transmission electron microscope as described previously (12). CXCR4-tropic NL4-3, CCR5-tropic 92TH014.12 and dual-tropic 81A and NL4-3 infectious HIV-1 clones were prepared as previously described [14]. The p24 antigen concentration was determined via ELISA, as previously described [15]. The enhancing effects of EPs on HIV-1 infectivity (both infectious clones and clinical isolates) were determined using TZM-bl cells [12]. To study the counteracting effect of EGCG on EP2-mediated enhancement of HIV-1 infection, EP2 was first incubated with EGCG at 37 °C for 48 h under constant agitation. The mixtures were then centrifuged at 5000 rpm for 15 min. The pellets were dissolved in medium and incubated with R5-tropic virus. Then, the mixtures were used to infect TZM-bl cells. To assess the EP-induced promotion of cell–cell HIV-1 transmission, HIV-1BaL-infected PBMCs [16] were mixed with peptide for 10 min at 37 °C. Then, the mixtures were added to CEMx174 5.25M7 cells and cultured at 37 °C for 3 h. The cells were then centrifuged and cultured for 3 days. The cells were collected and lysed for analysis of luciferase activity. The inhibitory activity of ARVs in the presence of 2 μM EP2 was determined as previously described [17, 18]. EPs were incubated with R5-tropic viruses at room temperature for 5 min. Subsequently, the mixture was added to the cells. After 3 h, cells were washed and replaced with fresh medium. The luciferase activity was measured 72 h later. Alternatively, the R5-tropic viruses were first added to the cells. After a 3-h binding period, the unbound virus was removed and fresh medium with or without peptides was added. Luciferase activity was determined 72 h later. The binding of HIV-1 to the EPs was determined using a virus pull-down assay [14]. Briefly, the peptides at graded concentrations were mixed with the R5-tropic virus (100 ng/ml p24) for 1 h at 37 °C, followed by centrifugation at 5000 rpm for 5 min to pellet the fibrils and the bound virions. The proportion of p24 and the infectivity of the HIV-1 bound to the pellet were evaluated. Graded dilutions of EPs were first incubated with TZM-bl cells at 37 °C for 1 h. Then, the unbound peptides were washed once with medium. The R5-tropic virus (100 ng/ml) was incubated with the cells at 37 °C for 1 h, followed by washing the cells once with medium. Then, the cells were either lysed in 5% Triton X-100 to measure p24 levels or cultured for 72 h to assay the luciferase activity. EPs were derived from HIV-1 MN gp120 co-receptor binding region. EP1 and EP2 are 15-amino acids in length, with an 11-amino acid overlap between the two peptides. EP3 covers the length of EP1 and EP2 (Fig. 1 A). All of the EPs could significantly enhance the infectivity of the R5-tropic, X4-tropic and dual-tropic HIV-1 infectious clones in TZM-bl cells (Fig. 1B, C and D). When applied at the same concentration, both EP1 and EP3 exhibited a more pronounced increase in HIV-1 infection than SEVI, the positive control. EP3 boosted HIV-1 infection more potently than EP1 or EP2. The scrambled sequence of EP1 displayed no ability to enhance HIV-1 infection (Fig. 1B, C and D). Cytotoxicity assay demonstrated that EPs at 40 μM displayed no cytotoxicity to the target cells (data not shown). It was found that the solutions of EPs were turbid. Thus, we speculated that EPs might form amyloid fibrils that were water insoluble. As shown in Fig. 2 , EPs could bind to the amyloid-specific dyes Congo red and ThT in a dose-dependent manner (Fig. 2A and B). Notably, unlike SEVI, which fibril formation requires specific conditions, including agitation, a suitable salt concentration and pH of the solution [19], EPs could spontaneously form amyloid fibrils. Next, we performed transmission microscopy to examine the morphology of amyloid fibrils composed of EPs. Interestingly, the amyloid fibril structures of EPs are different from that of SEVI (Fig. 2C). EPs form short and soft amyloid-like fibrils, while SEVI forms long and rigid amyloid fibrils. Next, we confirmed that the ultrastructure of the amyloid fibrils corresponded to the enhancement of HIV-1 infectivity. After centrifugation of the EP solution, the supernatants, containing soluble peptides, did not enhance HIV-1 infectivity, while the pellet, containing amyloid fibril aggregates, did enhance HIV-1 infectivity (Fig. 2D). Subsequently, we tested the potential enhancing effect of EPs on different primary HIV-1 isolates. As shown in Fig. 3 A–D, EPs substantially enhanced the infectivity of all the examined primary HIV-1 isolates. Importantly, these peptides also facilitated the transmission