Study On The Mechanism Of Metalloproteinase TRABD2A Suppressing HIV-1 Virus Replication

Objective:Human Immunodeficiency Virus?HIV?is the main pathogen that causes AIDS?Acquired Immune Deficiency Syndrome,AIDS?.After HIV infection in humans,the immune function is severely damaged,often combine with severe opportunistic infections that ultimately lead to death.HIV restriction factors,which refer to the proteins that exist in the host cells and can inhibit virus replication,are the body's first line of defense against HIV infection.Multiple restriction factors have been found at present,including SAMHD1?SAM and HD Domain Containing Deoxynucleoside Triphosphate Triphosphohydrolase 1,SAMHD1?,APOBEC3G?apolipoprotein B-editing catalytic polypeptide 3G proteins,APOBEC3G?,TRIM5?tripartite motif-containing protein 5,TRIM5?and Tetherin?BST-2/CD317/HM1.24?.The research on HIV restriction factors can explain the mechanisms of the body defense against viral infection and the mechanisms by which the viruses escape from the body's inhibition,and provides new research ideas and directions for the treatment of AIDS.Therefore,it is important to actively find new types of HIV restriction factors.In our earlier search for virus restriction factors that inhibited the release of infectious progeny virus by resting CD4⁺T cells,we found a gene that differentially expressed in resting CD4⁺T cells and activated CD4⁺T cells,TRABD2A?TRAB domain containing2A,TRABD2A?.TRABD2A is a metalloproteinase.The enzymatic activity is regulated by metal ions.It has been proven that TRABD2A can cleave Wnt proteins and maintain the normal structure of brain tissue by antagonizing the effect of Wnt proteins.There is no related research on TRABD2A in the HIV field and at the same time and the effect of TRABD2A on HIV replication has not been clarified.In addition,Real-time PCR was used to detect the expression level of TRABD2A in different cells.It was found that,in addition to expressing in resting CD4⁺T cells,TRABD2A expression was relatively high in monocyte-derived dendritic cells?MDDCs?,while it was extremely low or not expressed in other cells.Dendritic cells?DCs?are most important professional antigen-presenting cells in the human and play an important role in HIV infection.DCs are one of the important target cells of HIV because they express the CD4 and co-receptors of HIV.Although HIV can infect DCs,only about 1%of DCs are infected,and it is difficult for the infected viruses to release infectious progeny viruses due to the replication process is blocked.Therefore,there must be virus restriction factors that inhibits HIV replication in DCs.Although SAMHD1,APOBEC3G and Tetherin have been found to inhibit HIV replication in DCs at present,they have not been able to explain why HIV replication in DCs is blocked and it is difficult to release progeny viruses.Our study found that TRABD2A was relatively highly expressed in MDDCs.Can TRABD2A block HIV replication and release infectious progeny viruses in MDDCs?Our research aims to explore the effects and the specific molecular mechanisms of TRABD2A on HIV replication.At the same time,the effect of TRABD2A on HIV replication in MDDCs is explored and clarified whether TRABD2A is an HIV restriction factor that inhibits HIV replication and release progeny viruses in MDDCs.The study of TRABD2A and HIV will further reveal the mechanism of immune cells against HIV infection,and provide new research targets and directions for treatment and cure of AIDS.Study Subjects:293T cell lines and TZM-bl cell line were mainly used for the study of TRABD2A inhibiting HIV-1 virus replication.293T cell lines and TZM-bl cell line were mainly used for the study for the mechanism of TRABD2A to inhibit HIV-1replication.For the study of endogenous TRDB2A inhibiting HIV-1 viral replication in MDDCs,PBMCs from EDTA anticoagulated peripheral blood of