Study On The Mechanism Of LAPTM5 Inhibiting Env’s Synthesis In Resting CD4~+ T Cells

Objective:Acquired Immune Deficiency Syndrome（AIDS）,is an infectious disease caused by the Human Immunodeficiency Virus（HIV）infection.Since the first AIDS patient was identified,AIDS has become one of the world’s most prevalent major infectious diseases.According to the latest UNAIDS data,as of 2021,about38.4 million people were living with HIV worldwide,1.5 million were infected in2021,and 650,000 died of AIDS-related causes.Highly Active Antiretroviral Therapy（HAART）is a widely used anti-AIDS therapy,which can significantly inhibit HIV replication,but can not cure AIDS.This is mainly due to the formation of the so-called viral reservoir soon after HIV infection,and currently researchers believe that the main component of the reservoir is the dormant CD4⁺T cells.Generally,after HIV infects human cells,the surface of infected cells will express the HIV Envelope protein Envelope（gp120 and gp41）,which can be recognized and cleared by the immune system.However,there is almost no Env expression on the surface of dormant CD4⁺T cells,which cannot be identified and cleared,and then survive in infected patients for a long time,which is an important source of viral rebound after drug withdrawal in patients.Therefore,taking resting CD4⁺T cells as the study object,elucidating the mechanism of no Env expression on their surface will provide an effective means for the clearance of the reservoir.Previous research results of our laboratory show that:Lysosome Associated Protein Transmembrane 5（LAPTM5）can degrade Env proteins in Monocyte derived macrophage（MDM）.However,the effect and mechanism of LAPTM5 on Env in resting CD4⁺T cells have not been reported.This study analyzed the effect of LAPTM5 on Env expression in resting CD4⁺T cells and explored its mechanism to find new targets for the functional cure of AIDS.Methods:1.Research ObjectAll of the 82 subjects were collected from the First Affiliated Hospital of China Medical University.Among them,there were 22 Healthy Controls（HC）,14 patients who did not receive ART therapy（HIV group）,and 46 patients with rapid progression（RP group）.Inclusion criteria of HC group:age 18-60;HIV antigen and antibody were confirmed negative by experiment;Good physical condition,no bacterial or viral infection in the past month;No liver or kidney diseases and other metabolic diseases;No autoimmune disease;Women exclude pregnancy.2)Inclusion criteria for HIV group:age 18-60;HIV antigen and antibody were confirmed positive by confirmatory experiments;Never received antiretroviral drugs such as ART;No liver or kidney diseases and other metabolic diseases;No autoimmune disease;Women exclude pregnancy.The inclusion criteria of RP group are as follows:age 18-60;HIV antigen and antibody were confirmed positive by confirmatory experiments;CD4⁺T cell count in peripheral blood within 1-2 years of HIV infection.350 PCS/μL;No liver or kidney diseases and other metabolic diseases;No autoimmune disease;Women exclude pregnancy.All subjects involved in this study have given informed consent and signed informed consent.This study has been approved by the Ethics Committee of the First Affiliated Hospital of China Medical University.2.Isolation of human Peripheral blood mononuclear cells（PBMCs）and Sorting of CD4⁺T lymphocytes,B lymphocytes and NK cellsFresh peripheral blood of people who living with HIV and healthy blood donors were collected to contains EDTA（Ethylenediaminetetraacetic acid,EDTA）anticoagulant blood-collecting vessels.PBMCs were isolated by centrifugation of peripheral blood with Ficoll reagent according to the principle of density gradient centrifugation,and then treated with Easy Sep^TMHuman CD4 Positive Selection Kit II（STEMCELL Technologies,Canada）,Easy Sep^TMHuman B Positive Selection Kit II（STEMCELL Technologies,Canada）and Easy Sep^TMHuman NK Cell Isolation Kit（STEMCELL Technologies,Canada）to separate CD4⁺T cells,B cells and NK cells for the experiment according to the reagent specification.3.Extraction of Ribonucleic Acid（RNA）from primary cells and cell lines（TRIzol method）The primary cells,human embryonic kidney epithelial cells（293 T）,human cervical cancer cells（He La）,and human T lymphocyte（Jurkat）cell line samples were added