Study On The Role And Mechanism Of LAPTM5 In Macrophages In HIV-1 Replication

Objective:The infection of Human immunodeficiency virus（HIV）cause Acquired Immunodeficiency Syndrome（AIDS）.Current global AIDS epidemic is mainly caused by HIV-1.HIV-1 mainly infects CD4⁺T lymphocytes,resulting in the exhaustion of CD4⁺T cells,leading to the downregulation of the body’s immune function.Since the identification of HIV-1,there has been a lack of effective vaccines and treatments to prevent and completely eliminate the virus.Although highly active antiretroviral therapy（HAART）can inhibit the replication of HIV-1 and control the progress of AIDS,the problem of drug resistance caused by long-term medication has become increasingly prominent.New targets and new mechanisms are important tasks in the current field of AIDS treatment.In vivo,the target cells of HIV infection mainly include CD4⁺T cells and myeloid cells,and myeloid cells mainly include monocytes,macrophages（M?）and dendritic cells（DCs）,macrophages and dendritics are important shelters for HIV-1 replication in body,and both are vectors for virus transmission in the host.At the same time,macrophages and dendritics play important roles in the body’s immune response to HIV-1 infection,participate in the body’s non-specific and specific immune responses to HIV-1,and activate other immune cells,prompting the body to respond to pathogens.HIV-1 is an obligate intracellular parasitic pathogen,and the completion of most stages of its life cycle depends on the interaction between it and host proteins.After HIV-1 infects the body,it induces the body to produce a large number of cytokines including interleukin,interferon,and tumor necrosis factor,and promotes the expression of certain proteins in the body to exert antiviral functions.At the same time,there are certain unique cytokines in host cells that can resist the replication of viral pathogens including HIV-1,and these cytokines that interact with viral proteins are defined as HIV-1 host restriction factors.HIV-1 host restriction factors inhibit HIV-1 replication in different ways by interfering with different stages of the viral life cycles,prompting host cells to develop physical or functional defenses against viral replication.CD4⁺T cells and myeloid cells are important target cells for HIV-1 replication in vivo.Compared with CD4⁺T cells,myeloid cells are more able to resist HIV-1 infection,which may be related to the expression of host cells in myeloid cells.related to the limiting factor.The highly expressed restriction factors in myeloid cells that have been found so far include SAM domain HD domain-containing protein 1（SAMHD1）,Apolipoprotein B m RNA editing enzyme catalytic subunit 3G（APOBEC3G）etc.These host restriction factors interact with viral components and can also induce specific immune responses in the body.It is critical to the development of new HIV-1treatments.Through omics identification,our research group screened the host-restricted factors that can interact with HIV-1 in MDMs,and finally obtained Lysosomal-associated transmembrane protein 5（LAPTM5）.LAPTM5 is a lysosomal transmembrane protein with a molecular weight of 30 k D,which is mainly expressed in immune cells and hematopoietic cells.LAPTM5 contains five transmembrane segments,three PY motifs binding to Neural precursor cell expressed developmentally down-regulated protein 4（NEDD4）and one Ubiquitin interaction motif（UIM）,in which the PY motif can interact with NEDD4 to promote LAPTM5 sorting from the Golgi apparatus to lysosomes.Studies have shown that LAPTM5 is a positive regulator of multiple pro-inflammatory signaling cascades in macrophages,is required for Toll like receptor（TLR）-mediated inflammatory cytokine production in macrophages,and participates in the activation of macrophages.However,the role of LAPTM5 in HIV-1-infected macrophages has not been reported so