| Objective:Human Immunodeficiency Virus(HIV)is the pathogen of Acquired Immunodeficiency Syndrome(AIDS).It's one of the most serious epidemics in the world.Resting CD4+T cells are highly resistant to HIV-1 replication.The difficulty of HIV-1 replication in restingCD4+T cells is related to resting CD4+T cell specific host limiting factor.In fact,in the long-term struggle with the virus,the host gradually evolved some specific host cytokines to fight against the invasion of pathogens such as HIV-1.These host limiting factors form the natural barrier of host cells to virus replication by interfering with specific steps of virus life cycle.At present,it has been reported that TRIM5 a,MX2,samhdl and APOBEC3G are the early targets of HIV-1 replication,and tetherin blocks the release of HIV-1 infected cell offspring.It is worth noting that samhd1,as a limiting factor of HIV-1 reverse transcription process in resting CD4+T cells,plays an important role in limiting HIV-1 infection of resting CD4+T cells.Other host factors TRIM5a,MX2,tetherin and APOBEC3G have not been reported to be restricted in resting CD4+T cells.In a word,as an integral part of the inherent antiviral response,the restrictive factors provide the initial defense against infection even before the inherent antiviral response,and determining the mechanism of these restrictive factors is a key point in HIV researchCD4+T cells are the main target cells of HIV.Activated CD4+T cells are the main site of HIV-1 replication in human while most of the CD4+T cells(about 95%)in human are in a resting state.At present,many studies have proved that resting CD4+T cells can be directly infected by HIV in vivo,and constitute the main cells of host latent infection.The infected CD4+T cells can not be recognized and killed by the immune system for a long time;once activated,it will cause a sharp rebound of the virus.Therefore,HIV-1 latent infection of resting CD4+T cells is considered to be the most important virus reservoir.The existence of a repository is the fundamental reason why HIV cannot be completely cured.To study the mechanism of HIV replication in resting CD4+T cells is very important for the complete elimination of the reservoir and the complete cure of AIDSThe current situation of the research on the repository:Previous studies suggested that the repository of latent HIV infection was completely static and could not be transcribed and translated.Now,some researchers point out that at least some of the repositories are transcriptional or even translational.So far,no reservoir has been found that can produce HIV offspring.How to identify,detect and search for biomarkers is an important topic in the field of HIV cure researchThe most important reservoir is the resting CD4+T cells,resting CD4+T cells are highly resistant to the production of human immunodeficiency virus type 1(HIV-1)However,the mechanism by which resting CD4+T cells restrict such production in the late viral replication phase of infection has remained unclear.To study the host factor spectrum of HIV-1 Gag interaction in resting CD4+T cells,find out the key host factor and study its effect on HIV-1 replication,as well as its mechanism and application in HIV-1 infection to better understand the molecular mechanism and pathophysiological effects of HIV-1 infection by resting CD4+T cells,to provide new ideas and a potential target for anti-HIV therapiesMethods:1.Study SubjectsThe PBMCs were extracted from peripheral blood by Ficoll density gradient centrifugation,and the resting CD4+T cells(CD3+CD4+CD8-CD25-CD69-HLA-DR-)were further separated by magnetic bead method.The project was approved by the ethics committee,and the volunteers signed the informed consentSelection criteria for healthy donors:healthy adults,HIV negative,hepatitis B and C negative,syphilis negative;selection criteria for HIV untreated patients:HIV infection is definitely diagnosed,and they have not been treated with cART;selection criteria for HIV patients treated with cART:HIV infection is definitely diagnosed,long-term treatment of cART is more than one year,and viral load is less than 50 copies/ml.2.Isolation of host factor interacting with viral Pr55-Gag in resting CD4+T cellsResting CD4+T cell samples