| Objective:In the 1980s,scientific researchers discovered a serious threat to human health,Acquired Immune Deficiency Syndrome(AIDS),better known as AIDS;AIDS is a malignant infectious disease caused by the Human Immunodeficiency Virus(HIV),which causes severe defects in the functioning of the immune system.HIV mainly infects human CD4+T cells,macrophages,and dendritic cells,and the main clinical manifestation of the disease is the gradual decrease of CD4+T cells,which leads to death by concurrent severe infections.Myeloid cells include dendritic cells,monocytes,and macrophages.These cells have phagocytic and inflammatory responses to pathogens and play a protective role in maintaining tissue homeostasis,repairing damage and growth and development.Macrophages are widely distributed in all tissues and organs,including the central nervous system,they participate in the host’s antiviral innate immune response,and are also important target cells and reservoirs for HIV infection.Myeloid cells colonized in tissues are the first cells to be infected in the sexual transmission route and inhibit viral replication in the early stages of infection,so studying the role of myeloid cells in HIV infection has important implications for the treatment of AIDS.Several clinical studies have found that interferon is closely related to the disease progression of AIDS,so the relationship between interferon and HIV-1 infection has received increasing attention in the past decade.Acute HIV-1 infection in humans results in a rapid increase in interferon-α(IFN-α)in vivo,which is central to direct antiviral immunity and links innate and adaptive immune responses.At the same time,in vitro studies found that IFN-α had a significant inhibitory effect on the replication of HIV-1 in a cell culture model.Interferon-stimulated genes(ISGs)MX2,APOBEC3G,Trim22,Tetherin,and SAMHD1,which are upregulated by interferon,are cellular proteins that inhibit virus replication.Therefore,exploring the mechanism of interferon inhibiting HIV1 replication will provide new ideas for the treatment of AIDS.Otoferlin(OTOF)belongs to the Ferlins protein family,which is a relatively new large,multi-domain type Ⅱ transmembrane protein involved in a variety of biological functions centered on membrane fusion.OTOF is also known as Fer-l-like protein 2.Fer-1,a member of the Ferlin family,first discovered in elegans,is required for Ca2+-mediated fusion of membrane organelles with the plasma membrane of sperm during spermatogenesis.A homologous gene,misfre,was later found in Drosophila,which is related to sperm activation,egg patterning,and plasma membrane rupture in germ cells,while OTOF is the second member of a mammalian gene family related to Ferl,hence the name Fer-1 liked protein 2.OTOF encodes a predicted membrane protein isoform(1230aa)with three C2 domains and a carboxy-terminal transmembrane domain.The sequence homology and predicted structure of the OTOF-encoded protein suggest that it is involved in vesicle membrane fusion,and the OTOF protein encapsulates Ca2+,is Ca2+-dependent,and in many ways resembles Ca2+-dependent exocytosis.Immunofluorescence staining of cochlear cells in mice indicated that OTOF was mainly expressed in cochlear sensory hair cells.In the developing mouse cochlea,the localization of OTOF is localized to the site of formation of hair cells and afferent synapses;in the mature mouse cochlea,the localization of OTOF is restricted to inner hair cells.The localization results by immunoelectron microscopy also suggested that OTOF was located in the presynaptic membrane and synaptic vesicles of inner hair cells.The current research on OTOF mainly focuses on human hearing impairment,and there is no report in other fields.In this study,OTOF was screened by comparing the differentially expressed genes(DEGs)of PBMCs cells of HIV-infected patients and uninfected normal volunteers and found that OTOF was mainly up-regulated by interferon in myeloid cells.The effects of HIV-1 replication and the exploration of its mechanism provide new clues for the treatment of HIV-1 infection.Methods:1.Research objects and grouping criteriaA total of 62 samples were collected in this study,including 16 healthy