healthy donors were used,CD14⁺monocytes were sorted by magnetic beads form PBMCs and were induced into MDDCs.Adherent cell culture:293T cells and TZM-bl cells were cultured in DMEM medium containing 10%FBS and 100 U/mL penicillin-streptomycin.Cells were cultured at 37°C and 5%CO₂ and were passaged every two days.Plasmids transfection:The jet PRIME transfection reagent was applied in 293T cells for plasmids transfection,Lipofectamine 2000 transfection reagent was applied for siRNA transfection in 293T cells,and Lipofectamine 3000 transfection reagent was applied to transfect siRNA in MDDCs.shRNA lentivirus packaging:3×10⁶ 293T cells were plated in a 10 cm culture dish,and after 24 hours,the plasmids of 5?g sh RNA lentiviral expression plasmid,3.75?g p AX2 packaging plasmid and 1.25?g p MD2G were transfected with jetPRIME transfection reagent.After 6 hours,the cells were replaced with fresh medium.The cell supernatant was collected after 48 hours and were filtered through a 0.45?m filter after centrifugation,and the p24 content of the virus was detected by ELISA.HIVNL4.3,HIVAD8 and HIV89.6 virus packaging:3×10⁶ 293T cells were plated in a 10cm culture dish,and after 24 hours,the plasmids of 10?g HIV_NL4.3,HIV_AD8 and HIV_89.6plasmids were transfected with jet PRIME transfection reagent.After 6 hours,the cells were replaced with fresh medium.The cell supernatant was collected after 48 hours and were filtered through a 0.45?m filter after centrifugation,and the p24 content of the virus was detected by ELISA.Luciferase detection:After the TZM-bl cells or 293T cells were lysed with Passive Lysis Buffer,50?L of the lysed supernatant was pipetted to a 96-well blackboard,25?L of Promega luciferase substrate was added,and the value was read by a microplate reader to detect the luciferase reaction intensity.Real-time PCR:TRIzol reagent was used to extract cell RNA.TAKARA's Prime Script?RT reagent Kit with g DNA Eraser was used to reverse transcribe RNA into cDNA,and TB Green?Premix Ex Taq?II?Tli RNase H Plus?was used for Real-time quantitative PCR reaction.Western Blot:After the cells were lysed with RIPA,the lysate was sonicated to extract the proteins in the cells.The protein concentration was measured by BCA assay.5×SDS-PAGE loading buffer was added,and the mixture was denatured by boiling.Protein electrophoresis was performed at 100V for 1h,and transferred overnight at 35V4°C.BSA was blocked for an hour,and the primary antibody was incubated at room temperature for an hour.The PVDF membranes were washed three times with PBST for 5 min each.The secondary antibody was incubated for 30 min at room temperature,and then washed by PBST solution was washed for three times for 5 min each.Then,ECL chemiluminescence was performed.Immunofluorescence detection:Place glass slides in 12-well plates and 293T cells were cultured in the plates.Remove cell slides 24 hours later,wash the cell slides with PBS,and cells were fixed with fixative solution for 20 min at room temperature;wash3 times with PBS to wash away the fixative solution,Permeabilization Buffer was added to the slides to break the membrane for 15 min at room temperature.Wash the Permeabilization buffer with PBS for 3 times,and block with BSA for 60 min.After blocking,add diluted primary antibody and incubate overnight at 4°C;wash the primary antibody with PBS and add the fluorescence-labeled secondary antibody,and incubate in the dark at room temperature for 30 min;wash the secondary antibody,and antifade mounting medium with DAPI was added.Observe with Fluorescence microscope and take pictures.ELISA detection of p24 antigen:Microelisa Plate was prepared according to the sample numbers and equilibrated to room temperature.Diluted the wash buffer.Centrifuged the cell supernatant samples and diluted by 5000 times with the medium.Dilute the standard samples according to the instructions(the