Study On Antiviral Function And Mechanism Of Urokinase Plasminogen Activator Surface Receptor

Objective:Human immunodeficiency virus（HIV）is the pathogen that causes Acquired Immune Deficiency Syndrome（AIDS）.The main target cells of HIV-1 infection are CD4⁺T cells and myeloid cells,in which myeloid cells include macrophages and dendritic cells（DCs）.In vivo,the infection of macrophages and dendritic cells is particularly important,which can efficiently transmit HIV-1 virus to CD4⁺T cells in the process of antigen presentation of secondary lymphoid organs.At the same time,they are also an important bridge between innate immunity and adaptive immune response to invasive pathogens.Compared with activated CD4⁺T cells,myeloid cells are more resistant to HIV-1 infection.Therefore,it is of great significance to explore the specific host cell limiting factors that may exist in myeloid cells.Urokinase type plasminogen activator receptor（uPAR/PLAUR or CD87）is a member of the GPI anchored protein family.By binding to its ligand urokinase（uPA）,urokinase plays an important role in plasminogen activation,extracellular matrix degradation,tumor migration and invasion,inflammation and immunity.Some studies have found that the expression of uPAR on the surface of monocytes and T lymphocytes in patients with HIV-1 infection increases,and the interaction of uPA-uPAR can inhibit the replication of HIV-1 in monocyte-derived macrophages,suggesting that uPA or uPAR may be the host cell limiting factor in macrophages.At the same time,some studies have found that the level of serum soluble uPAR is closely related to the disease progression and survival time of HIV-1 infection,suggesting the correlation between uPA-uPAR and HIV-1 infection from the side,but the specific role and mechanism need to be further explained.In this study,the expression pattern of uPAR and its effect on HIV-1 replication were discussed for the first time（paper 1）,and the molecular mechanism of uPAR affecting HIV-1 replication was further explored（paper 2）.Finally,the effect of uPAR ligand uPA on its antiviral function was discussed（paper 3）,which provides a possible potential new treatment strategy for clinical treatment of AIDS.Methods:1.Research ObjectIn this study,15 healthy volunteer blood donors were recruited,including 8 males and7 females,requiring no tumor,pregnancy,metabolic diseases and autoimmune diseases,and no bacterial or viral infection in nearly one month.All the volunteers involved in this project informed consent and signed the informed consent form.This study was approved by the Ethics Committee of the first affiliated Hospital of China Medical University.2.Isolation and culture of primary cells and isolation of differentiated peripheral blood mononuclear cellsIsolation of peripheral blood mononuclear cells（PBMCs）:peripheral blood 50ml of healthy volunteers was collected and PBMCs was isolated by sucrose Ficoll（cytiva）density gradient centrifugation.Monocytes were sorted by Easy Sep^TM Human CD14⁺Positive Selection Kit II（STEMCELL Technologies）and CD4⁺T lymphocytes were sorted by Easy Sep^TM Human CD4⁺T Cell Enrichment Kit（STEMCELL Technologies）.Induction and differentiation of monocyte-derived macrophages（MDMs）:the selected monocytes were induced and cultured in RPMI1640 medium（Gibco）supplemented with 10ng/ml GM-CSF（R&D Systems）and 50ng/ml M-CSF（R&D Systems）.The fresh medium containing cytokines was changed every two days,and MDMs could be induced after 7 to 9 days of culture.Induction and differentiation of monocyte-derived dendritic cells（MDDCs）:the selected monocytes were induced and cultured in IMDM medium（Gibco）supplemented with 10ng/ml GM-CSF（R&D Systems）and 50ng/ml IL-4（R&D Systems）.The fresh medium containing cytokines was changed every two days and could be induced to differentiate into MDDCs after 5 to 7 days.Activation and culture of CD4⁺T lymphocytes:the selected resting CD4⁺T cells were cultured in RPMI1640 medium（Gibco）supplemented with 50U/ml IL-2（R&D Systems）,and activated CD4⁺T cells were obtained by adding Dynabeads^?Human T-Activator CD3/CD28（Thermo Fisher）at the ratio of 25μl/1×10⁶cells.All cells were cultured in 37℃incubator containing 5%CO₂.3.Culture of cell linesHuman embryonic renal epithelial cell lines（293T）and human cervical cancer cell lines（He La）were cultured in DMEM medium（Gibco）containing 10%inactivated fetal bovine serum（Viva Cell）and 100U/ml