with TRIzol and chloroform lysed cells,isopropyl alcohol precipitated RNA,washed with 70%ethanol,and dried at room temperature after discarded superclear.Dissolve with RNase-free water and promote dissolution at 55℃.4.Activation and culture of CD4⁺T cellsThe RPMI1640 culture-medium containing 10%inactivated FBS(Fatal bovine serun（FBS）was formulated,and chloromycin and streptomycin were added according to the concentration of 100 U/m L.The above medium was used for the culture of resting CD4⁺T cells.The medium was supplemented with Interleukin-2（IL-2）at 50 U/m L and Dynabeads^@Human T-Activator CD3/CD28（Thermo Fisher,USA）at 25μL/10⁶cells,and then it can be used to activate CD4⁺T cells.5.Cell line cultureDMEM culture-medium containing 10%FBS(Fatal bovine serum（FBS）,100U/m L concentration of cyan and streptomycin was prepared for 293 T and He La cell lines culture.The RPMI1640 medium containing 10%FBS and 100U/m L concentration of cyan and streptomycin was prepared for the culture of Jurkat cell line.All the above cells were cultured in an incubator containing 5%CO₂at 37℃,and passage was performed once in 2-3 days.6.HIV_NL4.3virus packagingThe density of 293 T cells was 5×10⁶cells/plate in a 10 cm²petri dish.16-24 h later,the plasmid p NL4.3 was transfected with Lipo D-293 transfection reagent according to the instructions at 10μg/petri dish.The fluid was changed 4-6 h after transfection.After 48-72 h,the supernatant was removed.At 1500 rpm and 5 min,the supernatant was centrifuged,and the virus supernatant was packaged as needed for easy use.7.HIV_NL4.3viral infectionAccording to the requirement of MOI value,calculate the amount of virus supernatant required.The cells were divided into the required number of groups,and the virus was diluted to the required volume with serum-free RPMI1640 medium at the amount of 200μL fluid per well,and polybrene was added at the concentration of0.5μL/m L.Add to the 96-well plate at the bottom of U one by one at the rate of 200μL/well.Centrifuge at room temperature 1200×g for 2 hours;After centrifugation finish,the 96-well plates were gently taken out and placed in an incubator at 37℃and5%CO₂for 2 hours.The centrifugally infected cells were sucked out and placed in6,12 or 24-well plates,and supplemented medium was added into the holes for culture according to 2×10⁶cell count/m L density.8.Real Time quantitative polymerase chain reaction,RT-q PCRRNA reverse transcription was performed in two steps using Prime Script RT Reagent Kit with g DNA Eraser（Perfect Real Time）（TAKARA）kit according to the operating instructions.1)Firstly,the dopant Deoxyribonucleic Acid（DNA）in RNA was removed by the reagent specification;2)reverse transcription of DNA-depleted RNA into c DNA;3)Quantitative real-time PCR was then performed using TB Green^?Premix Ex Taq^TMII（Tli RNase H Plus）（Ta Ka Ra）kit.9.Cell total protein extractionCell samples were removed to the EP tube and centrifuged to discard the supernatant.The cells were shaken to disperse cell precipitates.RIPA Lysis and Extraction Buffer（Thermo Fisher）were added to re-suspend cells and lysis was allowed to stand.Ultrasonic-assisted nucleus fragmentation;The supernatant was centrifuged at 12000 rpm for 15 min at 4℃.The supernatant was transferred to a new1.5m L centrifuge tube.Add 4×Protein SDS PAGE Loading Buffer（TAKARA）to the lysed supernatant at a ratio of 3:1,and mix well with a tipand then the loading buffer was blown and mixed with the tip.Boil the mixed liquid at 99℃for 5 minutes to denature the protein,then take out the boiled protein sample and leave it to cool down on ice.10.Western Blot（WB）Polyacrylamide gel was prepared by SDS-PAGE gel preparation kit（Kaibiol）according to experimental requirements.Take out the electrophoresis instrument,fix the rubber plate,add the electrophoresis solution,and take samples one by one.Concentrated glue 80 V constant pressure electrophoresis,separation glue 120 V constant pressure electrophoresis until the end of electrophoresis;0.3 A（or 60 V at constant pressure）4℃for 2 h transfer;After cutting the film,PBST washed the film once,and 5%BSA was closed at room temperature for 1 h;Incubate primary antibody at 4℃overnight（16h or more）;PBST washed the film three times,10 min each time,incubated the second antibody at room