| Objective:Acquired immune deficiency syndrome(AIDS)is a highly harmful infectious disease caused by the human immunodeficiency virus(HIV)attacking the human immune system,which seriously threatens human health.Highly active antiretroviral therapy(HAART)suppresses HIV replication to undetectable levels and thus delaying the progress of disease and prolonging the survival time of patients but cannot fully eradicate the virus because a small reservoir of various cells remains latently infected.The reservoirs are established early during infection,interruption of treatment inevitably results in a rapid rebound of viremia from the reservoirs,so the persistence of cells carrying a latent infection represents a major barrier to eradicationAlthough multiple reservoirs may exist,the persistence of resting memory CD4+T cells carrying a latent infection represents a major component of the reservoirs Although these latently infected resting CD4+T cells carry integrated HIV-1 genome,they do not spontaneously produce new virus particles,so they cannot be recognized and cleared by the immune system.The entrance into latency and its maintenance is a complicated phenomenon that is controlled and regulated by various mechanisms involving cellular host and virus factors.A better understanding of the mechanisms of postintegration latency will be necessary to uncover novel targets and methods for attacking and eradicating the latent reservoir.HIV transcription silencing(including transcription initiation and transcription elongation)in resting CD4+T cells is one of the main causes of virus latency.The mechanisms that have been reported to cause transcription silencing including transcription factor isolation and transcription interference.Therefore,understanding the mechanism of HIV-1 transcriptional silencing in resting CD4+T cells is critical to finding a way to clear the HIV reservoir and cure AIDSTranscription inhibitors such as COUPTF Interacting Protein 2(CTIP2),DRB-Sensitivity Inducing Factor(DSIF),Negative Elongation Factor(NELF),and TRIM protein family play an important role in HIV-1 transcription.In this study,by comparing the differentially expressed genes(DEGs)of activated and resting CD4+T cells,we screened out Period Circadian Regulator 1(PER1)as candidate.This gene is a core component of the circadian clock and a transcriptional repressor and expressed in a circadian pattern in the suprachiasmatic nucleus,which plays an important role in the regulation of circadian rhythm,and is of great significance for integrating the circadian rhythm of body activity.The circadian clock is an internal timing system that regulates various physiological processes by generating a circadian rhythm of gene expression for about 24 hours,and converts it into a rhythm of metabolism and behavior.PER1 is an important regulator of a series of physiological functions such as metabolism,sleep,body temperature,blood pressure,endocrine,immunity,cardiovascular and renal function.As a transcriptional repressor,whether PER1 regulates HIV-1 replication in resting CD4+T cells remains unclear.This study first analyzed the effect of PER1 on HIV-1 replication in resting CD4+T cells and explored its mechanism,providing new clues for latent infection caused by HIV-1 transcriptional silencing.In addition,the survey found that sleep disorders are very common among AIDS patients,and about 70%of adult patients have difficulty sleeping,such as insomnia and daytime sleepiness.Sleep disorders may exacerbate HIV-related complications,such as cognitive impairment,painful peripheral neuropathy,and chronic fatigue,leading to poor quality of life.PER1 is a core component of the regulation of biological rhythms.It is unclear how the expression of PER1 is in peripheral blood mononuclear cells(PBMCs)after HIV-1 infection.This study analyzes the expression of PER1 in PBMCs of HIV-1 infected patients,explores the relationship between its expression level and patient disease progression,and