isolated from healthy donors(n=10)were electroporated(4 × 106 cells per electroporation)with Myc-tagged Gag constructs and spinoculated 6 h later with HIV-1NL4-3.Five days after infection,cells were lysed using immunoprecipitation lysis buffer(50 mM Tris-HCl,pH 7.2,50 mM NaCl,1%NP-40,1 mM EDTA,2%glycerol,1 × cOmplete)and cell lysates were pooled.Lysates were incubated on ice for 30 min and centrifuged at 12,000 r.p.m.for 10 min at 4?Supernatants were transferred to fresh tubes and the pellets were mixed with cold immunoprecipitation lysis buffer before sonication followed by a second round of centrifugation at 12,000 r.p.m.for 10 min at 4?.The supernatants obtained from the two extraction steps were pooled and incubated with mixed protein A and protein G Dynabeads(Invitrogen),which had been pretreated overnight with antibody at 4?.The immunoprecipitation products were washed with cold immunoprecipitation buffer and phosphate-buffered saline 5-10 times(500 ?l per wash)3.HIV-1 spinoculationResting CD4+T cells were incubated with 100 ng of p24 virus centrifuged at 1,200g for 2 h at 25? as described previously 4.RNA-mediated interference in resting CD4+T cellsTo achieve siRNA-mediated silencing(Invitrogen),resting CD4+T cells(3 × 106cells per treatment)were directly electroporated using a Human T cell Nucleofector Solution and program U-14(Amaxa),as described previously.Resting cells were then spinoculated with HIV-1NL4-3;primary CD4+T cells were in the resting state and unstimulated during the experimental period5.Isolation and culture of primary cellsPBMCs obtained from healthy blood donors were purified via Ficoll-Hypaque gradient centrifugation.CD4+T cells or monocytes were isolated from PBMCs via negative selection with human CD4+T cells(STEMCELL Technologies).Resting CD4+T cells were isolated with a resting CD4+T cells isolation kit according to the manufacturer's instructions(STEMCELL Technologies)and cultured at a density of 2×106 cells ml-1 in RPMI 1640 medium supplemented with 10%heat-inactivated FCS,glutamine(2mM)and antibiotics(100Uml-1 penicillin and 100 mg ml-1 streptomycin)To activate CD4+T cells,Dynabeads Human T-Activator CD3/CD28(Invitrogen)were added to the culture medium for 2 d together with IL-2(50 U ml-1)(Biomol)according to the manufacturer's instructionsThe isolation and culture of monocytes,MDMs and MDDCs were performed as described.Briefly,monocytes were purified from total PBMCs after Ficoll-Hypaque gradient separation with CD 14+enrichment.MDMs were generated via stimulation of monocytes with 50 ngml-1 recombinant human granulocyte-macrophage colony stimulating factor(GM-CSF;R&D Systems)for 7d.MDDCs were generated by incubating CD14-purified monocytes in IMDM medium(Gibco)supplemented with 10%FCS,2 mM L-glutamine,100 IU ml-1 penicillin,100 mgml-1 streptomycin,10 mM HEPES,1%non-essential amino acids,1 mM sodium pyruvate,10 ngml-1 GM-CSF and 50 ngml-1 IL4(Miltenyi Biotec).On day 4,two-thirds of the culture medium was replaced with fresh medium containing GM-CSF and IL4.Immature MDDCs were collected and used for experiments on day 66.ELISA detection of virionsp24 protein levels in the cell culture were measured with ELISA according to the manufacturer's instructions(Advanced BioScience Laboratories).The HIV-1 virion genome RNA quantification COB AS AmpliPrep/COB AS TaqMan HIV-1 Test v.2.0 was purchased from Roche;HIV-1 genome levels were measured according to the manufacturer's instructions.To measure HIV-1 production in resting CD4+T cells,virions from the cell culture were purified and enriched(HIV Isolation Kit;Miltenyi Biotec)according to the manufacturer's instructions7.MicroscopyCells were overlaid on poly-1-lysine-coated glass slides and the images were collected using the EVOS FL Imaging System(Thermo Fisher Scientific).Stimulated and resting CD4+T cells from healthy donors or patients were fixed and stained with an anti-TRABD2A antibody(R&D Systems)and a FITC-conjugated anti-mouse secondary antibody(Invitrogen),according to the manufacturers'instructions(Image-iT Fixation/Permeabilization Kit;Invitrogen).8.Luciferase detection assay.Luciferase activity in the cell lysates was measured according to themanufacturer's instructions(Promega).9.Isolation of