far.Therefore,this study plan to investigate the role and mechanism of LAPTM5 in HIV-1 infection.Methods:1.Research object:In the study of the antiviral function of endogenously expressed LAPTM5 in monocytes-derived macrophages（MDMs）and monocytes-derived dendritic cells（MDDCs）,were isolated from healthy human peripheral blood mononuclear cells（PBMCs）.Healthy blood donors were all from the First Affiliated Hospital of China Medical University,and 100m L of whole blood was collected from each case.The cell lines used in the study of the antiviral function mechanism of LAPTM5 mainly include human embryonic kidney 293T cells,human cervical cancer He La cells etc.Recruitment criteria for volunteers:between 20-50 years old,in good health,negative for syphilis,HIV,hepatitis B and C,without other infectious diseases,without liver,kidney disease and other metabolic diseases,female non-pregnant status.This study has been approved by the Medical Science Research Ethics Committee of the First Affiliated Hospital of China Medical University,and the volunteers signed the informed consent.2.Induction and sorting of MDMs、MDDCsPBMCs were isolated by density gradient centrifugation using Ficoll^?Paque Plus.Use the Easy Sep?Human CD14 Positive Selection Kit II kit to sort cells according to the instructions,positively select CD14⁺monocytes from PBMCs,centrifuge the monocytes and resuspended in RPMI1640 medium containing 10%inactivated FBS,2m M L-glutamic acid,100U/m L penic-streptomycin,10m M HEPES,1%non-essential amino acids,1m M sodium pyruvate,10ng/m L GM-CSF and 50ng/m L M-CSF,Inoculate 4×10⁶cells/10cm dish in a cell culture dish,replace fresh culture medium every 3 days,and obtain MDMs after culturing for 7-9 days;obtained by centrifugation,the mononuclear cells were resuspended in IMDM medium containing 10%FBS,2m M L-glutamate,100U/m L penicillin-streptomycin,10m M HEPES,1%non-essential amino acids,1m M sodium pyruvate,10ng/m L GM-CSF and 50ng/m L Resuspend IL-4,inoculate 4×10⁶ cells/10cm dish in cell culture dishes,replace fresh culture medium every 2 days,and obtain MDDCs after 5-7 days of culture.3.Extraction and activation of human CD4⁺T cellsSort CD4⁺T cells from peripheral blood PBMCs by using the Easy Sep?Human CD4⁺T Cell Enrichment Kit kit,and CD4⁺T cells were activated with 10%FBS,100U/m L penicillin-streptomycin,50U/m L IL-2 in RPMI1640 medium;Dynabeads?Human T-Activator CD3/CD28 was added at a ratio of 12.5μL/10⁶cells,and culture all cells in an incubator at 37℃containing 5%CO₂.4.Cell lines culture293T cells were cultured in DMEM medium that contains 10%FBS and 100U/m L penicillin-streptomycin.He La cells were cultured in DMEM medium that contains 10%FBS,100U/m L penicillin-streptomycin and 25m M HEPES.Passage once every two days.5.Intracellular RNA extraction and RT-q PCRFirstly,use the Trizol method to extract the total RNA in the cells.Remove the doped DNA in the RNA by the Prime Script?RT reagent Kit with g DNA Eraser（Perfect Real Time）then reverse transcribe the RNA into c DNA,and use the TB Green?Premix Ex Taq?II kit for real-time quantification PCR.6.Western blot analysisThe cell samples were lysed with RIPA Lysis and Extraction Buffer,the cells were ultrasonically assisted,and then centrifuged to obtain the supernatant.The supernatant was mixed with 4×Protein SDS PAGE Loading Buffer,and placed in a dry heat oven at 99°C for 5 mins to denature the protein.SDS-PAGE gel preparation kit to make gel,after loading the sample,electrophoresis at 80V until the electrophoresis run out of the stacking gel,and120V electrophoresis run the separation gel.Place on ice and transfer membrane at constant voltage 60V for 2 h.Wash the membrane once with PBST.Blocked with 5%BSA for 1 h,incubated with primary antibody for 1 h at room temperature,washed 3 times with PBST,10 mins each time.Incubate the secondary antibody for 1 h at room temperature.Wash with PBST 3 