analyzes the correlation between its expression level and patient viral load and CD4+T lymphocytes count,providing new ideas for targeted treatment of AIDS.Methods:1.Research ObjectA total of 88 samples were collected in this study,including 23 healthy controls(16 in the Healthy group),28 HIV-infected patients(cART group)who received cART,and 31 HIV-infected patients who did not receive cART including 17 cases of rapid progressors(RPs group)and 14 cases of long-term non-progressors(LTNPs group)Selection criteria for the Healthy group:no tumor,pregnancy,metabolic disease and autoimmune disease,no bacterial or viral infection in the past 1 month;Selection criteria for cART group:receiving cART and plasma viral load<50 copies/mL;Selection criteria for RPs group:within 1-2 years after HIV infection,peripheral blood CD4+T cell count<350/?L;Selection criteria for LTNPs group:At least 8 years after HIV infection,the peripheral blood CD4+T cell count>500/?L.All volunteers participating in the study gave informed consent and signed the informed consent form.This study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University2.CD4+T cell absolute count and plasma viral load of patientsCD4+T cell absolute count:Collect 10 mL of peripheral blood of the patient with EDTA anticoagulated purple vacuum blood collection tube,and mix by inversion Take 50 ?L of anti coagulated whole blood into an absolute counting tube and add the corresponding reagents for staining.BD Calibur flow cytometer was used to detect the samples,and Multi SET software was used to automatically analyze the detection results,and the absolute value of CD4+T cells was finally obtainedDetection of plasma viral load:The collected anti coagulated whole blood was centrifuged to separate plasma,and 1.2 mL was taken for viral load detection.The instrument used was a Roche COBAS AMPLICOR automatic load meter,and the detection method was Real Time quantitative polymerase chain reaction(RT-qPCR)3.Isolation and differentiation of primary cellsIsolation of primary cells:Collect 50 mL of peripheral blood of the healthy controls with EDTA anti coagulated purple vacuum blood collection tube,and mix by inversion.Separation of PBMCs by Ficoll density gradient centrifugation.Monocytes were first sorted using Easy Sep TM Human CD 14 Positive Selection Kit ?(STEMCELL Technologies),and then CD4+T lymphocytes were sorted using EasySep TM Human CD4+T Cell Enrichment Kit(STEMCELL Technologies)CD4+T lymphocytes culture and activation:Resting CD4+T cells were cultured in RPMI1640 medium containing 10%inactivated FBS and 100U/mL penicillin-streptomycin;activated CD4+T cells were cultured in RPMI1640 medium containing 10%FBS,100U/mL of penicillin-streptomycin,50U/mL of Interleukin-2(IL-2),and Dynabeads(?)Human T-Activator CD3/CD28(Thermo Fisher)was added at a ratio of 25?L/1×106 cells Induction of CD14+ monocytes:Monocytes were cultured in RPMI1640 medium containing 10%inactivated FBS,2mM L-glutamic acid,100U/mL penicillin-streptomycin,lOmM HEPES,1%non-essential amino acids,1mM sodium pyruvate,10ng/ml GM-CSF(R&D Systems)and 50ng/ml M-CSF(R&D Systems),culture medium was replaced with fresh medium every two days.Monocyte-Derived Macrophage(MDM)were generated by 7-9 days of stimulation;Monocytes were cultured in IMDM medium containing 10%FBS,2mM L-glutamic acid,100U/mL penicillin-streptomycin,10mM HEPES,1%non-essential amino acids,1mM sodium pyruvate,10ng/ml GM-CSF and 50ng/ml IL-4(R&D Systems),culture medium,was replaced with fresh medium every 2 days.Monocyte-derived dendritic cells(MDDC)were generated by 5-7 days of stimulation4.RNA-sequenceDetection of RNA quality:Total RNA was extracted from cells using TRIzol(Invitrogen)reagent,and NanoDropTM One/OneC micro-ultraviolet-visible spectrophotometer was used for preliminary quantification of RNA samples.Aglient 2100 was used to accurately quantify the concentration of RNA samples.The total amount,concentration,integrity(RIN value,RNA Integrity Number),and