Vpr Counteracts The Restriction Of LAPTM5 To Promote HIV-1 Infection In DCs

Objective:Acquired Immunodeficiency Syndrome（AIDS）is a highly harmful infectious disease caused by the Human Immunodeficiency Virus（HIV）.The target cells of HIV are mainly CD4+T lymphocytes,macrophages and dendritic cells in the human immune system.The main manifestation is that due to the continuous replication of HIV,the number of CD4+T lymphocytes continues to decrease,which will eventually lead to deficiencies in human immune function and cause various opportunistic infections and tumors.The auxiliary proteins encoded by HIV-1 are not necessary for virus replication in vitro,but they are essential for the continuous infection of the virus in vivo.Vif,Nef and Vpu have been proved to be necessary to effectively complete virus replication by fighting against multiple host factors at different stages of the virus life cycle,and promote the infection and spread of the virus,especially in cell lines and CD4+T lymphocytes.Studies have shown that Vif antagonizes the antiviral effect of APOBEC3G protein in the process of HIV-1 reverse transcription.Nef resists the influence of SERINC3 and SERINC5 on the infectivity of the progeny virus and Vpu can antagonize the effect of Tetherin to block the release of viral particles.The host restriction factor that Vpr can antagonize has not yet been determined.According to reports,Vpr protein mainly promotes HIV-1 replication in myeloid cells.The replication of Vpr-deficient HIV-1 in human dendritic cells（DCs）is significantly defective,but the mechanism is unclear.The functions of Vpr that have been reported include affecting the fidelity of the reverse transcription process,promoting the entry of pre-integration complex（PIC）into the nucleus,inducing cell cycle arrest and apoptosis,and transactivating HIV-1 LTR.However,Vpr mainly plays these roles in non-myeloid cells.Recently,it has been reported that Vpr may antagonize an undiscovered host restriction factor in DCs through the DCAF1-E3 ubiquitination ligase pathway to promote HIV-1 infection.As one of the target cells infected by HIV-1 in vivo,dendritic cells are a significant link between innate immunity and adaptive immune response to invading pathogens,and play an important role in the initial stage of HIV-1 infection.Studies have shown that DCs can effectively transmit the virus to activated CD4⁺T lymphocytes during the antigen presentation process,and it can also transmit the virus by being infected by HIV-1.Therefore,identifying the host restriction factors counteracted by Vpr in DCs and elucidating its antiviral mechanism is essential for the treatment of HIV-1 infection.In the early stage of my research group,through the screening of HIV-1 restriction factors,a host protein LAPTM5 that can interact with Vpr was discovered.This study aims to explore the expression of LAPTM5 in DCs and its effect on HIV-1 replication in DCs and clear whether the interaction between Vpr and LAPTM5 will promote the replication of HIV-1in DCs.It provides a new mechanism for the key role of Vpr in HIV-1 infection,and provides some clues to the complex relationship between infection and anti-infection between the virus and the host.Methods:1.Research object:In the study of Vpr promoting HIV-1 replication in DCs by antagonizing endogenous LAPTM5,CD14⁺monocytes are sorted from PBMCs,which are extracted from healthy people,and induced into MDDCs.For the study of the mechanism of LAPTM5 inhibiting HIV-1 replication and the mechanism of Vpr antagonizing the antiviral function of LAPTM5,He La cells,TZM-bl cells and 293T cells are mainly used.Selection criteria for healthy people:No gender limit,age 18-55,HIV negative,hepatitis B and C negative,syphilis negative,no tumors,no metabolic diseases,and no autoimmune diseases,and no pregnancy for women.2.Induction of MDDCs:PBMCs were extracted by Ficoll density gradient centrifugation.Easy Sep^TMHuman CD14 Positive Selection Kit was used to sort CD14⁺monocytes which induce into MDDCs with GM-CSF and IL-4.3.Preparation of infectious clones:Jet PRIME Transfection Reagent was used to transfect 10μg pro-viral vector into 293T cells（5×10⁶/10 cm dish）,and medium was replaced with fresh medium 6 hours after transfection.The supernatant was collected after48 