The Discovery And Identification Of Naturally Degraded Amyloid Peptides From HIV-1 Gp120 And Their Effects On Promoting HIV-1 Infection

Background:In the process of HIV infection on targeted cells,the naturally existance of various amyloid peptide,such as SEVI(virus enhancer factor),Aβ(Alzheimer’s)can significantly improve the infection of HIV.In previous study,we found that the synthesized peptide from gp120MNβ region can form amyloid fibers,promote HIV infection and improve IC50 of ARV drugs.In the complex physiological environment of human,is there any factors improving the infection of HIV-1 virus onto targeted cell?Does the peptide really exist during the process of HIV-1 infection?Can gp120 be degraded into small fragments by various enzymes and do have some physiological effect?What is the factor contributing for the degradation of gp120 in vivo?Objective:We use highly resolution mass spectrometry(LC-MS/MS),searching for peptides from the HIV envelope gp 120 enzymatic digested product,the supernatant of viral-infection,the plasma or lympe leakage of HIV positive patients respectively.We then use immunofluorescent,immune colloidal gold to determine the formation and composition of amyloid fibril from viral-infection supernatant and lympe leakage from HIV positive patients.We next use immunohistochemical combined with Congo red staining to analyze the lymph nodes of HIV positive patients.We found these peptides could enhance viral infection onto targeted cells and increase IC50 of clinical antiviral drugs by forming into GEVI.This study may provide theoretical reference for a better understanding of a newly ungenetic drug resistant mechanism and optimizing the clinical treatment of HIV in the future.Methods and results:In order to detect the effect of enzymes on stability of gp120,four enzymes from human bodyfluid,Thrombin B,Plasmin,Cathepsin B and a-L-AFU were used to hydrolyze gp120JRFL protein at 37 ℃ in vitro.The hydrolysis products of gp120 protein at different time point were detected by 10%SDS-PAGE with Coomassie brilliant blue staining as well as(Transmission Electron Microscope,TEM),TEM.In order to verify the generation of gp120 derived peptides during infection,we simulate the infection environment in vitro.By using different tropism virus X4 tropism(HIVIIIB)virus infected with MT-2 cells or R5 tropism(HIVSF162)virus infected with CEMx1745.25M7 cells,the supernatant was collected after 7 days’infection after removing cells and cell fragments by centrifugation for 10 mins at 3000 rpm.The presence and composition of amyloid fibrils were detected by immunofluorescent and immune colloidal gold respectively.In order to verify the real existence of gp120 derived peptides of clinical samples,are there any factors promoting virus infection in lympy leakage?The inactivated lympy leakage could promote the(R5 tropism)clone virus infection onto Tzm-bl cells in a dose dependent manner.The amyloid fibrils from gpl20 was detected by immunofluorescence and immune colloidal gold.Immunohistochemistry and Congo red staining showed that the expression of gp120 protein in lymph nodes of HIV-1 positive patients was relatively high and partly composed of amyloid fibles.In order to identify the amino acid sequence of peptide,we use ultrafiltration to treat with hydrolysate of gp120.We also use OMC(ordered mesoporous carbons,OMC)material adsorption,immune affinity to deplete the highly,acetonitrile precipitation plus ultrafiltration method to treat with supernatant from HIV virus infected targeted cells,lympy leakage samples.A series of peptides from gp120 were identified by highly resolution LC-MS/MS.Selective synthesis of those peptides among 25 β domains derived from HIV-1 gp120.The peptide sequence of gp120 and the secondary structure were studied by using specific staining of Congo red,Thioflavine T,transmission electron microscopy(TEM)and circular dichroism spectrum(CD).Peptides can forming into β-sheet amyloid fibrils.GAP380-396;GAP229-244;GAP354-369 peptide against the clone virus,GAP380-396;GAP229-244 has enhanced effect on both clonal and clinical strain.GAP380-396;GAP229-244 increased the IC50 of four clinical antiviral drugs by 2 to 16 times more than that of control.In order to analyze the mechanism of peptide induced enhancement on virus infection,the Zeta potential of peptide was detected by Zetasizer Nano ZS potential instrument.CS has partial inhibitory effect on the virus infection of GAP229-244,but has no effect on GAP380-396 or EP2 for they carry negative or no charge.The infectivity of fibrin perception and supernatant were detected by virus pull-down assay,only the precipitate part could enhance virus infection,suggesting that the fiber can capture and bind to virus.Cell-binding and fluorescence virus experiments shown that peptides can bind to both viruses and cells.The fluorescence peptide combined with Confocal microscopy showed that different peptide can form hybrid peptide and contained the ability to enhance virus infection,which may have superposition effect.XTT showed that peptides and viruses have no effect on cell virability below the concentration of 10 μm.Conclusion:The GAPs peptide and GEVI can be found in enzymatic hydrolysate products,virus-infection supernatant and clinical samples of HIV-1 positive patient.The catalytic effect of protease in humoral on HIV gp120 is one of the possible reasons for degradation of gp120 in vivo.Peptides can promote HIV virus infection and antagonize the antiviral activity of ARV drugs,and do have enhancement effect among different peptides.The enhancement effect may be asscociated with the electrostatic and hydrophobic interaction of peptide on cell membrance and the HIV receptors(CD4,CXCR4,CCR5).This study may provide the theoretical basis for in-depth understanding of HIV ungenetic drug resistance and provide for the potential improvement on HIV therapy in the near future.