purity(OD260/OD280 ratio)meet the standards for library constructionlibrary construction:First remove rRNA and break the RNA into short fragments,synthesize the first-strand cDNA with six base random primer;Buffer,dNTPs(dUTP,dATP,dGTP and dCTP)and enzyme were added to synthesize the second-strand cDNA and purify the double-stranded cDNA;The purified double-stranded cDNA is first repaired at the end and connected to the sequencing adapter,and then the fragment size is selected to degrade the second-stranded cDNA containing U bases;Finally,perform PCR enrichment and purify the PCR products to obtain the final chain-specific library.Library quality inspection:Quantitative-PCR(Q-PCR)is used to accurately quantify the library.The effective concentration of the library>2nM can be used for sequencing.Sequencing:SE50 sequencing was performed after pooling each library according to the requirements of effective concentration and target offline data volume.5.Culture of adherent and suspended cell linesHuman kidney epithelial cell line(293T)and human cervical cancer cell line(HeLa)were cultured in DMEM medium containing 10%FBS and 100U/mL penicillin-streptomycin;Human T lymphocyte strain(Jurkat)and human monocyte leukemia cell line(THP-1)were cultured in RPMI1640 medium containing 10%FBS and 100 U/mL penicillin-streptomycin;Induction of THP-1 derived macrophages(THP-1-MA):THP-1 cells are differentiated into macrophages after cultured for 48 hours in a medium containing 100nM phorbol ester(Phorbol 12-Myristate 13-Acetate,PMA)(Promega).All cells were cultured at 37? 37? in a humidified incubator supplied with 5%CO2.6.RT-qPCRTotal RNA was extracted using TRIzol reagent and PrimeScript TM RT reagent Kit with gDNA Eraser(Perfect Real Time)(TAKARA)was used to remove DNA contamination of RNA samples and reverse transcribes RNA into cDNA;The first-strand cDNA was used as template for amplification via real-time PCR with TB Green(?)Premix Ex TaqTM II(TAKARA).GAPDH was used as an endogenous control.7.Cell transfection293T and HeLa cells were transfected with jetPRIME transfection reagent(Polyplus-transfection)according to the Manual;The primary resting CD4+T cells were transfected with Human T Cell NucleofectorTM Kit(Lonza)via Nucleofector 2b electroporation instrument by program U-14.MDM and THP-1-MA were transfected with Lipofectamine 3000 transfection reagent(Invitrogen).8.Virus production and infectionViral particles were produced from 293T cells.HIV-1NL4.3-Luc(VSV-G):Transfection of 6.7?g pNL4.3-Luc and 3.3?g pCMV-VSV-G plasmids into 5×106 cells with jetPRIME transfection reagent;VLP-vpx:Transfection of 4.5?g pSIV3+-vpx-,4.5?g pCMV-vpx and 1?g pCMV-VSV-G into 5×106 cells with jetPRIME transfection reagent;shRNA lentiviral particles:Transfection of 5?g shRNA lentiviral expression plasmid,3.75?g pAX2 packaging plasmid and 1.25?g pCMV-VSV-G plasmid into 5×106 cells with jetPRIME transfection reagent;Media was replaced 6h after transfection and viruses were harvested 48h later,filtered at 0.45?m.When required,p24 concentration was measured by ELISA(Advanced BioScience Laboratories,ABL).HIV-1NL4.3-Luc(VSV-G)infection:293T,HeLa,THP-1-MA and MDM were infected according to MOI=1.0;Resting CD4+T cells were infected with 100ng of p24 HIV-1NL4.3-Luc(VSV-G)/2×106 cells and centrifuged at 1200×g for 2h;ShRNA lentivirus infection:Infection was performed at a rate of 200ng p24 shRNA lentivirus/0.6×106 cells.After 3 days of infection,it was screened by puromycin(Sangon Biotech)at a concentration of 1?g/mL.9.Detection of luciferase activityPassive Lysis Buffer(Promega)was used to lyse the cells and luciferase activity in the supernatant was measured according to the manufacturer's instructions10.Alu-PCRGenomic DNA was extracted using QIAamp mini kit(QIAGEN)and amplified by nested PCR.The first round of amplification:the upstream primer is located in the Alu element of the human genome,the downstream primer is located in the HIV-1 gag,and the second round of amplification