penicillin-amphotericin（Viva Cell）,and human T lymphocyte lines（Jurkat）and human monocytic leukemia cell lines（THP-1）were cultured in RPMI1640 medium（Gibco）containing 10%inactivated fetal bovine serum（Viva Cell）and 100U/ml penicillin-amphotericin（Viva Cell）.THP-1-derived macrophages（THP-1-MA）were induced by culture in the medium containing 100n M phorbol ester（Promega）,and then adhered to and differentiated into macrophages 48 hours later.All the cells were cultured in an incubator containing 5%CO₂ at 37℃and subcultured every 2 days.4.RNA-sequenceDetection of RNA quality:The total RNA was extracted by TRIzol（Invitrogen）reagent,and the total RNA was preliminarily quantified by Biodrop ultramicro protein and nucleic acid analyzer.Agilent Aglient 2100 was used to accurately quantify the total RNA concentration,and the library was constructed after the total amount,concentration,integrity and purity of the extracted total RNA samples met the standard.Construction library:after removing the ribosomal RNA from the total RNA sample,the RNA was broken into short fragments.Using RNA as the template,the first strand c DNA;was synthesized by six base random primers,and then the second strand c DNA was synthesized by adding the reaction system to purify the double strand c DNA;.Then,the purified double strand c DNA was connected to the sequencing connector,and the fragment size was selected to degrade the second strand c DNA;containing U bases.Finally,the chain specific library was obtained by enriching and purifying the PCR products.Library quality inspection:The chain-specific library was accurately quantified by q-PCR method,and the effective concentration of the library>2n M could be sequenced on the computer.Computer sequencing:The obtained library is sequenced by SE50 after pooling according to the effective concentration and the target amount of data off the machine.5.Real time quantitative polymerase chain reaction（RT-q PCR）After the total RNA was extracted by TRIzol（Invitrogen）reagent,the genomic DNA was removed by Prime Script genome RT reagent Kit with g DNA Eraser（Perfect Real Time）（Ta Ka Ra）,then c DNA was synthesized by reverse transcription reaction,and then the DNA fragment was amplified and synthesized by TB Green^?Premix Ex Taq^TM II（Ta Ka Ra）kit using c DNA as template.With GAPDH as the internal reference,the relative expression of target gene was calculated by 2^-△△Ct method.6.Cell transfection and preparation of virus particles.293T and He La cells were transfected with plasmids using Lipo D293 transfection reagent（Signa Gen Laboratories）,and the specific operation was carried out according to the instructions of the kit.HIV-1_{NL4-3.Luc.R-E-}（VSV-G）was used to transfect p NL4.3-Luc-R-E-6.7μg and p CMV-VSV-G 3.3μg in 293T cells.48 hours after transfection,the supernatant of cell culture was collected and centrifuged to obtain virus particles.Sh RNA lentivirus was transfected with sh RNA lentivirus expression plasmid 5μg,p AX2packaging plasmid 3.75μg and p CMV-VSV-G plasmid 1.25μg in 293T cells by Lipo D293.The virus particles were collected 48 hours after transfection and centrifuged filter.The concentrated virus particles can be obtained by sucrose pad density gradient high-speed centrifugation.7.Virus infection.HIV-1_{NL4-3.Luc.R-E-}（VSV-G）infection:293T,Jurkat and MDMs cells were infected according to MOI=1.0,Jurkat cells and activated CD4⁺T cells were infected according to HIV-1 _{NL4-3.Luc.R-E-}（VSV-G）of 100ng p24 every 2×10⁶ cells,and centrifuged for 2 hours.Sh RNA lentivirus infection:cells were infected according to the proportion of 200ng p24sh RNA lentivirus per 0.6×10⁶cells after VLP-Vpx treatment.4 days later,puromycin was added to screen,the concentration was 2μg/ml.8.Detection of virus particlesThe level of p24 in cell culture supernatant was detected by human immunodeficiency virus antigen antibody diagnostic kit（enzyme-linked immunosorbent assay）（Wantai）,the level of p27 in cell culture supernatant was detected by SIV p27 Antigen Capture Assay（ABL.Lnc）,and the content of HIV-2 virus was detected by Reverse Transcriptase Assay（Roche）.The specific operation is carried out according to the instructions of the kit.MLV virus content:genomic DNA was extracted from mouse 3T3 cells infected with viral particles and then RT-q PCR was performed.The late reverse transcription products were detected by specific primers.9.HBV