Myricetin Antagonizes Semen-derived Enhancer Of Viral Infection(SEVI) Formation And Inhibits SEVI-mediated Enhancement Of HIV-1 Infection

OBJECTIVE:Microbicides are gels,creams,suppositories,films or sponges containing anti-HIV-1 ingredients,generally used in vagina or anus of patients before sexual intercourse to prevent the spread of HIV-1 and other sexually transmitted pathogens.The development of a topical microbicide might be a hopeful way to control the spread of HIV/AIDS because it can be used by women themselves.Unfortunately,two decades of researches on topical microbicides are limited success and further researches for searching new and ideal microbicides are urgently needed.It is difficult to understand that several polyanionic microbicides are effective to preventing HIV-1 in preclinical animal models,but have failed in clinical trials.Continual failures of those microbicide candidates in clinical trials imply that there should be some unknown reasons to affect their efficacy during application in human beings.Generally,a microbicide candidate in clinical trials is administrated by vagina or rectum.During sexual intercourse,the influence of female genital tract secretions and male semen in the host environment on the antiviral efficacy of those microbicides should not be ignored.Notably,it has recently been reported that semen boosted HIV-1 infectivity and impaired the antiviral efficacy of candidate microbicides in vitro.Some endogenous amyloid aggregates have been detected in healthy human semen samples,which partially consist of prostatic acidic phosphatase(PAP)fragments and increase HIV-1 infectivity by trapping viral particles.The first discovered one is semen-derived enhancer of viral infection(SEVI);an amyloid fibril formed from a naturally occurring peptidic fragment derived from the residues 248 to 286 of PAP(PAP248-286).Some researches confirmed that SEVI fibrils could capture virus and promote viral attachment to the target cells by its cationic property.Other fragments of prostatic acid phosphatase(PAP85-120)derived from the N-proximal fragment of PAP and semenogelins(SEMI and SEM2)form amyloid fibrils in seminal fluid,which also promote HIV-1 infection.That may explain the contradiction between the low infectious in vitro and global high morbidity of HIV-1.Therefore,SEVI or other seminal amyloid fibrils play a critical role and serve as a novel target in identifying the best microbicide candidates for the prevention of the sexual transmission of HIV-1.Theoretically,a compound may reduce the enhancement of viral infection by blocking the binding of HIV-1 to seminal amyloid fibrils or preventing the formation of seminal amyloid fibrils.This study intends to SEVI as a major drug target for further researches due to its stable structure and quite rich in semen.It is well known that some fatal brain diseases,such as Parkinson’s disease,are closely related to the formation of amyloid fibrils in the brain.There are many studies on the inhibitors of those amyloid fibrils.Because of low price and safety,Chinese herbal medicines obtained by the present modern science and technology are generally to be used to treat many diseases.Myricetin,an active ingredient of flavonoids from Myrica rubra,is reported to inhibit the formation of various amyloid fibers,such as Aβ,Tau protein and some other amyloidogenic peptides.Therefore,we hypothesize that myricetin might have the same inhibitory activities on the formation of SEVI amyloid fibrils.In this study,we evaluated the effects of myricetin on SEVI amyloid fibrils formation and its function through a series of biophysical and biochemical methods.The mechanisms of myricetin on inhibition of SEVI amyloid fibrils formation and SEVI-mediated HIV-1 infection enhancement are studied in detailed.It will be helpful to the development of new target microbicide candidates or antiviral drugs research.Since the myricetin itself has antiviral activity,it is likely to be developed into a dual functional antiviral drug with both anti-HIV activities and anti-SEVI enhancing activities of viral infection for preventing HIV-1 sexual transmission.METHODS:1.Effects and the possible mechanism of myricetin on the formation of SEVI fibrils.1)The effects of myricetin on the formation of SEVI amyloid fibrils were detected by thioredin T fluorescence assay,Congo