is carried out using the PCR amplification product of the first round as a template;the upstream and downstream primers are located in the HIV-1 LTR.GAPDH was used as an endogenous control.11.immunoblottingCollect cell samples,resuspend the cell samples by RIPA Lysis and Extraction Buffer(Thermo Fisher).Centrifugation and transfer the supernatant to a new tube Add 4×Protein SDS PAGE Loading Buffer(TAKARA)and boil to denature the protein.Load equal amounts of protein into the wells of the SDS-PAGE gel,along with molecular weight marker.Run the Separation gel at 80V and Concentrated gel at 120V;Transferring the protein from the gel to the membrane for 2h at 60V on ice;Block the membrane for 1h at room temperature with 2%BSA;Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer for 1h at room temperature;Wash the membrane in three washes of PBST,5 min each;Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 30min;Wash the membrane in three washes of PBST,5 min each;Incubate the membrane with ECL solution for lmin for signal development.GAPDH was used as an endogenous control12.Flow cytometryAfter the cell culture and corresponding treatments were completed,the cell suspension was transferred to a flow tube,washed with FACS Buffer(PBS with 2%FBS)twice,and then added fluorescently labeled antibodies:aCD69-PE(BD Pharmingen)and aCD25-BB515(BD Horizon),stained on ice in the dark for 30min,washed twice with FACS Buffer and analyzed by BD FACSCelesta flow cytometry CellTrace cell proliferation staining was performed according to the kit instructions13.Statistical analysisStatistical analysis was performed using SPSS19.0 and GraphPad8.0.Two-tailed,unpaired Student's t-tests were used for between-group statistical comparison;One-way analysis of variance was used for comparison between groups,SNK test was used for pairwise comparison between groups,and Pearson correlation test was used for correlation analysis.The difference was statistically significant with P<0.05Results1.Identification of PER1 and its role in HIV-1 replicationSequencing and analyzing of transcriptomes of resting and activated CD4+T cells,with padj<0.05&log2 | FoldChange |>1 as the screening criteria,combined with GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis and Bioinformatics website's role of various differentially expressed genes in physiological processes such as immunity,nerves,secretion and regeneration and the results of basic screening tests,we finally screened PER1 as the protein of interest.RT-qPCR was used to verify the results of RNA-seq.The results showed that with the extension of the activation time of CD4+T cells,the expression level of PER1 mRNA gradually decreased.At the same time,the expression levels of PRE1 mRNA in other cell lines and primary cells were detected.It was found that in addition to activated CD4+T cells,the relative expression levels of PER1 mRNA in primary monocytes,MDM and THP-1-MA were higher,and relative expression levels of PER1 mRNA were lower in 293T,HeLa and MDDC.Therefore,we performed overexpression experiments in cells with low PER1 content and knockdown experiments in cells with high PER1 content.Bioinformatics website search results show that PER1 has two isoforms(PER1-201 and PER1-202)PER1-201 and PER1-202 were overexpressed in 293T and HeLa cells,respectively,and infected with the HIV-1 replication reporter virus HIV-1NL4.3-Luc(VSVG).The results showed that PER1-202 can inhibit HIV-1 replication(P<0.01),and PER1-201 had no effect on HIV-1 replication(P>0.05);SiRNA knockdown of PER1 expression in resting CD4+T cells,MDM and THP-1-MA and infection with HIV-1 replication reporter virus HIV-1NL4.3-Luc(VSVG).The results showed that Knockdown of PER1 expression could promote HIV-1 replication(P<0.01).These results suggest that PER1 can inhibit HIV-1 replication in resting CD4+T cells and MDM2.The Mechanism of PER1 Inhibiting HIV-1 ReplicationAccording to information provided in previous literature and bioinformatics