nucleocapsid-associated DNA purificationThe viral particles were purified from the supernatant of cell culture by PEG8000precipitation,and the relaxed circular DNA was purified from nucleocapsid related DNA.After plasmid removal by DNase I,restriction enzyme digestion with protease K and sodium dodecyl sulfate（SDS）,phenol-chloroform extraction and isopropanol precipitation,HBV DNA was quantified by q PCR with specific primers.10.Luciferase detection assayLuciferase activity detection cells were cleaved with Passive Lysis Buffer（Promega）and the supernatant was detected by Bright-Glo Luciferase assay system（Progema）.The specific operation was carried out according to the instructions of the kit.11.Isolation of membrane-associated proteinsMembrane-associated protein separation experiment uses Mem-PER^TMPlus Membrane Protein Extraction Kit（Invitrogen）to separate membrane-associated proteins,and the specific operation is carried out according to the instructions of the kit.12.Protein extraction and western blotThe cells were resuscitated with RIPA Lysis and Extraction Buffer（Thermo Fisher）,then centrifuged with ultrasound-assisted breakage,the supernatant was added with 4×Protein SDS-PAGE Loading Buffer（Ta Ka Ra）and denatured at high temperature;SDS-PAGE gel was sampled and electrophoretic until bromophenol blue reached the bottom of the gel;the first antibody was incubated overnight at room temperature after 5%BSA was closed at room temperature for 1 hour or 60V cross-flow membrane for 2 hours;the second antibody was incubated at room temperature for 1 hour after PBST washing.After washing the film with PBST,chemiluminescence reaction was performed and imaging was performed..13.Cell and virion imagingZeiss LSM 980microscope was used for cell imaging:after the cells were fixed,rabbit anti-p24 antibody,mouse anti-CD81 antibody and corresponding fluorescent labeled secondary antibody were stained and imaged after sealing.The virus particles were imaged by JEM-1400 flash electron microscope.14.Statistical analysisSPSS20.0 software was used for statistical analysis,and Graph Pad Prism9.0 was used to map the data.The comparison between groups was analyzed by unpaired double-tailed t-test,and when multiple groups were compared,one-way analysis of variance（one-way ANOVA）was used.（*P<0.05,**P<0.01,***P<0.001,****P<0.0001）.Each experiment was carried out three times independently,and the data were expressed as mean±standard deviation.Results:（一）Expression pattern of uPAR and its effect on HIV-1 replication.1.High expression of uPAR in macrophages and dendritic cells.The expression of uPAR in primary cells and cell lines was compared by sequencing analysis,RT-PCR and western blot assays.It was found that uPAR was highly expressed in THP-1 cells,macrophages,dendritic cells and monocytes,but almost no expression in activated or resting CD4⁺T lymphocytes,293T,Jurkat and He La cells.2.uPAR inhibits the production of HIV-1 progeny virus.Overexpression of uPAR in 293T cells was followed by infection of HIV-1replication reporter virus HIV-1_{NL4-3.Luc.R-E-}（VSV-G）.Compared with the control group,uPAR did not affect luciferase activity,suggesting that uPAR did not inhibit the early infection of HIV-1.Further analysis of the effect of uPAR on the production of HIV-1progeny virus showed that uPAR significantly decreased the content of p24 in the supernatant of cells in a concentration gradient dependent manner,but had no effect on the protein levels of Gag and Env,indicating that uPAR inhibited the production of HIV-1progeny virus.3.Effect of Endogenous uPAR on HIV-1 replication in macrophages and dendritic cells.Furthermore,the level of p24 in the supernatant of cell culture was detected after MDMs and MDDCs knocked down the endogenous uPAR and infected with HIV-1_AD8.It was found that the endogenous uPAR had antiviral function in inhibiting the production of offspring virus.4.uPAR has broad-spectrum antiviral function against lentivirus.In order to investigate the inhibitory effect of uPAR on different tropism of HIV-1,we co-transferred uPAR overexpression plasmid with different tropism HIV-1 provirus plasmid in 293T cells.It was found that uPAR could inhibit the production of different tropism HIV-1（X4 tropism,R5 tropism and ditropism）offspring virus production.In order to investigate whether uPAR specifically inhibits HIV-1,the overexpression plasmid of uPAR was co-transferred with HIV-2,SIV,MLV and HBV virus plasmids in 293T cells.It was found that uPAR had a significant inhibitory effect on HIV-2,SIV and MLV virus replication,but had no significant effect on HBV virus replication,indicating that uPAR has broad-spectrum antiviral function against lentivirus.