Effects Of HIV-1 Vpr Protein From HAD And Non-HAD Patients On Cell Cycle And Apoptosis Of Mouse Neuroblastoma Cell Line N2a

OBJECTIVEHIV associated dementia(HAD)is an important neurological complication in HIV-1 infected patients.The symptoms include memory impairment,mental retardation,attention loss,apathy and cognitive impairment.With the aggravation of the disease,severe dementia,coma and paralysis will occur in the later stage.With the application of highly active antiretroviral therapy(HAART),on the one hand,it improves the survival time and quality of life of HIV patients,On the other hand,AIDS has gradually become a kind of chronic disease,which increases the incidence rate of AIDS related chronic neurological complications.At present,it is believed that the pathogenesis of HAD is related to the injury and apoptosis of brain neurons,and the occurrence and development of the disease is also related to the neuropathogenicity of HIV-1 and the immune level of patients.After HIV-1 invaded the central nervous system(CNS),due to the characteristics of self height variation and the unique physiological environment of CNS,the variation of virus in the central and peripheral was different.Vpr protein is an auxiliary protein of HIV-1.It can affect the expression of host gene,induce G2 phase stagnation of nerve cells,and induce apoptosis of nerve cells directly or indirectly through interaction with various proteins in cells.It is one of the factors to induce the occurrence of HAD.To explore HIV-1 The variation of vpr gene in CNS and peripheral of patients and the relationship between the change of Vpr protein biological activity and HAD caused by the variation of vpr gene amino acid sites were studied.The sequence variation of central and peripheral HIV-1 vpr gene in a HAD and a non-HAD patient was studied,and the eukaryotic expression vectors of Vpr from different sources were constructed to explore the effect of Vpr protein on N2a cells of mouse neuroblastoma mother cells In order to provide a new way for the pathogenesis of HAD,the effects of activity,cycle arrest and apoptosis and their mechanism of action were studied.METHODS1.Cloning and sequence analysis of HIV-1 vpr gene(1)Cloning of HIV-1 vpr gene The lymph node(LN)and spleen(SPL)in peripheral area of a HAD patient(H)and a non-HAD patient(N),The gene group DNA of 14 autopsy samples including SPL,liver(LIV)and central nervous system meninges(MG),white matter from frontal cortex(WM),temporal cortex(TC)and basal ganglia(BG)were amplified by nested PCR Vpr gene sequence,and the amplified target gene was connected to the pMD19-T clone vector,transformed into the DH-5 ?.Through ampicillin screening and blue-white spot test,positive colonies(5 each)were screened out and sent to sequencing company for sequencing.(2)The sequence analysis of HIV-1 vpr The phylogenetic tree,gene distance,synonymous/non synonymous substitution value and amino acid mutation sites of the sequenced sequences were analyzed by using the biological software such as MEGA and Bioedit.2.The effect of HIV-1 Vpr protein on the activity,cycle arrest and apoptosis of neural cells(1)Construction of eukaryotic expression vector of pEGFP-N 1-vpr According to the sequencing results,four vpr gene fragments(H-MG,H-SPL,N-MG,N-SPL)with larger central and peripheral variation were selected as templates.PCR amplified products were recovered by agarose gel electrophoresis.The recovered products were digested by restriction endonuclease Xho ? and Hind ? respectively.The product was recovered again and connected overnight at 16?.The conjugated products were transformed into E.coli DH5 ? sensitive cells,inoculated into LB solid medium containing ampicillin,cultured at 37? for 16-18 hours,and then the recombinant plasmids were extracted and sequenced for identification.(2)Expression and identification of HIV-1 Vpr in N2a cells The eukaryotic expression vector pEGFP-N1-vpr was transfected into N2a cells by liposome method.The blank control group and negative control group were normal cell group and pEGFP-N1 empty carrier group respectively.Observe,track and record the expression of green fluorescent protein at different time points under a fluorescent microscope to preliminarily judge the expression of HIV-1 Vpr protein,and identify the expression of HIV-1 Vpr in N2a cells by Western blot.(3)Effects of HIV-1 Vpr protein on the activity,cell cycle arrest and apoptosis of mouse neuroblastoma cell line N2a After 48 hours of transfection,CCK8 kit was used to detect the effect of Vpr protein on the activity of N2a cells;single standard method of flow cytometry(PI)was used to detect the effect of Vpr on the cell cycle arrest of N2a cells;double standard method of annexin V-7-AAD/PE was used to detect the apoptosis rate of N2a cells.3.Statistical analysisSPSS 18.0 software package was used for data analysis.The t-test was used to compare the gene distance;one way ANOVA was used to compare the activity,cycle arrest rate and apoptosis rate of N2a cells in each group.If the data were normal and the variance was homogeneous,LSD method was used to compare between groups;if the data were normal but the variance was uneven,Welch correction was used,and Dunnett's T3 method was used to compare between groups.The measurement data is expressed in(?)