Study On The Role Of Eukaryotic Initiation Factor 3 In The Progression Of HIV-infected Diseases

Objective: Human immunodeficiency virus type 1(HIV-1)mainly infringes on CD4+T cells.The HIV-infected people lost their immune function and eventually die due to various opportunistic infections and tumors.At present,there are no effective vaccines to prevent and cure,that led to a very serious global epidemic.There are different disease progression in HIV-infected people,and about 10%-15% of HIV-infected people show a rapid progression.A large number of studies have shown that the genetic background of the host,the immune response state of the body,the type of HIV virus infected,and the immune escape in the interaction between the virus and the host all affect the disease progression.However,these are not enough to fully explain the reasons for the progression in HIV-infected people.It is necessary to seek out the factors that affect the rapid progression for delaying the progression of the disease.The immune regulation mechanism plays an important role in inhibiting the replication of HIV virus,while CD8+ T cells are important effector cells.Studies have shown that the function of CD8+ T lymphocytes is significantly negatively correlated with disease progression in the early stages of infection and CD8+ T lymphocytes are important immune protection factors,but the extent of these protections is very different in terms of duration and intensity.The basis of this difference depends on the quality and quantity of CD8+ T lymphocytes.When CD8+ T cells encounter antigen,the degree of activation determines the strength of their effect.In addition,their ability to survive long-term through stable proliferation is also very important for immune protection,the ability of memory CD8+ T cells to maintain a stable self-renewal capacity through slow,stable cell turnover.Therefore,activation,proliferation,and apoptosis after stimulation of antigens by CD8+ T cells determine the quality of immune protection provided by it.Rapid progressor(RP)have a damage in CD8+ T cells in the early stages of infection may be due to defects in CD3 and CD28 receptor expression on CD8+ T cells in infected individuals,or synthesis of antiviral molecules(IFN-?)defects.However,this is not sufficient to explain thedifferences in the function of CD8+ T cells among different infected individuals.Finding the host factors that affect the immune response of CD8+ T cells and effectively intervening them to maintain the CD8+ T cell function at a high level will limit the maximum virus immune escape,so that the virus in the infected person maintained at a low level,to achieve long-term disease-free.Eukaryotic initiation factor 3(eIF3)is the largest and most complex eukaryotic translation initiation factor,a complex composed of 13 subtypes.In the process of protein translation initiation,it mainly acts as a scaffold protein.Previous studies on eIF3 s in HIV have focused on direct interventions for viral replication in cell line models,and no research in the HIV-infected people.Recent studies have shown that eIF3 s not only function as scaffold proteins,but some of these subtypes can also directly promote or inhibit the translation of mRNA by binding to the 5'-end stem-loop structure of certain mRNAs in the transcriptome and regulate cell functions,such as cell proliferation,activation,apoptosis etc.Subsequent studies found that different subtypes of eIF3 s have different expression levels in different tumors,and changes in some subtypes are not only related to tumor prognosis but also can be used as markers for evaluating tumor prognosis.In addition,studies have shown that eIF3 molecules have an effect on cell function.When eIF3 b is knocked out in renal cancer cells,the proliferation and activation of cells decline,cell cycle arrests and apoptosis increases.Apoptosis of tumor cells with high expression of eIF3 d in prostate cancer tissues is reduced,and the invasion ability is increased,indicating that the prognosis of the disease is poor.Knockdown of eIF3 a in keloid fibroblasts(KFs)results in decreased cell proliferation,reduced production of extracellular matrix(ECM),and reduced scarring.The above studies strongly suggest that eIF3 s have an effect on cell function.At present,studies have shown that eIF3 s are involved in the inhibition of HIV virus replication,but the relationship between the expression levels of eIF3 s and the disease progression in infected individuals has not been reported.In HIV infection,CD8+ T lymphocytes play an important anti-HIV role,CD8+ T lymphocyte function is significantly negatively correlated with disease progression,and the response function of HIV-1 specific CD8+ T lymphocyte in RPs is significantly decreased.Thereason for the decline is not yet completely clear.In addition to the explicit direct intervention of viral replication,it remains to be explored whether eIF3 s will lead