websites,PER1 is a transcriptional repressor.Therefore,we overexpressed PER1-201 and PER1-202 in 293T and HeLa cells,respectively,and infected with HIV-1 replication reporter virus HIV-1NL4.3-Luc(VSVG)to detect HIV-1 integration and transcription levels.The results showed that:PER1-201 and PER1-202 did not affect the integration of HIV-1(P>0.05),and overexpression of PER1-202 resulted in a significant decrease in Gag RNA expression level(P<0.01),that is,decrease in HIV-1 transcription level;Knockdown of PER1 expression by siRNA or shRNA in resting CD4+T cells,MDM and THP-1-MA can lead to a significant increase in Gag RNA expression levels(P<0.01),which means increased HIV-1 transcription levels,suggesting that PER1 affects HIV-1 replication by inhibiting transcription.To further clarify this mechanism,we co-transfected the PER1-202 expression plasmid with LTR-Luc or CMV-Luc reporter plasmids in 293T cells.The results showed that overexpression of PER1-202 can lead to a significant reduction in LTR-Luc expression levels(P<0.01)without affecting the expression of CMV-Luc(P>0.05);Co-transfection of siPERl and LTR-Luc or CMV-Luc reporter plasmids in resting CD4+T cells,the results showed that knockdown of PER1 can promote the expression level of LTR-Luc(P<0.01)without affecting the expression of CMV-Luc(P>0.05)The above results suggest that PER1-202 directly inhibits the transcription initiated by the HIV-1 promoter(LTR),thereby inhibiting HIV-1 replication in host cells.Tat as a regulatory protein can significantly increase the level of viral transcription,so can Tat ameliorates the inhibition of LTR transcription by PER1?We co-transfected PER1-202 with pHIV-1NL4.3-Luc Tat+or pHIV-1NL4.3-Luc-Tat-plasmid in 293T cells and found that HIV-1 inhibition increased from 5.28-to 12-fold in the absence of Tat,thereby indicating that Tat can ameliorate PER1-202-mediated suppression of the HIV-1 promoter.We then co-transfected 293T cells with PER1-202 vectors and an HIV-1 LTR-Luc reporterconstruct,with or without an HA-tagged Tat expression vector.Subsequently,PER1-202 inhibition of the LTR promoter gradually decreased with the increasing levels of Tat,therefore,Tat can counter PER1-202 inhibition of HIV-1 transcription3.Analysis of the relationship between PER1 expression and disease progression in HIV-1 infected patients.From the above results,we have confirmed that PER1 can inhibit HIV-1 replication in both resting CD4+T cells and MDM,but it is unclear how PER1 is expressed in HIV-1 infected patients and how it affects HIV-1 replication in vivo Therefore,we first tested the expression levels of PER1 mRNA in PBMCs of Healthy group,cART group,RPs group and LTNPs group and found that the expression level of PER1 mRNA in PBMCs of patients in RPs group was significantly lower than those in other groups(P<0.05 or P<0.01),and the expression level of PER1 mRNA in PBMCs of patients in LTNPs group was not significantly different from that in cART group and Healthy group(P>0.05).Correlation analysis results showed that in RPs,the expression level of PER1 mRNA was negatively correlated with viral load and positively correlated with the number of CD4+T cells,but not in LTNPs.The above results suggest that PER1 may be related to the disease progression of RPs.SiRNA knockdown of PER1 expression in CD4+T cells in untreated patients can increase Gag RNA content(P<0.05),which promotes HIV-1 transcription,indicating that PER1 can inhibit HIV-1 replication in patients.Conclusions1.PER1 is highly expressed in resting CD4+T cells and MDM and exerts antiviral effects;2.PER1 inhibits the transcription of HIV-1 promoter(LTR),thereby inhibiting the replication of HIV-1 in host cells and HIV-1 Tat can counter the inhibition of PER1;3.In RPs,there is a correlation between PER1 expression level and viral load and CD4+T cell count in patients,and it plays a role in controlling HIV-1 infection and RPs disease progression. |
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