times,10 mins each time.Incubate with ECL luminescent solution for1 min,and develop the image in an image analyzer.7.Transfection and viral packagingTransfection was carried out according to the kit instructions:Lipo D293^TMtransfection kit was used for transfection of 293T and He La cells and packaging of retroviral vector,and Lipofectamin3000^TM kit was used for packaging of sh LAPTM5lentiviral vector.8.Luciferase expression level detectionAfter using Passive Lysis Buffer to lysed the cells,the supernatant was taken,the luciferase substrate was added according to the kit instructions,and the luciferase activity was detected with a microplate reader.9.Detection of HIV-1 p24 and gp120 by ELISAAccording to the recommended operation steps of the ELISA detection kit manual,set the detection wavelength of the microplate reader to 450nm during the measurement.10.Statistical AnalysisGraph Pad Prism 6.0 and SPSS19.0 were used for graphing and statistical analysis.The measurement data was represented by mean±standard deviation.Analyze data by unpaired t test when comparing two groups.One-way analysis of variance（One-way ANOVA）was used when comparing multiple groups,P<0.05 was considered statistically significant.Results$1.The expression of LAPTM5 in various cellsIn order to explore the expression level of LAPTM5 in cells,we first analyzed the expression level of LAPTM5 in cells by RT-q PCR and Western blot.The results showed that LAPTM5 was highly expressed in MDMs and MDDCs,and it was also expressed at a low level in monocytes and activated CD4⁺T cells,but it was not expressed in 293T,Hela,Jurkat and other cell lines.2.Effect of LAPTM5 on HIV-1 replicationSince LAPTM5 is not expressed in He La cells,we next selected He La cells that do not express LAPTM5 for overexpression experiments to explore the role of exogenous LAPTM5 in HIV-1 replication and detect the effect of LAPTM5 on the replication of different lentiviruses.We co-transfected LAPTM5 expression plasmids and HIV-1,HIV-2,SIV,FIV and MLV proviral expression plasmids in He La cells to detect the effect of LAPTM5 on the replication of different lentiviruses.The results showed that LAPTM5could significantly inhibit the replication of HIV-1,HIV-2,SIV（P<0.05）,but did not affect the replication of FIV and MLV（P>0.05）.Since LAPTM5 is highly expressed in MDMs,in order to explore the effect of endogenous LAPTM5 in MDMs on HIV-1replication.We knocked down endogenous LAPTM5 by transducing sh-LAPTM5lentivirus in MDMs,and infected HIV-1_AD8vpr-after puromycin selection.Clearly detect the production of virus p24.The results showed that after knocking down LAPTM5 in MDMs,the replication level of HIV-1 was significantly increased（P<0.05）,indicating that endogenous LAPTM5 in MDMs has antiviral function.3.Research on the mechanism of LAPTM5 affecting HIV-1 replicationCo-transfect LAPTM5 and HIV-1 lentiviral plasmids in He La cells,collect cells and cell culture supernatants,detect the expression of intracellular viral proteins by Western blot,and detect the contents of p24 and gp120 in the supernatants by ELISA;at the same time,an equal amount of p24 virus infect TZM-bl reporter cells.The results showed that compared with the control group,the overexpression of LAPTM5 had no significant effect on the generation of HIV-1 progeny virus,but the expression level of Env protein in the supernatant and intracellular was significantly reduced,and LAPTM5 significantly reduced the infectivity of the progeny virus（P<0.05）,suggesting that LAPTM5 may inhibit the infectivity of progeny viruses by reducing the expression of the viral envelope protein Env.Conclusion:1.LAPTM5 is highly expressed in macrophages.2.Exogenous LAPTM5 inhibits HIV-1 replication in cell lines.3.Endogenous LAPTM5 inhibits HIV-1 replication in macrophages.4.LAPTM5 can reduce the infectivity of HIV-1 progeny virus by reducing the expression of HIV-1 Env.