hours,filtered with a 0.45μm filter after centrifugation,treated with DNase,detected by p24 ELISA,and stored at-80°C until use.4.si RNA transfection:Lipofectamine 3000 Transfection Reagent was used to transfect si RNA into the cells.Change the medium 6 hours after transfection,and then perform subsequent experiments.5.Transfection of overexpression plasmid:Each expression vector was transfected into the cells with jet PRIME Transfection Reagent.Change the medium 6 hours after transfection,and then perform subsequent experiments.6.Production of sh RNA lentiviral particles:Jet PRIME Transfection Reagent was used to transfect 5μg sh RNA lentiviral expression vector,3.75μg p AX2 packaging plasmid and1.25μg p MD2G plasmid into 293T cells（5×10⁶/10cm dish）.6 hours after transfection,the medium was replaced.After 48 hours,the supernatant was collected,centrifuged and filtered with a 0.45μm filter.The virus was detected by p24 ELISA,and stored at-80°C until use.7.Preparation of VLP-vpx:Jet PRIME Transfection Reagent was used to transfect4.5μg p SIV3+-vpx^-,4.5μg p CMV-vpx and 1μg p CMV-VSV-G plasmids into 293T cells（5×10⁶/10cm dish）.Change the medium 6 hours after transfection.The supernatant was collected after 48 hours,centrifuged and filtered with a 0.45μm filter.The obtained supernatant was concentrated 100 times and stored at-80°C until use.8.sh RNA lentivirus infection of MDDCs:On the 5th day of monocyte induction into MDDCs,cells were treated with the obtained VLP-vpx for 2-4 hours,then infected with sh RNA lentivirus（200ng/0.6×106 cells）for 2 days.Then 1.5μg/m L puromycin was used for screening,and puromycin-resistant MDDCs were obtained for subsequent experiments.9.Western blot:After lysing the cells with RIPA Lysis and Extraction Buffer,protein was extracted.The concentrated gel and separating gel are subjected to constant voltage electrophoresis at 80V and 120V,respectively.The protein was transferred from gel to membrane for 2 hours on ice at Constant current 0.3A（or constant voltage 60V）.The protein was blocked with 2%BSA at room temperature for 1 hour.The membrane was incubated with the primary antibody overnight at 4°C,incubated the secondary antibody at room temperature for 30 minutes and incubated in the ECL luminescent solution at room temperature for 1 minute and then was placed on the ECL luminometer for imaging.10.Luciferase activity detection:Adherent cell culture medium was discarded,and add 1×Passive Lysis Buffer 300μL/well into the 6-well plate（or 1×Passive Lysis Buffer200μL/well to the 12-well plate）.After fully lysing,50μL of cell lysate was took and added into a 96-well microplate.Then add 25μL of luciferase substrate,shake well and place in a microplate reader for detection.11.Immunofluorescence:Cells to be stained were placed on a cell slide and washed twice with PBS.Cells were fixed at room temperature for 15 minutes,and blocked with the blocking solution after treating with Permeabilization solution.Cells were incubated with the primary antibody overnight at 4°C,incubated wigh the secondary antibody at room temperature for 30 minutes,and then stained with DAPI.12.Immunoprecipitation:He La cells were lysed with IP buffer.The cell lysate was placed on ice for 30 minutes,and centrifuged at 12000 rpm at 4°C for 10 minutes.Transferring the obtained supernatant to a new centrifuge tube,and pre-chilled IP buffer was added into the obtained pellet.mix well,sonicate,and then centrifuge at 12000 rpm for 10 minutes at 4°C.The two supernatants obtained in the above steps were mixed and incubated with Dynabead protein A and Dynabead protein G.Wash the obtained product with pre-cooled IP buffer and PBS for 5-10 times,and then perform subsequent experiments.13.Statistical analysis:Use Graph Pad Prism8.0 and SPSS19.0 for statistical analysis and mapping.The measurement data are expressed as±.The comparison between the two groups of measurement data was analyzed by unpaired t test.P<0.05 was considered as statistically different.