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@#Abstract：Long interspersed element⁃1（LINE⁃1）is the only known active and autonomously transposable retroelement in human cells，which is related to autoimmune diseases and plays important roles in activating and regulating the antiviral innate immunity of cells，especially the level of interferon（IFN）. This paper reviews the mechanisms of several non ⁃ structural proteins from human immunodeficiency virus（HIV），hepatitis B virus（HBV）and other viruses participating in the regulation of LINE ⁃ 1 activity. These mechanisms not only ensure the normal expression of viral genome，but also participate in the cellular innate immunity regulation，the inhibition of which may provide new strategies to develop treatments of diseases caused by viruses.
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Objective@#To investigate the toxic effect of HIV-1 Vpr protein on neurons.@*Methods@#HIV-1 vpr gene was amplified by nested PCR in four parts of peripheral spleen (SPL) and central nervous tissue meninges (MG) of HIV-associated dementia (HAD) patients and non-HAD patients. Eukaryotic expression vector pEGFP-N1-vpr was constructed. The gene sequence and key amino acid sites were analyzed by BLAST and MEGA6. The expression of Vpr protein in N2a cells was detected by Western-blotting. The effects of Vpr proteins from different sources on the activity and cell cycle of N2a cells were studied by flow cytometry.@*Results@#HIV-1 vpr gene was successfully amplified by PCR. Sequence analysis showed that the vpr gene sequence belonged to HIV-1B subtype. There were amino acid mutations at C-terminal 84, 86 and 87 sites of central Vpr protein from HAD and non-HAD patients. Vpr protein could inhibit the activity of nerve cells, leading to G2 phase arrest. Different sources of Vpr had different intensity of action. Compared with other groups, Vpr protein from the meninges of HAD patients showed stronger inhibition of cell activity and G2 phase arrest ability.@*Conclusions@#Variations in key amino acid sites of Vpr protein could cause significant changes in its biological functions, and its significance in the pathogenesis of HAD remains to be further studied.
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Objective@#To study the sequence characteristics and variation of HIV-1 Vpr gene in different parts of an AIDS dementia complex (ADC) patient and provide basis for the study of the neurologic pathogenesis of HIV-1-assciatd dementia.@*Methods@#Genomic DNA was extracted from peripheral samples (lymph nodes, spleen, liver) and central nervous system (meninges, frontal lobe, temporal lobe gray matter, frontal white matter, basal ganglia cortex) of an ADC patient, The Vpr gene was amplified with nested polymerase chain reaction (PCR). PCR products were cloned into the pMD19-T vector. After transformation into DH5α competent E. coli, five positive clones were sequenced. The phylogenetic tree was built and genetic distance was calculated through MEGA6, and the values of ds/dn was calculated through SNAP, then the changes of the amino acid sites were analyzed.@*Results@#HIV-1 Vpr genes isolated from different tissues of the ADC patient had variations. Vpr HIV-1 gene sequences from central nervous system and peripheral tissues were intercrossed together in the phylogenetic tree. Central nervous system and peripheral HXB2 Vpr had no significant differences in genetic distance. The ds/dn of all the HIV-1 Vpr gene sequences were 3.3749.@*Conclusions@#The HIV-1 Vpr sequences were different in the ADC patient, and there were different variations in different parts of the peripheral and central regions. Whether these variations are related to the pathogenesis of ADC remains to be further studied.
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Objective To observe the expression of apoptosis related protein c-inhibitor of apoptosis protein (IAP)2 by RNA interference of human immunodeficiency virus-1 (HIV-1)vpr gene and analyze the apoptosis of Jurkat cells.Methods Vector (NC),HIV-1vpr (vpr),vpr+ pRNAT-U6.1/Neo-vpr-56 (Si56) and vpr+ pRNAT-U6.1/Neo-vpr-160 (Si160) were transfected to Jurkat cells and cultured for 48 hours.The total RNA and protein were extracted.Expression of vpr gene was detected by reverse transcription (RT)-polymerase chain reaction (PCR) to confirm the success of transfection.Expression of c IAP2 was detected by RT-PCR and Western Blot.The apoptosis of Jurkat cells was observed by flow cytometry.Results Expression of vpr gene was detected in vpr,Si56 and Si160 groups.The mRNA expression levels in Si56 and Si160 groups were significantly lower than that in vpr group,which declined 87.2％ and 82.2％,respectively (P＜0.05).The levels of c IAP2 mRNA expression in vpr,Si56 and Si160 groups were increased by 3.75,2.49 and 2.65 folds,respectively,compared to that in NC group.However,the c-IAP2 mRNA expressions in Si56 and Si160 groups were lower than that in vpr group,which declined 33.7％ and 29.5％,respectively (P ＜ 0.05).The c-IAP2 protein expression was consistent with mRNA by immunoblotting,and those in Si56 and Si160 groups were declined 42.2％ and 46.8％,respectively,compared to that in vpr group (P＜0.05).The apoptosis of J urkat cells was detected in all groups.Compared to NC group,the apoptotic rate in vpr group was increased by 1.76 folds.However,the differences of apoptosis rate among NC,Si56 and Si160 groups were not statistically significant (all P＞0.05).Compared to vpr group,the apoptotic rates in Si56 and Si160 were significantly decreased by 19.26％ and 18.05％,respectively (P＜0.05).Conclusions The expression of c-IAP2 could be downregulated by knockdown of HIV-1 vpr gene in transcription and protein levels,and the apoptosis of Jurkat cells is inhibited.
