| AIDS,acquired immunodeficiency syndrome(acquired immunodeficiency syndrome,AIDS),is caused by the infection of the human immunodeficiency virus(Human Immunodeficiency Virus,HIV).After HIV-1 invades the body,it mainly destroys CD4+ T cells and immune system which leads to the decrease in immunity,thereby causing various opportunistic infections.The emergence of highly active antiretroviral therapy(HAART)delays the onset time of most infected people and leads to a significant reduction in mortality.However,this method cannot eliminate HIV-1 in latented infected cells such as resting CD4+ T cells,monocyte-macrophages,dendritic cells and hematopoietic stem cells.Monocyte-macrophages can resist virus-induced apoptosis and become an important site for HIV-1 virus replication and latency.In addition,HIV-infected monocyte-macrophages can spread the virus to CD4+ T cells,promoting the latent infection of HIV and the expansion of latent pool.Recent studies have found that in the early stages of HIV infection,peripheral blood monocytes can express high levels of CD 169 which can bind to sialic acid on HIV gp120 and promote the combination of virus and cell.Some researchers believe that CD169 can be used as a marker for viral infections or serious disorders of the immune system.In order to study the role of CD 169 in infecting monocytes by HIV,we conducted the following three parts of the study.Part ?:Changes of the expression of CD169 in CD14+ monocytes of SIV-infected monkeys.Siglecs is the largest sialic acid binding protein in the host.CD 169,also known as Siglec-1,is the first member of the Siglecs family found to be expressed mainly in monocytes-macrophages and dendritic cells.CD 169 binds to certain glycoproteins on the surface of the virus and enhances the binding of the virus to cells,thereby promoting viral infection.In the HIV-1 study,the tissue macrophages and peripheral blood monocytes express high levels of CD169 after infection with HIV-1.So,does SIV-infected rhesus monkey peripheral blood monocytes also express high levels of CD 169?This section mainly studied the changes of the ratio of peripheral blood monocytes and the expression of CD 169 before and after infection with normal rhesus monkeys;The results showed that after SIVmac239 infection,the percentage of CD14+ monocytes of the normal rhesus monkeys was decreased and the expression of CD 169 on their surface was increased.Meanwhile,the expression of CD 169 on the surface of different subsets of peripheral blood monocytes was significantly increased,and the expression of CD 169 on the CD 14+CD16++ monocytes was increased more obviously.The above experimental results show that SIVmac239 infection can lead to the increase of CD 169 expression on the surface of peripheral blood monocytes in rhesus monkeys.Part II:Study on the internalization of HIV-1 by CD169.The binding of CD169 to sialic acid on HIV gp120 mediates virus and cell adsorption.pCD169 binds to the sialic acid on the surface of the PRRSV virus and mediates the internalization of the virus by cells.Previous studies have shown that the Fab segment of monoclonal antibody can induce the internalization of hCD169.Then,Can the binding of CD 169 and sialic acid on HIV gp120 cause internalization of the virus?In this section,the cells expressing CD 169 were co-cultured with the virus,and the localization of CD 169 and HIV gag-iGFP was observed by laser scanning confocal microscopy.The results showed that the transfected CHO-K1 cells can express CD 169 molecules.After adsorption,virus particles can be observed on the cell surface.After the cells were co-cultured with the virus,virus particles were observed in some of the cells which expressing the CD 169 molecule.With the prolongation of the culture time,the virus particles in the cells were gradually reduced to be completely unobservable.THP-1/IFN-?-THP-1 was infected with HIV gag-iGFP virus,respectively.After cells were infected,real-time PCR was used to detect the expression of HIV p24 in the cells.The results showed that HIV p24 expression was significantly higher in HIV gag-iGFP infected IFN-?-THP-1 cells than in THP-1 cells.The above experimental results indicate that CD169 can bind to the HIV-1,thereby promoting the internalization and replication of the HIV-1.Part ?:Interaction between CD169 and TBK1 and Its effect on viral budding.The virus needs to be assembled and released after it has replicated within the host cell.The budding of most enveloped viruses is dependent on the host cell's ESCRT complex(formed by multivesicular bodies).HIV-1 budding requires the participation of ESCRT-?,-? and Vps4,but does not require ESCRT-0,-?.After HIV-1 infects cells,TBK1 binds to VPS37C,the component of ESCRT-?,and phosphorylates it,thereby inhibiting viral budding.In the study of HSV/VSV,the virus infected macrophages can cause cells to express CD169 which can induce the degradation of TBK1,and negatively regulate viral infection triggerring the formation of type ? interferon.However,studies have shown that HIV-1 infection does not cause cells to produce type ? interferons.What is the expression of CD 169 on the cell surface after HIV-1 infection?After adding the cytokine IFN-? to induce the cell to express CD 169,can CD 169 promote viral budding by degrading TBK1?In this part of the study,the expression of CD 169 and cytokine IFN-a in the HIV-1-infected THP-1 and SIV mac239-infected CD14+ monocytes were detected by flow cytometry.The changes in the expression of CD 169 on other cytokins-stimulated and cytokine IFN-a-stimulated monocytes were detected by flow cytometry.The expression of mRNA and protein of TBK1 in IFN-?-stimulated THP-1 cells was detected at different times.The HIV gag-iGFP virus infected THP-1/IFN-?-THP-1 cells and the expression of HIV p24 was detecd in the supernatant at the end of the infection.The results showed that there was no changes of the expression of CD 169 on the surface of HIV-1-infected THP-1 cells.In IFN-?-stimulated THP-1 cells,as the stimulation time prolonged,the expression of CD 169 on the cell surface increased,and the expression of TBK1 protein showed a certain downward trend,but the decrease was not statistically significant.There was no change in the expression level of TBK1 protein in the HIV-1-infected IFN-?-THP-1 compared with THP-1,and there was no significant difference of virus in the supernatant.The results showed that the expression of CD 169 is not associated with the direct infection of virus,but is related to the cytokine IFN-?.The expression of CD 169 caused a certain decrease in the expression of TBK1 protein(no significant difference).CD 169 can not promote viral budding by degrading TBK1. |
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