to disease progression and its mechanism of action through the function of CD8+ T lymphocytes.Methods: 1.we examined the expression levels of eIF3 in peripheral blood mononuclear cells(PBMCs)from 15 HIV-infected patients and 11 healthy controls for the first time.Fifteen HIV/AIDS patients were from MSM,Liaoning Province,China and were infected for <180 days without antiviral treatment.All patients were confirmed positive for HIV antibody by Genelab's Western blot kit.According to the number of CD4 + T lymphocytes on the detection date,the patients were divided into two groups: Typital progressors(TPs)(CD4 + T cells> 500cells/uL)and Rapid progressors(RPs)(CD4 + T cells <350 cells/uL).Eleven healthy controls(HCs)were randomly selected HIV-negative healthy men and matched with HIV/AIDS patients.And analyzed the correlation between eIF3 s expression level and the progress of HIV infection,CD4 + T lymphocyte count and viral load.2.After screened eIF3 related to disease progression,knocked out 293 cells,JURKAT cell line and healthy male PBMC,and detected intracellular viral replication.3.We recruited 5 healthy men whose age matched that of the infected person.We collected 30 ml of peripheral venous blood and performed sorting by flow cytometry to extract the selected CD8+T,CD4+T,NK,B,and mononuclear cells.Cell cDNAs were used to detect the expression level of eIFs.STEMCELL was used to sort 18 CD8+ T lymphocytes from HIV-infected patients(MSM pathway).cDNA was extracted to detect the expression level of eIFs,and the correlation with absolute CD4 count was analyzed.4.In order to clarify the effect of eIF3 d on CD8+ T lymphocyte function,we selected 4 cases of HIV-negative healthy male PBMCs,sort out CD8+ T lymphocytes,knock out eIF3 d and stimulate with PHA,and culture for 2-4 days after proliferation,activation,apoptosis,IFN-? secretion and other functional tests.5.In order to find out the reason why eIF3 d affects the function of CD8+ T lymphocytes,we used the second generation sequencing method to perform transcriptome detection on the knockout eukaryotic cells of JURKAT.The differential genes were analyzed using the IPA database and the relevant pathways were screened.The gene was verified for SOCS7 expression in JURKAT and functionally verified in CD8+ T lymphocytes.Results: 1.The level of eIF3 d in PBMC of HIV-infected persons was significantly lower than that in healthy controls,and the rate of eIF3 d was the lowest among those with rapid progression.The level of eIF3 d was negatively correlated with disease progression and viral load.There was a significant positive correlation between eIF3 d and CD4 + T lymphocyte counts.2.Kaplan-Meier survival analysis showed that the expression level of eIF3 d directly affects the CD4 + T lymphocyte count down to 500 cells / ul in untreated patients with HIV infection(P <0.01).3.Infection of 293 cells,JURKAT cell line and healthy human PBMC with eIF3 d knocking down NL-43 virus at 48 hours and 72 hours respectively increased the level of virus replication(P <0.05).4.The level of eIF3 d in CD8+ T cells from HIV-infected patients was significantly lower than that in healthy controls and was positively correlated with CD4+T-lymphocyte counts.5.The knockdown of eIF3 d gene in CD8 + T lymphocytes and JURKAT cells showed that the proliferation and activation of cells were significantly decreased,the secretion of IFN-? was significantly decreased(P <0.05),and the apoptosis was significantly increased(P <0.05).6.The knockdown of eIF3 d gene JURKAT cell transcriptome results showed that with the decline of eIF3 d gene expression a total of 211 genes changed significantly,including 76 up-regulated genes and 135 down-regulated genes.211 differentially expressed genes were analyzed by IPA database for the signal pathway JAK-STAT associated with activation and proliferation of cells.SOCS7 locus was found by KEGG analysis.SOCS7 gene expression was significantly increased by real-time PCR(P <0.05).7.However,when both eIF3 d and SOCS7 were knocked out in CD8 + T lymphocytes and JURKAT cells,the proliferation of cells(P <0.05),the activation and the recovery of IFN-?secretion partially recovered,and the apoptosis partially decreased.Conclution: 1.The expression of eIF3 d in PBMC of HIV-infected patients was decreased,while the expression of eIF3 d was a protective molecule.2.The expression level of eIF3 d could predict the progression of HIV-infected patients.3.Decreased eIF3 d in HIV-infected individuals may affect CD8+ T cell function.4.After silencing eIF3 d gene in CD8 + T lymphocytes,the cellular antiviral function decreased.5.Deletion of eIF3 d gene can negatively regulate the JAK-STAT signaling pathway byinhibiting the expression of SOCS7,thereby inhibiting the anti-viral function of CD8+ T lymphocytes and further affecting the progress of HIV-infected diseases.Our research provides a new direction for controlling the progression of HIV disease.