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Objective To understand the rule of vpr gene variance of HIV-1 strains.Methods RT-PCR was used to amplify vpr gene of HIV-1 strains in Shenzhen. PCR products were sequenced and used for gene phylogenetic analysis and the 32-46 amino acids of Vpr protein were compared. The difference of 77 amino acid polymorphism distribution between domestic region and foreign region was analyzed. Results 01_AE was the major HIV-1 subtype in Shenzhen. The gene distance among subtype B was larger than in other subtypes. 77-amino acid of Vpr protein had three polymorphism forms as Arginin, Glutamine and Histidine, with Glutamine as the wild form. There were no significant differences in the three amino acid distributions between HIV-1 strains from domestic region and foreign region. Conclusion vpr genes of different HIV-1 strains belonged to 01_AE subtype. There was polymorphism seen in the vpr gene which was consistent with both domestic and international HIV-1 strains.
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Objective To package the recombinant lentivirus containing HIV-1 Vpr gene and detect the effect of Vpr protein expression on the latent infection and lyric replication of KSHV.Methods The fragment of Vpr gene from expression plasmid pCI-neo-Vpr was cloned into the lentivirus vector pHAGE-CMV-MCS-IzsGreen,then the recombinant plasmid pHAGE-Vpr,package vector psPAX2 and envelope vector pMD2.G were cotransfected into 293T cells.GFP expression was observed by fluorescent microscopy.Culture media of 293T cells were harvested and filtered through 0.45 μm filter.After 293T cells were infected by a series of diluted lentivirus,the virus titer was checked by observing GFP expression.Vpr mRNA transcripts in 293T cells were detected by RT-PCR.Then BCBL-1 cells were infected by the recombinant lentivirus with 1 MOI,GFP expression was observed by fluorescent microscopy,and the mRNA transcripts and protein expression of Vpr in BCBL-1 cells were detected by RT-PCR and Western blot.Meanwhile,the mRNA transcripts and protein expression of KSHV lytic cycle gene Rta were detected by RT-PCR and Western blot,respectively.Results The recombinant lentivirus carrying HIV-1 Vpr was packaged successfully with the virus titer of 4 × 107 TU/ml.After infected with lentivirus,BCBL-1 cells could express GFP,and the exact band of Vpr was detectable by RT-PCR and Western blot.Moreover,the expression of KSHV Rta mRNA and protein were downregulated by Vpr protein.Conclusion Overexpression of HIV-1 Vpr mediated by the recombinant lentivirus could inhibit KSHV lytic replication and enhance KSHV latent infection.