membrane-associated protein.Isolation of membrane-associated protein was performed according to themanufacturer's instructions(Mem-PER Plus Membrane Protein Extraction Kit;Thermo Fisher Scientific).Briefly,cells were collected from the cell suspension at 300g for 5 min.The obtained cell pellets were washed with 3 mL of Cell Wash Solution(Thermo Fisher Scientific)and centrifuged at 300g for 5 min.After the supernatants were removed,the cell pellets were resuspended with 1.5 ml of Cell Wash Solution and transferred to a new tube.Further centrifugation at 300g for 5 min was done to discard the supernatants.The resultant cell pellets were added to 0.75 ml permeabilization buffer to obtain a homogeneous cell suspension and incubated for 10 min at 4?.The permeabilized cells were centrifuged for 15 min at 16,000g;the resultant supernatants containing cytosolic protein were removed and transferred to a new tube for detection.The pellets were added to 0.5 ml solubilization buffer and resuspended by pipetting up and down with a further incubation for 30 min at 4?with constant mixing.The resultant solution in the tubes was centrifuged at 16,000g for 15 min at 4?.The supernatants containing the solubilized membrane and membrane-associated protein were moved to a new tube for detection.10.Flow cytometry.CD4+T cells were cultured and treated with a CellTrace Proliferation Kitaccording to the manufacturer's instructions(Invitrogen)and stained in FACS buffer with ?CD69-PE(BD Biosciences),?CD25-BB515(BD biosciences),HLA-DR-APC(BioLegend).For HIV-1 p24 intracellular detection,cells were fixed in 1%paraformaldehyde and stained in FACS buffer with anti-p24-FITC(Beckman Coulter)HIV-1 core antigen,clone KC57 according to the manufacturer's instructions(BD Cytofix;BD Biosciences).Data were collected on a FACS LSRII(BD Biosciences)and analyses were performed using the FlowJo software11.Mouse monoclonal anti-TRABD2A blocking antibody generationTo make the plasma membrane TRABD2A blocking antibody,we did not use TRABD2A peptides to immunize mice because blocking antibody may recognize the extracellular domain of the membrane metalloprotease TRABD2A.Recombinant full-length human TRABD2A-201 protein was expressed and purified through the prokaryotic system(purity>90%).The immune procedure was as follows.BALB/c mice were treated for primary immunization with complete Freund's adjuvant and immunization was strengthened using incomplete Freund's adjuvant.Equal volumes of antigen and adjuvant were mixed into an emulsion and subcutaneously injected into the mice.Each immunization interval was 20 days three times.The serum titer was detected after 14 d via ELISA.After the serum titer exceeded 1:16,000,mice with the best immune response were selected and their spleens were isolated.Spleen cells were fused with SP2/0 cells at a ratio of 5:1 with PEG 1500(Sigma-Aldrich).After screening and cloning,the positive monoclonal cell lines were obtained via indirect ELISA.The cell culture supernatants were subjected to neutralization testing and immunoblotting to verify the positive clones.Positive clones were selected to stimulate the production of ascites and for antibody purification.The selected positive clones were inoculated into mice via intraperitoneal injection to produce ascites.After confirmation using ELISA,immune ascites were further purified using protein G affinity chromatography to obtain antibody(IgG).The Research and Ethics Committee of the Beijing Biosynthesis Biotechnology Company approved this protocol to generate mouse-derived material(antibody)12.qPCRTotal RNA was extracted from cells using TRIzol reagent(Invitrogen)according to the manufacturer's instructions.RNA was dissolved in 100 ?l of diethyl pyrocarbonate-H2O;1 ?g of the purified RNA was treated with DNase I(amplification grade;Invitrogen)for 10-15 min at room temperature according to the manufacturer's instructions.RNA was immediately primed with oligo-deoxythymine and reverse-transcribed using Superscript ? Reverse Transcriptase(Invitrogen);rtPCR analysis was performed using the ??CT method.The results were normalized against the amplification results for the internal control(GAPDH)13.Patient viral load measurementPlasma HIV-1 RNA was measured using rtPCR,with