±S,P=0.05.RESULTS1.Analysis of vpr gene polymorphism and amino acid sequence of HIV-1(1)Cloning of HIV-1 vpr geneThe amplified products of nested PCR showed obvious bands at 300bp after 1.2%agarose gel electrophoresis,which was consistent with that predicted(288bp).After sequencing,it was identified as vpr gene sequence of HIV-1B subtype by blast.(2)Phylogenetic tree analysis of HIV-1 VprIt can be seen from phylogenetic tree that HIV-1 vpr gene sequences isolated from CNS and peripheral different parts of non-HAD patients cross less in phylogenetic tree,most of which are located on their own evolutionary branches;while vpr gene sequences of HAD patients cross more in phylogenetic tree,especially in the peripheral sequence,which is very obvious in the phylogenetic tree,and there is only a cross between TC and BG in the central part.(3)Gene distance analysisThe results of gene distance analysis showed that in the same patient,the variation of vpr from the central source was smaller than that of its peripheral sequence,indicating that the selection of different environmental factors in the body would also affect the variation of the virus.At the same time,the distance of vpr gene sequence of HAD patients is larger than that of non-HAD patients,which indicates that different patients will affect the variation of HIV due to the difference of virus infection,immune status and disease progress.(4)ds/dn value analysisThe value of ds/dn is often used to describe the mutation rate and selection pressure in molecular evolution.The results of this study showed that the value of ds/dn values of the two patients were greater than 1,indicating that the vpr gene variation was under negative selection pressure in HIV infected patients,and the ds/dn values of the genes from the central part of non-HAD patients were significantly lower than those of other groups,the difference was statistically significant(P?0.05).(5)Amino acid sequence analysis of HIV-1 VprThe analysis of Vpr amino acid sequence showed that the variation of Vpr amino acid sites in central nervous system and peripheral Vpr of the same patient was different.Compared with non-HAD,there are less differences in the central part,only T84I,Q86R and R87S amino acid variations.There are 12 amino acid variations in the peripheral part,D7N,Q11P,R36M,I37V,H45Y,A55T,I67M,R77H,T84I,R85P,Q86R and R87S.2.The effect of HIV-1 Vpr protein on the activity,cycle arrest and apoptosis of neural cells2.(1)Construction of eukaryotic expression vector of pEGFP-N1-vprAfter amplification of agarose gel electrophoresis with a concentration of 1.5%,a clear band of PCR appeared at 300bp,which was consistent with the expected size.The PCR products were sequenced and identified as HIV-1 vpr by blast sequencing.(2)Expression and identification of HIV-1 Vpr protein in N2a cellsThe transfection results showed that the green fluorescent protein in the empty vector group was very bright,which indicated that the transfection efficiency of the plasmid was high,and the other expression vectors also showed green fluorescent protein.The results of Western blotting showed that there was no expression of Vpr in blank cell control group and plasmid control group,and Vpr protein could be expressed in all experimental groups,and the expression amount was similar,no statistical difference.(3)The effect of HIV-1 Vpr protein on the activity of N2a cellsThere was no significant difference between blank cell control group and plasmid control group.There was no significant difference in cell activity between the N-MG group and the empty carrier group.However,the cell activity of H-MG,H-SPL and N-SPL groups decreased significantly(P?0.001),suggesting that Vpr protein may cause the decrease of cell activity.The results showed that H-MG had a stronger ability to inhibit cell activity than N-MG(P?0.05).(4)Effect of HIV-1 Vpr protein on cell cycle arrest of N2a cellsThere was no significant difference in cell cycle arrest rate between blank cell control group and plasmid control group.The G2 cycle arrest rate of the four groups of cells expressing Vpr protein,H-Mg,H-SPL,N-SPL and N-MG,was significantly higher than that of the plasmid control group(P?0.001).The protein of H-MG showed the strongest G2 cycle blocking ability(P?0.05).(5)Effect of HIV-1 Vpr protein on N2a cell apoptosisThere was no significant difference in apoptosis between blank cell control group and plasmid control group.Compared with the plasmid control group,the apoptosis level of H-MG,H-SPL,N-SPL and N-MG group which expressed Vpr protein was significantly higher,the difference was statistically significant(P?0.05),suggesting that Vpr protein can cause apoptosis.In the comparison of the experimental groups,H-MG had stronger ability of inducing apoptosis than N-MG,the difference was statistically significant(P?0.05).CONCLUSION1.There are significant differences in phylogenetic tree,gene distance analysis,the ds/dn value and amino acid site variation between HIV-1 vpr gene from HAD and non-HAD patients,which may be related to the occurrence and progress of HAD.2.HIV-1 Vpr protein can induce G2 phase arrest,apoptosis and cell activity of N2a cells.Moreover,Vpr protein from the central source of HAD patients showed stronger neurotoxicity,which indicated that the amino acid mutation or enhanced the neurotoxicity of viral protein,and may be related to the neuropathogenicity of HIV-1.