controls(Healthy group),20 HIV-1 infected patients who received cART treatment(ART+group),and 26 HIV-1 infected patients who did not receive cART treatment(ART-group),including 14 patients with long-term non-progression(LTNPs group)and 12 patients with rapid progression(RPs group)in the ART-group.Inclusion criteria for the Healthy group:healthy without serious diseases such as tumor diseases,pregnancy,metabolic diseases and autoimmune diseases,and no bacterial and viral infections in the past 1 month;ART+group inclusion criteria:have received cART treatment,and have no blood Medium viral load was less than 50 copies/mL;LTNPs group inclusion criteria:patients infected with HIV-1 for more than 8 years,CD4+T cell count in peripheral blood greater than 500 cells per microliter;RPs group inclusion criteria:patients infected with HIV-1 CD4+T cell count in peripheral blood was less than 350 cells per microliter for 1 to 2 years after the virus.This study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University.All volunteers who participated in this experimental research topic were informed about the relevant experimental information,and they all gave informed consent and signed an informed consent form.2.Plasma viral load detection of patientsFirst,the anti coagulated peripheral blood of the recruited patients was collected,the whole blood of the patients was centrifuged,the upper layer of fresh plasma was taken,and 1.2 mL was drawn for viral load detection.The Roche COBAS AMPLICOR automatic load analyzer was used to detect the patient’s plasma viral load.The detection principle was real-time fluorescence quantitative polymerase chain reaction RT-qPCR.3.Isolation and culture of primary PBMCsCollect 50 mL of anti coagulated peripheral blood from recruited patients and healthy volunteers,gently invert and mix 7 times.Use Ficoll method to separate PBMCs,mix 15 mL of whole blood with 15 mL of PBS,add to 15 mL of Ficoll,and use density gradient centrifugation.The cloud layer between the uppermost layer and the middle layer is PBMCs.According to the kit instructions,the EasySepTM Human CD14 Positive Selection Kit Ⅱ was used to positively select CD 14+monocytes,and then the Easy SepTM Human CD4+T Cell Enrichment Kit was used to negatively select CD4+T lymphocytes according to the kit instructions.4.Cell CultureTHP-1-induced macrophages(THP-1-MA)Untreated induced THP-1 cells were induced to become macrophages after 48 hours of culture in RPMI1640 medium containing 100 nM phorbol ester PMA.Culture conditions:37℃,cultured in an incubator containing 5%CO2,and subcultured once every 2 days.Human cervical cancer cell line(HeLa)and human embryonic kidney cell line(293T)were cultured in DMEM medium containing 10%FBS and 100U/mL penicillinstreptomycin,culture conditions:37℃,in an incubator containing 5%CO2 Culture,subculture every 2 days.Human leukemia T lymphocyte line(Jurkat)and human leukemia mononuclear cell line(THP-1)were cultured in RPMI1640 medium containing 10%FBS and l00U/mL penicillin-streptomycin,culture conditions:37℃,containing 5%CO2 cultured in an incubator and subcultured every 2 days.5.RT-qPCRUse the PrimeScriptTM RT reagent Kit to remove residual DNA contamination from RNA samples and reverse-transcribe RNA to cDNA.Using cDNA as a template,RT-qPCR test was performed using TB Green? Premix Ex TaqTM II(TAKARA)reagent;GAPDH was used as an internal reference,and the 2-ΔΔCt method was used to calculate the relative expression of the target gene.6.RNA sequencing(RNA-seq)Total RNA from cells was extracted with TRIzol reagent,and RNA samples were initially quantified using a micro UV-Vis spectrophotometer.The concentration of extracted RNA samples was precisely quantified using an Agilent 2100.The library was constructed when the RNA concentration,total amount,purity and RIN value met the standards.The rRNA was removed,then broken into short fragments,and the first-strand cDNA was synthesized using six-base random primers using the RNA as a template.Add buffer,dNTPs and enzymes to synthesize second-strand cDNA and purify into doublestranded cDNA.The purified double-stranded cDNA is end-repaired and ligated with sequencing adapters to degrade the second-strand cDNA