the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test.The detection range of the assay was 20-10,000,000 copies ml-114.Determination of HIV-1 production from CD4+T cellsCD4+T cells from patients were isolated from their PBMCs via Ficoll-Hypaque gradient centrifugation with negative selection using a human CD4+T Cell Enrichment Cocktail.Purified CD4+T cells were washed three times with cold PBS and once with culture medium to eliminate possible HIV-1 particle contamination The culture medium from the last wash was used as technical control 1 to infect TZM-bl reporter cells(background 1).The washed CD4+T cells were aliquoted and seeded at a density of 0.8×106 to 1.2×106 cells ml-1 in R10 medium in the presence or absence of TRADBD2A inhibitors and blocking antibodies or stimulation with CD3/CD28 activators plus IL-2.Cell culture supernatants were used to infect TZM-bl reporter cells and measure the produced levels of viral progeny;TZM-bl reporter cells without infection served as technical control 2(background 2).The relative luminescence units(RLUs)of the background of control 1 were subtracted from each data point.15.ImmunoblottingIn RIPA buffer,protease inhibitor was added in proportion and precooled on ice at 4?;cells were discarded and washed with PBS for 1-2 times;PBS was discarded and precooled RIPA buffer was added to mix well and split cells on ice for 30min;samples were transferred into tubes at 4?,12000g and centrifuged for 10min.After centrifugation,the supernatant was transferred to a new tube.The precipitate was added to RIPA buffer and centrifuged at 12000g for lOmin.The supernatant obtained by two centrifugations was mixed.Extract 3?l from the original solution,add 27?l PBS,dilute 10 times and use BCA method to measure the protein concentration.The protein sample was mixed with loading buffer and boiled at 95? for 5min.-20? for storage.After SDS-PAGE gel electrophoresis,5%BSA PBST blocked 1h.The first antibody was incubated overnight,and PBST was washed three times for 10min each time;the second antibody was incubated for one hour,and PBST was washed three times for 10min each time.The film was immersed in luminescent solution for 10s and then tested.16.Statistic analysisThe results were analyzed by GraphPad Prism 8.3.0 and SPSS 25.0.IGVsoftware was used to analyze the RNA-Seq transcription level of different proteins in resting cells and activated CD4+T cells.Flow cytometry results were analyses were performed using the FlowJo software10.5.Two-tailed,unpaired Student's t-tests were used for between-groupstatistical comparisons and p values under 0.05 were considered statistically significant.The correlations between variables were evaluated using the Spearman correlation.Results:1.Identification of host factor TRABD2A metalloproteinase in resting CD4+T cells.1.1 Metalloproteinase TRABD2A interacts with HIV-1 Gag protein in resting CD4+T cells.We performed immunoprecipitation assays to pull down cellular proteins interacting with Gag during the late phase of HIV-1 replication.We found that only 25 cellular proteins can bind specifically to Gag which includes the host protein TRABD2A.1.2 TRABD2A metalloprotease is expressed mainly in resting CD4+T cells.The results of RNA sequencing(RNA-seq)and real-time PCR showed that only TRABD2A was differentially expressed in resting and activated CD4+T cells,and TRABD2A was highly expressed in resting CD4+T cells.1.3 TRABD2A also localized to the plasma membrane of resting CD4+T cells and rapidly disappeared following T cell activation.In immunofluorescence,TRABD2A was stained in healthy blood donors and HIV-1 infected persons who had been treated by cART for a long time.It was found that TRABD2A was distributed on the resting CD4+T cell membrane,and the content of TRABD2A decreased rapidly 24 hours and 48 hours after activation1.4 We observed that TRABD2A transcript levels were notably lower in both PBMCs(p<0.0001)and CD4+T cells(p<0.0001)from untreated patients relative to their levels in combined antiretroviral therapy(cART)-treated patients or healthy donors.Moreover,in the presence of cART treatment,TRABD2A transcript levels reverted to levels similar to those observed in healthy donors.1.5 TRABD2A expression inversely correlates