Effect of HIV-1 Nef protein on autophagy of U87 cells in HAD and non-HAD patients
10.3760/cma.j.issn.1003-9279.2020.01.002
- VernacularTitle:HAD及非HAD患者中枢神经系统来源的HIV-1Nef蛋白对U87细胞自噬的影响
- Author:
Xiaoyu SHAN
1
;
Xinyue CAO
;
Wenhui ZHENG
;
Shuaizhi GUO
;
Hongling WEN
;
Zhiyu WANG
;
Tao HUANG
;
Li ZHAO
Author Information
1. 山东大学公共卫生学院卫生微生物检验学系 山东省“十三五”高校重点实验室
- Keywords:
HIV-associated dementia;
HIV-1 nef;
Autophagic marker protein LC3-Ⅱ;
Autophagic marker protein p62
- From:
Chinese Journal of Experimental and Clinical Virology
2020;34(1):7-11
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the effect of amino acid site variation of HIV-1 Nef protein on its inhibition of neuronal autophagy and explore the mechanism of Nef protein-induced central nervous system injury.Methods HIV-1 nef genes were amplified and cloned from the temporal cortex (TC) of the central nervous system in 1 case of HIV-associated dementia (HAD) H and 1 case of non-HAD AIDS patient N.The amino acid sequences were aligned by NCBI BLAST tools and MEGA6.0 software to study the variation of amino acid sites.The eukaryotic expression vectors pEGFP-N1-nef derived from H-TC and N-TC were constructed and transfected into U87 cells to observe green fluorescence.At the same time,the expression of Nef protein,LC3-Ⅱ and p62 protein in U87 cells were detected by Western blot,and the effects of different sources of Nef on autophagy of U87 cells were analyzed.Results The nef genes were amplified by PCR and clone vectors pMD-19T-nef of H-TC and N-TC were successfully constructed.The sequencing confirmed that they were HIV-1B subtypes.The amino acid sequence analysis showed that there were differences between H-TC and N-TC key amino acid sites.The recombinant plasmid pEGFP-N1-nefwas successfully constructed and expressed Nef protein in U87 cells.Western blot analysis showed that the expression of LC3-Ⅱ protein was significantly different among groups (F =11.764,P =0.001).There was no significant difference in the expression of LC3-Ⅱ between cell control and plasmid control(P=0.169).The content of LC3-Ⅱ was low in the two groups of cells,which could not be detected by Western blot.The expression of LC3-Ⅱ in H-TC,N-TC and positive control CQ group increased,compared with blank control or blank vector.The difference was statistically significant (P=0.017,P=0.039,P=0.031),and the expression of LC3-Ⅱ in H-TC group was higher than that in N-TC group (P =0.023);the expression of p62 protein in H-TC,N-TC and positive control CQ group was higher than that in blank control or blank vector group,but there was no significant difference between groups (F=2.049,P =0.163).Conclusions HIV-1 Nef amino acid sites in the central nervous system of patients with HAD and non-HAD were different,and their effects on autophagy of U87 cells were different.The expression of LC3-Ⅱ,an autophagic marker protein,was more strongly induced by Nef from H-TC.