containing U bases.The purified products were enriched by PCR,and finally a specific library was obtained.Quantitative PCR is used to accurately quantify the library,and the effective concentration of the library is greater than 2nM,and it can be sequenced on the machine.Each library was pooled according to the effective concentration and the target loading requirements,and SE50 sequencing was started.7.Cell Transfection293T and HeLa cells were transfected with LipoD293TM transfection reagent(SignaGen Laboratories)for overexpression plasmids.The plasmids were dissolved in serum-free DMEM medium,and LipoD293 reagent was dissolved in another tube of serum-free DMEM medium.Diluted LipoD293 was added to the diluted plasmid,vortexed and mixed and incubated for 10 minutes,and finally added dropwise to the cells,which can be used for subsequent studies after 24 hours.8.HIV-1NL4-3-Luc(VSVG)reporting viral packagingVirus packaging was performed in 293T cells,and 6.66 μg pNL4-3-Luc and 3.34 μg pCMV-VSV-G plasmids were transfected into 5 ×106 cells using LipoD293TM transfection reagent,and fresh medium was replaced 6 hours after transfection 10 mL,the supernatant was collected after 48 hours,and cell debris was filtered using a 0.45 μm filter.The virus was quantified by ELISA using HIV-1 p24 Antigen Capture Assay reagent and stored at80℃ for subsequent use.9.shRNA lentiviral packaging5×106 cells were transfected with 5μg shRNA lentiviral expression plasmid,3.75μg psPAX2 packaging plasmid and 1.25μg pMD2.G plasmid using LipoD293TM transfection reagent.After 6 hours of transfection,10 mL of fresh medium was replaced,and 48 hours later the supernatant was collected and cell debris was filtered using a 0.45 μm filter.The virus was quantified by ELISA using HIV-1 p24 Antigen Capture Assay reagent and stored at-80℃ for subsequent use.10.Luciferase activity assayLyse cells with 300 μL of 1x Passive Lysis Buffer and place on a shaker for lysis at 150 rpm for 10 minutes.Aspirate 50 microliters of supernatant,avoiding aspirating cell debris,add 25 microliters of luciferase substrate,and perform luciferase activity detection by microplate reader.11.Western blot analysisCollect cell samples,add 300 microliters of RIPA Lysis and Extraction Buffer reagent to resuspend the lysed cell samples,and place the samples on ice for 10 minutes to precool.The remaining uncleaved components were disrupted with a ultrasonicator.After centrifugation,suck the supernatant into a clean new tube,add the corresponding volume of 4×Protein SDS PAGE Loading Buffer,and boil to denature the protein.Use the SDS-PAGE gel preparation kit to prepare a gel suitable for the size and concentration of the target protein and use 80V for electrophoresis to pass through the stacking gel and 120V for electrophoresis to pass through the separating gel.The PVDF membrane was activated by methanol,and the membrane was transferred at a constant pressure of 60 V on ice for 2 hours.Block with 2%BSA at room temperature for 1 hour,incubate with primary antibody overnight at 4℃,and wash 3 times with PBST for 10 minutes each time to remove unbound primary antibody;incubate with secondary antibody for 30 minutes at room temperature,and wash membrane 3 times with PBST,each 10 minutes,to wash away unbound secondary antibody.The washed membranes were placed in ECL luminescent solution,incubated for 1 minute,and then developed and imaged on a gel imaging analyzer.GAPDH protein was used as an internal reference to analyze the results.12.Flow CytometryAfter cell culture and corresponding treatment,the cell suspension was transferred to a flow tube,washed twice with 1 mL of FACS Buffer containing 2%serum PBS,and finally resuspended with 100μL of FACS Buffer,and incubated with the corresponding concentration of fluorescently labeled antibodies.30 minutes.Wash twice with FACS Buffer,and finally resuspend with 300 microliters of FACS Buffer,and then it can be detected on the machine.Select the corresponding laser and detection channel on the flow cytometer to analyze the cells.For the specific operation,refer to the Sony ID7000 manual.13.HIV Fusion AssayThe virus was packaged in 293T cells,transfected with LipoD293TM transfection reagent,6μg