with viral loads in vivo.We correlated TRABD2A expression and viral loads in both PBMCs and total CD4+T cells isolated from patients and healthy donors.We discovered that TRABD2A expression in PBMCs(r=-0.764p=0.0014)and CD4+T cells(r=-0.743 p=0.001)inversely correlated with viral loads in vivo in untreated HIV-1-infected individuals.2.The effect of TRABD2A on HIV-1 replication and the mechanism in resting CD4+T cells2.1 TRABD2A restricts HIV-1 production in resting CD4+T cells.We aime to deplete TRABD2A in resting CD4+T cells using an RNA-mediated interference strategy.TRABD2A depletion resulted in the substantial production of infectious HIV-1 progeny in resting CD4+T cells after infection,whereas viral Gag transcript levels remained unchanged.2.2 Inhibition of TRABD2A protease activity allows HIV-1 progeny production in resting CD4+T cells.Ni2+?Co2+?1,10-phenanthroline inhibited the anti HIV-1 activity of TRABD2A in a dose-dependent manner,but did not affect the transcription level of TRABD2A and viral gag.?2.3 TRABD2A actively degraded HIV-1 Gag in resting CD4+T cells at the cell membrane.The resting CD4+T cells infected with HIV-1NL4-3 centrifugally were treated with TRABD2A antibody 8B6,9E7 and 18B6 respectively.The results showed that TRABD2A blocking antibodies 8B6 and 18B6 could up regulate the level of HIV-1 Gag protein.2.4 The anti-HIV-1 activity of TRABD2A can be compromised by blocking antibodies.The proteins of CD4+T cell membrane infected with HIV-1 were purified,and the Gag protein was combined with immunoprecipitation.The results showed that TRABD2A could effectively degrade HIV-lgag protein in resting CD4+T cells.2.5 TRABD2A restricts HIV-1 production in patients' CD4+T cells ex vivo.PBMCs and CD4+T cells were isolated from HIV-1 infected patients and treated with TRABD2A inhibitor or its blocking antibody.The results showed that Ni2+,Co2+ and 1,10-phenanthroline and blocking antibody 8b6 could enhance the production of HIV-1 progeny of PBMCs and CD4+T cells compared with untreated group.2.6 Re-expressing TRABD2A reinforced the resistance of activated CD4+T cells to the production of HIV-1 progeny.3.Inhibition of metalloprotease TRABD2A facilitates the study of HIV-1 replication inresting CD4+T cells3.1 The absence of SAMHD1 promotes HIV-1 production in resting CD4+T cells only when TRABD2A is inhibited.Regardless of the presence of SAMHD1,TRABD2A inhibitors upregulate the protein levels of Gag.The use of Vpx to degrade SAMHD1 in resting CD4+T cells makes the increase of TRABD2A inhibitors and blocking antibodies in HIV progeny more obvious.3.2 By inhibiting the activity of membrane metalloproteinase TRABD2A and RNA interference technology,we can screen out the related factors involved in HIV-1 replication in resting CD4+T cells.Inhibition of TRABD2A provides an effective way to identify HIV-1 replication related host cytokines in resting CD4+T cells.Conclusion:1.TRABD2A metalloprotease is expressed mainly in resting CD4+T cells.Metalloproteinase TRABD2A interacts with HIV-1 Gag protein in resting CD4+T cells.TRABD2A expression inversely correlates with viral loads in vivo.TRABD2A transcript levels were notably lower in both PBMCs and CD4+T cells from untreated patients relative to their levels in combined antiretroviral therapy(cART)-treated patients or healthy donors.TRABD2A is highly correlated with HIV-1 replication of late phases in resting CD4+T cells.2.TRABD2A restricts HIV-1 production in resting CD4+T cells.TRABD2A potently restricts HIV-1 production by degrading the viral Gag polyprotein at the host cell membrane for blocking progeny production in resting CD4+T cells.TRABD2A protease activity is also differentially regulated by metal ions.Ni2+?Co2+and 1,10-phenanthroline and TRABD2A blocking antibody can inhibit protease activity,Mn2+ enhanced the anti HIV-1 effect by enhancing TRABD2A protease activity.3.By inhibiting TRABD2A protease activity combined with siRNA technology,we can provide an effective method to identify HIV-1 replication related host factors in resting CD4+T cells;by inhibiting TRABD2A activity,we can form a new experimental technology to study HIV-1 replication in resting CD4+T cells. |
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