（二）Study on the Molecular Mechanism of uPAR affecting HIV-1 replication.1.uPAR localized on the cell membrane.After 293T cells were transfected with GFP-labeled uPAR,the subcellular localization and imaging of uPAR were observed by laser confocal microscope,and it was found that uPAR was located in the cell membrane,while western blot analysis was performed after the membrane separation of MDMs and MDDCs,which indicated that the endogenous uPAR was located in the cell membrane.2.uPAR exerts antiviral function on cell membrane.In order to further explore whether uPAR functions on the cell membrane,we constructed a mutant plasmid（uPAR-Δ1-22）with the deletion of uPAR signal peptide,so that it could not be located on the cell membrane.The results showed that after the absence of signal peptide,uPAR could not target to the cell membrane and lost its antiviral function,suggesting that uPAR needs its membrane localization to exert its antiviral function.3.uPAR inhibits the release of HIV-1 progeny virus on cell membrane.uPAR and Gag plasmids were co-transferred in He La cells,and the co-localization of uPAR and Gag protein was analyzed by laser confocal microscopy.Since uPAR did not affect the intracellular gag protein,but p24 in the supernatant decreased,we further examined the effect of uPAR on virus release from offspring.The virions of HIV-1offspring in 293T cells were observed by electron microscope and photographed.It was found that uPAR inhibited the release of virions from HIV-1 progeny on the cell membrane.4.uPAR induces virions to enter the virus inclusion interval（VCC）in macrophages.In MDMs cells,mature but unreleased virions are temporarily stored in the chamber VCC formed by invagination and extension of the cell membrane.Under some conditions,virions can be released out of the cell again,or directly transmitted to the target cells through viral synapses.Therefore,we further explore whether uPAR induces virions to enter VCC.Immunofluorescence staining was used to analyze the co-localization of p24and VCC marker molecule CD81 in the presence or absence of uPAR.It was found that after knockout uPAR,the intracellular p24 decreased significantly,while in the control group,there were more p24 and co-located with CD81,indicating that uPAR induced virions to enter VCC.（三）Study on antagonistic effect of uPA on antiviral function of uPAR.1.uPA promotes the release of HIV-1 progeny virus from macrophages and dendritic cells in a certain concentration range.In view of the fact that uPAR plays an important role in plasminogen activation,extracellular matrix degradation,tumor migration and invasion,inflammation and immunity by binding to its ligand uPA,we further studied the effect of uPA on HIV replication and found that uPA promoted the release of HIV-1 progeny virus from MDMs and MDDCs in a certain concentration range.2.uPA treatment did not affect the expression of uPAR in macrophages and dendritic cells.In order to explore whether uPA promotes the release of HIV-1 progeny virus by degrading uPAR expression,we detected the expression of uPAR after uPA treatment in MDMs and MDDCs,and found that uPA treatment did not affect the expression of uPAR m RNA and protein.3.uPA increases the release of HIV-1 by antagonizing the antiviral function of uPAR.The endogenous uPAR in MDMs was knocked out,and the virus release in the culture supernatant was detected after uPA treatment.It was found that the control group treated with uPA could promote the release of viral particles from the supernatant to some extent,but after the knockout of uPAR,uPA treatment had no significant effect on the release of viral particles from the supernatant,indicating that uPA increased the release of HIV-1from macrophages by antagonizing the antiviral function of uPAR.Conclusion:1.High expression of uPAR in macrophages and dendritic cells and inhibition of HIV-1 replication.2.uPAR inhibits the release of viruses on the cell membrane by inducing virions to enter the viral inclusion interval（VCC）in macrophages.3.uPA antagonizes the antiviral function of uPAR in macrophages and dendritic cells.