concentration of the standard samples was 100 pg/m L,50 pg/mL,25 pg/m L,12.5 pg/m L,6.3 pg/m L,3.1 pg/m L.After the samples and standards were prepared,operated according to the kit instructions.After adding the peroxidase substrate and incubating,read the value and standard curve at 450nm on the microplate reader,and calculated the concentration of p24 in each sample according to the times of volume dilution.Statistical analysis:Graph Pad Prism 8.0 was used for statistical analysis and mapping of the data.Measurement data were expressed as±.Comparison of measurement data between the two groups was analyzed by unpaired t test,and P<0.05 was considered statistically significant.*P<0.05,**P<0.01,***P<0.001,****P<0.0001.Results:1.Study of TRABD2A inhibited HIV-1 virus replication1.1 TRABD2A-myc/Mock?1.5?g,1.0?g,0.5?g?plasmid and HIV-1 virus plasmid p NL4.3?0.5?g?were transfected at the same time in 293T cells at different ratios?3:1,2:1 and 1:1?.48 hours after transfection,the cell supernatant and the cells were collected,200?L of the virus supernatant was taken to infect TZM-bl cells,and the virus p24 and viral RNA in the supernatant were detected at the same time;total RNA was extracted from the cells,and gag RNA was detected by real-time PCR.From the Luciferase results,we can see that the produced infectious HIV particles from 293T cells transfected with TRABD2A were significantly lower than the control group,indicating that after transfection of TRABD2A,the infectious virus production was significantly reduced,and p24 was also significantly produced.The viral RNA in the supernatant was significantly lower than that in the control group,indicating that TRABD2A can effectively inhibit the replication of HIV-1 virus.Gag transcript level was basically unchanged,which indicated that TRABD2A does not affect the transcription of HIV-1virus.After transfecting 293T cells with 1.5?g and 1.0?g TRABD2A-myc/Mock plasmids,293T cells were infected with HIV-1 pseudotyped virus HIV_NL4.3-Luc?VSVG?.24 hours later,293T cells were lysed to detect changes in Luciferase.The expression of Luciferase after overexpression of TRABD2A in 293T cells was not significantly different from that of the control group,indicating that TRABD2A does not affect the early process of HIV-1 virus replication and virus transcription,but exerts antiviral functions in the late stage of HIV virus translation.1.2 The TRABD2A variants TRABD2A-201 had the strongest antiviral function,TRABD2A-201 inhibited HIV-1 production by 1,087-fold reductions,TRABD2A-202also inhibited HIV-1 virus replication and antiviral function of TRABD2A-202 was significantly lower than TRABD2A-201,whereas TRABD2A-203 exhibited no anti-HIV-1 activity.Another TRAB family member,TRABD2B also inhibited of HIV-1production significantly compared to controls,TRABD2B inhibited of HIV-1production by 186-fold reductions.Chimpanzee TRABD2A-201 also inhibited HIV-1 production,although in a weaker manner than TRABD2A-201,suggesting a conserved anti-HIV-1 function of the TRAB family of metalloproteases in primates.1.3 TRABD2A can effectively inhibit the replication of different HIV-1 strains including HIV_BH10 X4 strains,HIV-1_{Ba L} R5 strains,HIV _AD8 R5 strains and X4/R5 dual-tropic HIV_89.6 as well as HIV-2 wild type strain HIV-2_ST and SIV strain 239?SIVmac239?virus,suggesting the broad anti-HIV-1 activity for the TRABD2A.1.4 The anti-viral function of TRABD2A depended on its metalloproteinase activity.Ni²⁺,Co²⁺and metal ion chelator 1,10-phenanthroline can inhibit the antiviral ability of TRABD2A,while Mn²⁺promoted anti-viral functions of TRABD2A.2.Study on the Mechanism of TRABD2A Inhibiting HIV-1 Virus Replication2.1 We first transfected 293T cells with a TRABD2A-GFP plasmids construct together with proviral pNL4.3 vectors.Cells were collected and total protein were extracted 48h later.Western Blot was used to