of HIV-1BaL from PBMCs to CEMx174 5.25M7 cells (Fig. 3E). These results suggested that EPs effectively enhanced the infectivity of both cell-free and cell-associated HIV-1 of different subtypes and co-receptor tropisms. Using a virus removal assay, we found that EPs enhanced HIV-1 infection only when the peptides were added to the cells either before or at the same time as the virus attached to the cells. No post-entry enhancement of HIV-1 infection was detected when peptides were added after the virus bound to the cells (Fig. 4 A). Therefore, EPs might target the virus entry process to boost virus infection. Next, we examined the mechanism of action by which EPs enhance HIV-1 infection. Using a virus pull-down assay as reported previously [14], we found that the levels and infectivity of HIV-1 treated with EPs or SEVI in the pellets were much higher than those of viruses treated with PBS (Fig. 4B and C). However, the infectivity of the viruses treated with SEVI was significantly lower than the viruses treated with EPs, while the levels (p24 concentration) of the viruses bound to SEVI and EPs were in a similar range. These results suggested that although these peptides exhibit a similar potency to bind to HIV-1, SEVI was not as efficient as EPs in enhancing viral infection, possibly because these peptides form amyloid fibrils exhibiting different properties. Next, we sought to determine whether EPs could also bind to target cells and enhance HIV-1 infection. When the peptides were first incubated with TZM-bl cells, we detected increased virus binding and subsequent higher viral infectivity (Fig. 4D and E). The EP-induced increase in HIV-1 infection antagonised the anti-viral activity of T-20 [1]. Thus, we investigated whether EPs could also counteract the inhibitory effect of current clinically available ARVs. We evaluated the effect of EP2 on the antiviral activity of four classes of ARVs against infection using two laboratory-adapted HIV-1 strains, IIIB (subtype B, X4) and BaL (subtype B, R5). Surprisingly, we found that all of the examined ARVs exhibited reduced anti-HIV-1 activity in the presence of 2 μM EP2 (Supplementary Table S1). This low concentration of EP2 caused a general decrease in the in vitro effectiveness (i.e., an increase in the IC50 value) of the ARVs from approximately 2–15 fold. EP2 showed less potency to enhance HIV-1 infection, it could be concluded that EP1 and EP3 displayed stronger ability to antagonise the antiviral activities of ARVs. We examined whether EGCG, a common amyloid fibril inhibitor and breaker, could exert any influence on EPs. To be consistence with the above result, we continued to use EP2 as a representative EP in this study. We incubated freshly dissolved EP2 with EGCG, and the formation of the β-sheet structures was monitored. The freshly dissolved peptide could readily bind to ThT, implying a spontaneous process of fibril formation (Fig. 5 A). During the incubation period of EP2 and EGCG, the ThT fluorescence intensity decreased in time- and dose-dependent manners, while the signals slightly increased during incubation of EP2 alone (Fig. 5A). Next, we exposed preformed EP2 fibrils to various concentrations of EGCG and monitored the degradation of the fibrillar structures over time via ThT staining. The preformed EP2 amyloid fibrils (550 μM) were highly stable in PBS for a period of 72 h. In contrast, the preformed amyloid fibrils were clearly degraded in the presence of EGCG (Fig. 5B). Based on transmission electron microscopy, large EP2 fibril aggregates (275 μM) disappeared after the addition of EGCG (1375 μM) during the 48-h incubation period (Supplementary Fig. S1), while the EGCG control displayed no fibril structures, as expected. Next, we investigated whether EGCG could counteract the EP2-mediated enhancement of HIV-1 infection. EP2 alone exhibited a potent enhancement of HIV-1 infectivity, while EGCG counteracted the EP2-mediated increase in HIV-1 infectivity in a dose-dependent manner. At a concentration approximately 10-fold higher than that of EP2, EGCG could completely abrogate EP2-mediated enhancement of HIV-1 infectivity (Fig. 5C). EGCG could also effectively rescue the EP2-weakened antiviral activities of the ARVs (Supplementary Table S2). In this study, we found that EPs derived from the HIV-1 gp120 co-receptor binding site could greatly enhance HIV-1 infectivity (Fig. 1). These peptides form amyloid fibrils, as determined via ThT staining assay, Congo red assay and electron microscopic analysis (Fig. 2A, B and C). EPs could substantially enhance the infectivity of both cell-free and cell-associated