temperature for 1 h;PBST was washed three times,10 min each time,and ECL was used for luminescent imaging.The results were analyzed with GAPDH as the internal parameter.11.Flow cytometryThe treated cells were collected into flow tubes and washed twice with Phosphate Buffered Saline（PBS）,and the supernatant was discarded.According to the flow dye instructions,the flow antibody dye system was prepared and stained at4℃for 20 min.Cytofix/Cytoperm^TM,AB＿2869008)was added to the cells after surface staining and washed twice with PBS buffer containing 2%FBS to fix the broken membranes.After the membrane breaking,the supernatant was centrifuged and the cells were washed twice with the diluted membrane breaking lotion.Flow antibody dye system was prepared and stained at 4℃for 20 min.After dyeing,it was washed twice with PBS buffer containing 2%FBS,and an appropriate amount of PBS buffer was added for machine detection.12.si-RNA electrotransfer assay in resting CD4⁺T cellsThe Nucleofector 2b and Human T Cell Nucleofector ^TMKit（Lonza）were used to convert si RNA,and the program U-14 was used to convert resting CD4⁺T cells.The specific operation of this experiment was in accordance with the instrument and reagent instructions.13.Statistical analysisFlowjo X was used for flow analysis,SPSS 19.0 and Graph Pad 8.0 were used for statistical analysis.Measurement data±,the comparison between the two groups using the paired t test,comparison between multiple sets of using single factor analysis of variance,correlation analysis using the Spearman correlation statistics,P<0.05 credited with for statistically.（*P<0.05,**P<0.01）Results:1.Expression of LAPTM5 in resting CD4⁺T cellsFirstly,we analyzed the expression of LAPTM5 in resting and activated CD4⁺T cells in healthy subjects.The results of RNA sequencing showed that LAPTM5 was highly expressed in resting CD4⁺T cells.The sequencing results were further verified by RT-q PCR.It was found that the expression level of LAPTM5 in resting CD4⁺T cells was 166 times that of activated CD4⁺T cells,and the expression level decreased with the extension of the activation time of CD4⁺T cells.In addition,LAPTM5 was low or not expressed in NK cells,B cells and 293 T cell lines.The expression of LAPTM5 in resting CD4⁺T cells in healthy individuals was significantly lower than that in untreated HIV patients,and was negatively correlated with plasma viral load in rapidly progressive patients.These results suggest that LAPTM5 is highly expressed in resting CD4⁺T cells and may be involved in disease progression.2.LAPTM5 inhibits Env expression in resting CD4⁺T cells through lysosomesAfter the use of si-RNA knockdown of LAPTM5 expression in resting CD4⁺T cells,the level of gp120 in HIV was significantly higher than that in the control group,indicating that LAPTM5 could inhibit the synthesis of Env.The intracellular distribution of LAPTM5 was detected by immunofluorescence staining,and it was found that LAPTM5 was colocalized with the lysosome marker LAMP1,that is,LAPTM5 was localized in the lysosomes of resting CD4⁺T cells,suggesting that LAPTM5 may play a role through the lysosomes.Therefore,we co-transcultured LAPTM5 and HIV_NL4.3plasmids in 293 T cells after treatment with lysosome inhibitors,and found that LAPTM5 no longer inhibited Env expression after treatment with lysosome inhibitors.These results suggest that LAPTM5 reduces the amount of HIV Env in resting CD4⁺T cells through lysosomes.3.LAPTM5 inhibits Env synthesis by promoting lysosome AMPK phosphorylationNext,we further explored the molecular mechanism of LAPTM5’s inhibition of Env expression.Flow cytometry and western blotting showed that there was no significant change in m TOR phosphorylation but increased AMPK phosphorylation in293 T cells after co-transmutation of LAPTM5 and HIV_NL4.3,suggesting that LAPTM5 may inhibit Env synthesis by promoting lysosomal AMPK phosphorylation.Conclusion:1.LAPTM5 was highly expressed in resting CD4⁺T cells,and its level was negatively correlated with the plasma viral load of rapidly progressing HIV patients;2.LAPTM5 located in lysosome,resting CD4⁺T cells through the lysosome inhibit the expression of Env;3.By promoting AMPK phosphorylation,LAPTM5 inhibit the synthesis of Env,thus play a role of inhibition of HIV infection.