（*P<0.05,**P<0.01）Results:1.Study on the effect of endogenous LAPTM5 on HIV-1 replication in DCs1.1 MDDCs were infected with 100ng wild-type or Vpr-deficient HIV-1_AD8 for 12days.The supernatant was collected at 3d,6d,9d and 12d after infection and tested for p24.The results indicated that as the infection time increases,p24 in the supernatant produced by wild-type HIV-1 _AD8 is significantly higher than that of Vpr-deficient HIV-1 _AD8.The results suggesting that Vpr can promote HIV-1 infection in MDDCs.After extracting protein and RNA from various cells,we detected that LAPTM5 has a higher expression level in MDDCs by Western blot and q PCR.Next,we used 100 ng wild-type or Vpr-deficient HIV-1_{Ba L}or HIV-1_AD8 to infect MDDCs.We found that Vpr can significantly reduce the expression level of endogenous LAPTM5 in MDDCs by Western blot detection.1.2 After knocking down LAPTM5 in MDDCs with sh RNA lentivirus,100ng wild-type or Vpr-deficient HIV-1_AD8 were infected for 10 days,and the supernatant was collected at 2,4,6 and 10 days after infection.Accumulated virus p24 in the cell supernatant was detected by ELISA.Data shows that Vpr promotes HIV-1 infection by antagonizing LAPTM5 within 10 days of continuous infection.2.Study on the Mechanism of LAPTM5 Inhibiting HIV-1 Replication2.1 The LAPTM5 and Vpr-deficient HIV-1 proviral vector were co-transfected in He La cells.After 48 hours,the supernatant was collected to infect TZM-bl cells and p24in the supernatant was detected.The results showed that LAPTM5 significantly reduced the infectivity of X4-tropic,R5-tropic or bitropical Vpr-deficient HIV-1.The production of p24 has no significant effect.2.2 The LAPTM5 and Vpr-deficient HIV-1 proviral vector were co-transfected in He La cells,and p24 and gp120 in the supernatant were detected 48 hours later.The results showed that LAPTM5 did not affect the level of p24 in the supernatant,but gp120decreased significantly.This indicates that LAPTM5 down-regulates the Env of progeny viruses and reduces the infectivity of the progeny virus.2.3 Overexpression of LAPTM5 in He La cells and immunostaining of the lysosomal marker LAMP1 protein showed that LAPTM5 and LAMP1 were co-localized on the lysosome.The LAPTM5 and Vpr-deficient HIV-1 proviral vector were co-transfected in He La cells,treated with lysosomal inhibitors bafilomycin A1（Bafilomycin A1,BFLA1）and chloroquine（CLQ）.Lysosomal inhibitors can inhibit the degradation of Env in cells,and the expression level of Gag is not affected.And through immunofluorescence analysis,it was determined that Env that was not degraded co-localized with LAPTM5 under the action of lysosomal inhibitors.3.Study on the mechanism of Vpr antagonizing LAPTM5 antiviral function3.1 The vector of LAPTM5,Vpr-deficient HIV-1 proviral vector,and HIV-1_NL4-3Vpr plasmid were co-transfected into He La cells.After 48 hours,the infectivity of the virus particles in the supernatant was detected by TZM-b1 cells.The results showed that Vpr Significantly reduces the restriction of LAPTM5 on HIV-1.After lysing the cells,it was detected by Western blot that Vpr could mediate the degradation of LAPTM5.3.2 The over-expression plasmids of LAPTM5 and Vpr were co-transfected in He La cells,treated with 1.5μM MG132.It was found that MG132 can inhibit the effect of Vpr on the level of LAPTM5,indicating that Vpr mediates the degradation of LAPTM5through the proteasome pathway.3.3 The over-expression vector of LAPTM5 and Vpr were co-transfected into He La cells,treated with MG132 for 10 hours.Cells were subjected to immunoprecipitation.The result suggested that in addition to Vpr,its cofactor DCAF1 also can interact with LAPTM5 and only in the presence of Vpr,DCAF1 can interact with LAPTM5,which shows that Vpr-induced degradation of LAPTM5 is mediated by DCAF1.LAPTM5 and Ubiquitin expression vector were co-expressed in He La cells.DCAF1 was knocked down and the ubiquitination of LAPTM5 was inhibited.In summary,Vpr relies on DCAF1 to promote the ubiquitination of LAPTM5,thereby inducing its degradation in the proteasome.Conclusions:1.LAPTM5 has a high level of expression in DCs.2.HIV-1 Vpr protein can significantly reduce the expression level of endogenous LAPTM5 in DCs.3.LAPTM5 takes an antiviral effect in DCs,but Vpr can promote HIV-infection and transmission by antagonizing LAPTM5.4.LAPTM5 limits the infectivity of HIV-1 by inducing Env protein degradation in lysosomes,and has no effect on the p24 production of viral particles.5.Vpr induces the degradation of LAPTM5 in the proteasome through the DCAF1-E3 ubiquitination ligase pathway.