HIV-1 of various subtypes and coreceptor tropisms (Fig. 3). Notably, EPs self-assembled and displayed an ability to immediately form amyloid fibrils. As soon as the peptide was dissolved, it could readily bind to amyloid fibril-specific dyes, ThT (Fig. 5A), implying a spontaneous process of β-sheet formation. In mechanistic studies, we demonstrated that EPs exhibited a strong potency to bind to HIV-1 and target cells, leading to a remarkable increase in HIV-1 infectivity (Fig. 4). EPs enhanced HIV-1 infection by targeting the viral entry process (Fig. 4A). Amyloid fibrils appear to share a common feature to enhance HIV-1 infectivity by targeting the viral entry process. This feature may be explained by their aggregated structures, which could serve as an arm to connect the virus to the target cells. In addition, the properties of amyloid fibrils, such as their cationic property [14], may act like a hand to grab the viruses and cells tightly, thus fulfilling the process of directing the viruses to the cells. However, as found in our study, different amyloid fibrils exhibited different potencies of HIV-1 infectivity enhancement. This result may be due to differences in fibril structure, flexibility, density, and binding properties. Aromatic residues, such as tryptophan in EPs, might provide sufficient hydrophobicity to facilitate interactions between EPs and the membrane [20, 21]. Thus, EPs could bind to viruses and enrich the binding of viruses to the cellular membrane surface to enhance viral infection. In addition to cell-free virions, amyloid fibrils might attract neighbouring HIV-1-infected cells to uninfected target cells, leading to facilitated infection of the newly budding viral particles [22]. It has been reported that the anti-HIV-1 activity of some ARV-based microbicides was impaired in the presence of certain HIV-1 infectivity-enhancing factors, such as SEVI and semen [18, 23]. Our result suggests that the antiviral efficacy of ARVs become significantly decreased in the presence of EPs (Supplementary Table S1). For example, the addition of 2 μM EP2 to the virus resulted in an increase in the IC50 values, ranging from 1.9 to 15.9-fold, of all the examined ARVs. EGCG, an effective antioxidant and anti-infective agent, was reported to inhibit amyloid fibril formation and to disrupt preformed amyloid fibrils derived from various proteins [10, 11, 24-26]. We found that EGCG also effectively inhibited amyloid fibril formation and disrupted the preformed fibrils composed of EP2 (Fig. 5A and B), thus counteracting the EP2-mediated enhancement of HIV-1 infectivity (Fig. 5C). Free gp120 exists in vivo [27] and it is subjected to many degrading factors in vivo, including lysosomal or ubiquitin-mediated intracellular degradation [28, 29], and catalytic antibodies [30-32]. Therefore, gp120 protein and peptide products are detected in the serum and tissue in HIV-1/AIDS patients [33-35]. Fragments of EP2 have been reported to be present in hepatocyte culture medium exposed to gp120, implying the physiological presence of EPs in vivo. One limitation of our study is the lack of a description of the physiological significance of EPs. Both the existence of EPs in vivo and their biological function warrant further investigation. EPs are virus-derived amyloid fibrils, which are distinct from semen-derived amyloid fibrils in that EPs self-assemble and display a higher potency to enhance viral infectivity. These properties warrant the potential biological applications of these amyloid fibrils. For example, EP may be used to promote the retroviral gene transfer. It has been reported that SEVI could be applied to promote retroviral gene transfer [36]. Since EPs display higher potency than SEVI to enhance retroviral infection, EPs might be more effective in promoting retroviral gene transfer. We express our special thanks to Frank Kirchhoff and Jan Münch at the University of Ulm for their helpful discussion regarding this project. This work was supported by Grants from the National 973 Program (2012CB519001 to S.J.), the (31370781 to S.L., 81102482 to S.T., 81102476 to L.L., and 81273560 to L.L.), and the Natural Science Foundation of Guangdong Province (S2011020005207 to S.L.). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2014.03.016. Supplementary data 1. Supplementary material Supplementary data 2. Supplementary material Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W2032706681 title "Peptides derived from HIV-1 gp120 co-receptor binding domain form amyloid fibrils and enhance HIV-1 infection" @default.
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