HIV NL4-3 proviral DNA,2μg of pCMV-BlaM-Vpr,and 10μg pAdVAntage vector were transfected into 5×106 293T cells,replaced with fresh 6h Culture medium,collect the supernatant after 2 days,filter the cell debris with a 0.45μm filter,ultracentrifuge at 72,000×g,90 min,4℃,concentrate the virus 10 times,quantify the P24 concentration by ELISA;infect 400ng of the virus with 0.5×106 TZM-bl cells were washed for 4 h,and the virus was washed away;stained with CCF2 dye at room temperature for 1 h,washed away the dye,and incubated with development media at room temperature for 16 h;fixed in FACS Buffer containing 1.2%paraformaldehyde,fixed at 4℃ for 24 h before use For flow cytometry,cleaved CCF2 was detected using Pacific Blue,and uncleaved CCF2 dye was detected using BV510.14.Statistical analysisSPSS 26.0 was used for statistical analysis of the data.The measurement data were expressed as x±s.Pearson correlation test was used for correlation analysis,one-way ANOVA analysis was used for comparison between multiple groups,students’ t-test was used for comparison between two groups,and SNK test was used for comparison between two groups.Differences were considered statistically significant when P<0.05.Statistics were plotted on the data by GraphPad 9.0.Flow Cytometry analysis was performed by FlowJo 10.4 software for analysis and graphing.Results:1.Identification of OTOF and its role in HIV-1 replication(1)The transcriptomes of PBMCs cells from five HIV-infected patients and five healthy volunteers were sequenced and analyzed with padj<0.05&log2|FoldChange|>1 as screening criteria,combined with KEGG(Kyoto Encyclopedia of Genes and Genomes)and GO(Gene Ontology)enrichment analysis and bioinformatics analysis website search of the role of each differentially expressed gene in neural,secretory,immune and regenerative physiological processes and the results of basic screening tests,we finally screened OTOF as the protein of interest.(2)The results of the RNA-seq screening were validated by RT-PCR,and it was found that as the expression of OTOF increased,the serum viral load of patients was lower,while comparing the expression of OTOF in healthy individuals with and without cART patients revealed that the expression of OTOF in patients without cART was higher,while the expression of OTOF in healthy individuals and patients with cART levels were relatively low.(3)The expression levels of OTOF mRNA in other cell lines and primary cells were also examined,and it was found that OTOF was highly expressed in interferon-stimulated MDMs,MDDCs and THP-1-MA with resting CD4+T cells,while OTOF expression was lower in 293T,HeLa,Jurkat,activated CD4+T cells and non-interferon-stimulated MDMs and MDDCs.Therefore,subsequent experiments will be performed in OTOF low expression cells for overexpression experiments and in OTOF high expression cells for knockdown experiments.(4)A search of bioinformatics websites revealed that there are seven isoforms of OTOF,including isoforms 201,202,203,204,and 205,which encode proteins,and these five isoforms were overexpressed in 293T and HeLa cells and infected with HIV-1 replication reporter virus HIV-1NL4-3-Luc(VSVG),respectively.The results showed that only OTOF-204 was able to inhibit HIV-1 replication(P<0.01),and OTOF-204 was found to have a strong antiviral function.(5)Overexpression of OTOF-204 in 293T cells infected with HIV-1NL4-3-Luc(VSVG),HIV-2Rod-Luc(VSVG)and MLV-Luc(VSVG)reporter viruses,respectively,showed that OTOF was able to inhibit HIV-1 and HIV-2 replication(P<0.01),and no significant inhibitory effect on MLV(P>0.05).After infection with MDM and CD4+T cells screened with viruses packaged with OTOF-204 lentiviral expression vector and infected with HIV1AD8 and HIV-1NL4-3 viruses respectively for continuous infection,the supernatant of culture fluid was collected continuously to detect p24 for comparison with the Lenti-Ctrl group,and the results showed that the difference in viral replication in both groups was increasing with time,indicating that OTOF was effective in MDM as well as in CD4+T cells can inhibit HIV-1 replication.2.The effect of OTOF on the anti-HIV-1 virus function of interferon(1)In the first part of the study by RT-PCR analysis we found that IFN-a can induce upregulation of OTOF expression in myeloid cells,so we decided to study the effect of OTOF on interferon inhibition of HIV-1 replication in myeloid cells.We tested the expression of OTOF in MDMs,MDDCs,THP-1-MA,and CD4+T cells after stimulation by different interferon isoforms,and the results showed that IFN-α and IFN-β could induce elevated OTOF expression in MDMs,MDDCs,and THP-1-MA,while IFN-γ could not induce increased OTOF expression in CD4+T cells various isoforms of interferon only mildly upregulated OTOF expression.