test whether TRABD2A can directly degrade the HIV-1 Gag protein.Compared to controls,TRABD2A promoted viral Gag protein degradation in a dose-dependent manner whereas the envelope glycoprotein was not affected indicating specific TRABD2A-mediated Gag precursor degradation.With the increase of Mn²⁺concentration,the protein expression of Pr55-Gag gradually decreased.When the concentration of Mn²⁺increased to 200?M,the Pr55-Gag protein band almost disappeared.To sum up,TRABD2A mainly inhibited virus packaging by degrading HIV-1 Gag protein,thereby inhibiting HIV-1 virus replication.2.2 We employed phosphoinositide-specific inositol polyphosphate 5-phosphatase IV?5ptase IV?overexpression to reduce the levels of the phospholipid phosphatidylinositol 4,5-bisphosphate?Ptd Ins?4,5?P2?at the cell membrane,which controls the plasma membrane localization of a number of cellular proteins and is the host cell determinant of Gag binding to the plasma membrane.We observed that 5ptase IV overexpression significantly reduced the anti-HIV-1 activity of TRABD2A and reduced TRABD2A-mediated degradation of the viral Gag precursor.Subsequently,we purified the cell membrane-associated viral Gag protein and observed that it was dramatically reduced by membrane-bound TRABDs,whereas the cytosolic Gag remained intact in the presence of cytosolic TRABD2A.TRABD2A did not degrade a Pr55-Gag mutant that lacked the membrane-targeting determinant of the N-terminal basic sequence?GARASVLS?for myristoylation??Myr?,suggesting that TRABDs only degrade membrane-bound viral Gag.In summary,TRABD2A mainly inhibited HIV-1 virus replication by degrading Pr55-Gag on the cell membrane.3.Endogenous TRABD2A in MDDCs inhibited HIV-1 virus replication3.1 The results of RNA-Seq showed that there was almost no TRABD2A expression in monocytes,and the expression of TRABD2A increased significantly after induction into MDDCs.Immunofluorescence results showed that TRABD2A was mainly distributed on the cell membrane of MDDCs.3.2 After silencing TRABD2A in MDDCs by siRNA,MDDCs was infected with high-inoculums?50ng?and low inoculums?5ng?HIV_AD8,and p24 in the supernatant was increased 6-8 times 72 hours later,indicating that TRABD2A can inhibit the replication of HIV-1 viruses.sh RNA lentivirus vectors were also used to silence TRABD2A in MDDCs and p24 expression in supernatant was 8-10 times higher.We explored HIV-1spreading infection in MDDCs for 15 days in the presence or absence of TRABD2A.Consequently,silencing of TRABD2A by sh RNA lentivirus vectors enhanced both CCR5-trophic proviral AD8 and dual-trophic 89.6 infection in MDDCs during viral spreading infection,particularly at low inoculums.In summary,the endogenous TRABD2A in MDDCs can inhibit the replication of HIV-1 virus.Conclusions:1.TRABD2A can effectively inhibit the HIV-1 virus replication in 293T cells,has obvious antiviral functions,and is a new type of HIV restriction factor.2.TRADB2A does not affect the process of reverse transcription,nucleation,integration,transcription,and translation of the virus,but affects the production of p24 of HIV-1virus,indicating that TRABD2A plays an antiviral function in the late stage of HIV-1virus infection3.Metal ions and metal ion chelator 1,10-phenanthroline can inhibit the metalloprotease activity of TRABD2A,increase virus replication,and significantly reduce the antiviral effect of TRABD2A,indicating that antiviral effect of TRABD2A depends on its Metalloproteinase activity.4.TRABD2A mainly degrades the Gag protein by binding to the HIV Gag protein on the cell membrane,so that HIV progeny virus cannot complete the packaging on cell membrane,thereby inhibiting the virus replication.5.After knocking down the endogenous TRABD2A in MDDCs,HIV-1 virus replication increased,indicating that TRABD2A also plays an antiviral function in MDDCs and is a new type of virus restriction factor of MDDCs.