red staining and transmission electron microscopy.2)The effects of myricetin on the P-sheet secondary structure of SEVI fibrils were analyzed by circular dichroism.3)The SEVI-mediated enhancement of HIV-1 infection treated by myricetin was detected using HIV-1 infected clones and TZM-bl cells.4)Exploring the effects of myricetin on SEVI formation stages by western blot and photo-induced cross-linking of unmodified proteins(PICUP)assay.5)The possible binding sites between PAP248-286 and myricetin were speculated by molecular docking.2.The effects of myricetin on SEVI-mediated enhancement of HIV-1 infection1)The effects of myricetin on SEVI-mediated enhancement of HIV-1 infection were observed by flow cytometry and immunofluorescence microscopy.2)The effects of myricetin on the binding of HIV-1 virions to SEVI fibrils were analyzed by virus pull down and zeta potential assay.3.The effects of myricetin on the formation of seminal amyloid fibrils and semen-mediated HIV-1 infection enhancement1)The effects of myricetin on the formation of seminal amyloid fibrils were detected using ThT fluorescence assay and HIV-1 infection assay.2)The synergy of myricetin and various types of ARVs drugs in the presence of semen.4.The researches of myricetin on the remodeling of mature SEVI fibrils1)The effects of myricetin on the remodeling of mature SEVI fibrils were detected by ThT fluorescence,Congo red staining and transmission electron microscopy.2)The effects of myricetin on β-sheet secondary structure of SEVI were measured by circular dichroism.3)The mature SEVI-mediated enhancements of HIV-1 infection treated by myricetin were detected using HIV-1 infected clones and TZM-bl cells.RESULTS:1.Myricetin inhibited the formation of SEVI fibrils in a dose-dependent manmer as assessed by both ThT and Congo red assay.At the high concentrations(75 μg/ml),myricetin could completely abrogated the formation of SEVI fibrils in vitro even at 48 h after agitation.The results were further observed directly by TEM.Of note,the spectra of PAP248-286 in the presence of myricetin at 48 h after agitation did not adapt to any stable conformation,indicating that β-sheet aggregation had not formed,which demonstrated that the addition of myricetin failed to induce any cross-β-sheet structure of PAP248-286.Myricetin might be affected the whole SEVI formation stages including the early oligomer formation and fiber extension.The results of molecular docking showed that myricetin might specifically bindto PAP248-286 by the covalent interaction on the sites of Leu-258,Gln-259,Val-264,Leu-268,Met-271 and Arg-273 of PAP248-286.2.Myricetin lowered the positive electrostatic potentials of the surface of SEVI fibrils,and then inhibited the assembly and attachment of HIV-1 virions to SEVI fibrils.Therefore,myricetin could block the enhancement of SEVI-mediated HIV-1 infection.3.Myricetin inhibited the formation of seminal amyloid fibrils in a dose-dependent manner.The semen-mediated HIV-1 infection enhancement was abrogated by 5μg/ml of myricetin specifically.Notably,myricetin had great synergistic anti-HIV-1 activities with a series of ARVs in semen,including zidovudine(AZT),raltegravir(RAL),maraviroc(MAR),tenofovir(TNF),nevirapine(NVP)and efavirenz(EFV).The CI values referred to less than 0.5.4.Myricetin rapidly remodeled perfonned mature SEVI fibrils,thereby undermining SEVI-mediated viral infection enhancement.CONCLUSION:Myricetin significantly inhibited the whole stages of SEVI formation including the early oligomerization and fiber extension.Myricetin lowered the positive electrostatic potentials of the surface of SEVI fibrils,and then inhibited the assembly and attachment of HIV-1 virions to SEVI fibrils.Therefore,myricetin could block the enhancement of SEVI-mediated HIV-1 infection.The semen-mediated HIV-1 infection enhancement was also abrogated by myricetin specifically.Notably,myricetin had great synergistic anti-HIV-1 activities with a series of ARVs in semen.Furthermore,myricetin rapidly remodeled performed mature SEVI fibrils,thereby undermining SEVI-mediated viral infection enhancement.Since the myricetin itself has antiviral activity,it is likely to be developed into a dual functional antiviral drug with both anti-HIV-1 activities and anti-SEVI enhancing activities of viral infection for preventing HIV-1 sexual transmission.