(2)According to the literature,IFN-α is the main isoform of the interferon family that exerts anti-HIV-1 replication function,so subsequent studies have mainly focused on IFNα.The expression of OTOF was detected in MDMs,MDDCs,THP-1-MA,and CD4+T cells after stimulation with IFN-α for 24/48/72 h.It was found that the expression of OTOF in MDMs,MDDCs,and THP-1-MA cells increased with the prolongation of IFN-a stimulation time.(3)Using siRNA to knock down OTOF in MDMs,and subsequently taking half of the cells using IFN-α stimulation,infected with HIV-1NL4-3-Luc(VSVG)reporter virus to detect luciferase activity,and the ability of IFN-α to inhibit HIV-1 replication was reduced in the knockdown OTOF group compared to the control group(P<0.01);MDM knockdown OTOF was infected using shRNA-packed lentivirus,and half of the cells were taken after puromycin screening using IFN-α stimulated and infected with HIV-1NL4-3-Luc(VSVG)reporter virus to detect luciferase activity,the ability of IFN-α to inhibit HIV-1 replication was decreased in the group with knockdown of OTOF compared with the control group(P<0.01);knockdown of OTOF using siRNA in the THP-1-MA cell line,followed by half of the cells taken and stimulated with IFN-α,infected with HIV-1NL4-3-Luc(VSVG)reporter virus assayed for luciferase activity,and the ability of IFN-α to inhibit HIV-1 replication was decreased in the group with knockdown of OTOF compared with the control group(P<0.01);the results suggest that OTOF is an important factor for the anti-HIV-1 viral function of interferon in macrophages.3.The mechanism of OTOF against HIV infection(1)Transfection of OTOF-GFP plasmid in 293T cells,along with immunefluorescence staining for the membrane protein Caveolin,revealed that the localization of OTOF and Caveolin largely overlap,indicating that OTOF is localized to the cell membrane and is a membrane protein.(2)By HIV-1 Attachment Assay assay and HIV-1 Fusion Assay assay,the results showed that OTOF did not affect the contact adhesion between HIV-1 and target cells,but inhibited HIV-1 replication by inhibiting HIV-1 viral membrane fusion.(3)Because OTOF is a calcium ion sensor,we investigated the effect of calcium ions on OTOF inhibition of HIV-1 replication was investigated by overexpressing OTOF-204 in 293T cells and subsequently treating the cells with Ca2+,while treating with Mg2+ as ions control,the results showed that calcium ions had no significant effect(P>0.05).(4)Subsequently,we constructed deletion plasmids of OTOF-204 in order to find the site of action of OTOF to inhibit HIV-1 replication,making one deletion per 100 base pairs,with two adjacent deletion plasmids with Subsequently,these deletion plasmids were overexpressed in 293T cells infected with HIV-1NL4-3-Luc(VSVG)reporter virus to detect luciferase activity and compared with full-length OTOF-204,and the results showed that the plasmids overexpressing deletion 540-640aa、810-910aa and 1170-1230aa lost the antiHIV-1 replication ability.These deletion sites were the sites we were looking for the ability of OTOF-204 against HIV-1.Conclusions1.OTOF can be up-regulated in HIV-1 infected patients in vivo.2.OTOF can be highly up-regulated by interferon in myeloid cells.3.OTOF can inhibit the replication of HIV-1 and HIV-2 but has no obvious inhibitory effect on MLV.4.OTOF is an important factor for interferon to exert antiviral function in myeloid cells.5.OTOF inhibits HIV-1 replication by inhibiting HIV-1 membrane fusion;calcium ions do not affect the antiviral function of OTOF and OTOF does not inhibit HIV-1 replication through receptors. |
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