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Objective To explore ability of the vpr gene of human immunodeficiency virus type 1 ( HIV-1 vpr) to induce cell G_2 arrest and apoptosis, and the influence when it mutated, the relationship between Vpr-induced G_2 arrest and apoptosis inductions. Methods Fourteen mutant vpr fragments selected from Chinese patients with HIV. Both eukaryotic expression vector pcDNA3.1( + ) and PCR products purified, double-cut by Hind Ⅲ and BamH Ⅰ and the cut products legated and transformed into competent cells JM109. The 14 reconstructed plasmids electronically transfected into Jurkat-cells, and established cells with pcDNA3. 1-vpr , pcDNA3. 1-vpr-Fs and pcDNA3. 1 blank cells, and without pcDNA3. 1 cell. Cells were harvested after 24 h. mRNA expression was detected by RT-PCR, the DNA content and percentage of apoptosis were monitored by flow cytometry. Results Transfected with 14 mutant HIV-1 Vpr protein, cells display different G_2 percentage and apoptosis ratio. HIV-1 vpr induce cell cycle G_2 arrest and apoptosis, wherase Vpr Fs with a C-terminal end truncation, vector pcDNA3.1( + ) and the blank cells can not. The G_2 percentage and apoptosis ratio reduced when transfected with vpr expressing mutating of 70V, 85P, 86G, 94G compared to the wild type. Subtype AE has a weaker potential to induce cell cycle G_2 arrest and apoptosis. Preliminary, we find that the higher G_2 percentage followed the higher ratio of apoptosis. Conclusion HIV-1 vpr can induce cell cycle G_2 arrest and apoptosis, wherase Vpr Fs with a C-terminal end truncation can not. We firstly found that mutated sites of 70V, 85P, 86G, 94G may reduce the ability of Vpr to induce cell cycle G_2 arrest and apoptosis, subtype AE of vpr in Chinese HIV-1 patients has a weaker potential to induce cell cycle G_2 arrest and apoptosis. Analysis of various mutations in the vpr gene revealed that the extent of Vpr-induced G_2 arrest correlated with the levels of apoptosis. And investigate the pathegenesis of HIV vpr. This can also make a good foundation for further study on gene therapy.
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Objective To compare the mutation sites in human immunodefieiency virus type 1 (HIV-1) vpr gene via of HIV-1 infected individuals from different regions in China with the previous studies, and to provide information for the further study on the relationship between HIV-1 vpr gene mutations and clinical conditions of the patients. Methods Reverse transcription-polymerasc chain reaction (RT-PCR) and nested PCR were used to amplify HIV-1 vpr gene of 398 HIV-1 infected individuals. The amino acid sequences were analyzed to determine polymorphisms, deviation rate and common mutation sites of HIV-1 vpr gene. Meanwhile, the viral load, subsets of lymphocytes and clinical course of patients infected with mutated HIV-1 were analyzed. Results One hundred and fifty three positive samples which were obtained from 398 HIV-1 infected individuals were available for further analysis. The amino acids sequence typing of HIV-1 Vpr were showed that CRF01 AE was 51.63%, subtype C 24.84%, subtype B 17.65%, CRF03_ AB 3.92% and CRF08 BC 1.31%. Eighty four point three percent of 77th amino acid of HIV Vpr sequence was glutamic acid which was significantly different from what overseas researches reported that the R77Q mutation was correlated with long-term non-progression (LTNP) of AIDS. The mutations of the, 63th, 70th, 85th, 86th, 89th and 94th amino acids of HIV Vpr were likely related to the clinical remission of HIV-1 infected individuals. Conclusions M group is the main type of HIV Vpr typing in China, and CRF01 AE is predominant. Some amino acid mutation sites of HIV-1 Vpr are possibly correlated with clinical manifestations of HIV-1 infected individuals.
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Objective To investigate the T cell cytotoxicity induced by recombinant adenovirus carrying HIV-1 vpr gene.Methods C8166 cells infected with rAd-vpr or negative control rAd-vector,were analyzed for cell cycle distribution and cell death by flow cytometry.The discrimination of living cells,apoptotic and necrotic cells were differentiated with Hoechst-PI double staining under the confocal microscopy.Changes of mitochondrial membrane potential(△ψm)were monitored by JC-1 staining method.Results Annexin V-PI and Hoechst-PI staining indicated the death effects of HIV-1 Vpr on C8166 cells.PI flow cytometric analysis showed that cell cycle arrested in G2 phase.C8166 cell△ψm collapse mediated by Vpr was detected by JC-1 fluorescent staining.Conclusion The ability of recombinant adenovirus carrying HIV-1 vpr gene to induce mitochondria dysfunction,cell cycle G2 phase arrest and cell death was confirmed in C8166 cells.
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Objective To establish cell strain expressing the genes of HIV vpr and mutant HIV vpr-FS, and to explore cell apoptosis ability by HIV Vpr and Vpr-FS. Methods The recombinant plasmids were constructed by cloning HIV vpr and HIV vpr-FS genes into the eukaryotic expression vector pcDNA3.1respectively. To determine the primary structures of HIV vpr and HIV vpr-FS, plasmids were cleaved by restriction enzymes. After the plasmids were transfected into HeLa cells by liposome, the HeLa cells were selected with G418 selective medium, mRNA expression of HIV vpr or HIV vpr-FS of transfected cells was detected by RT-PCR, and Vpr and Vpr-FS protein expression were detected by Western blot assay respectively. The DNA content and the percentage of apoptosis in HeLa HIV vpr cell, HeLa HIV vpr-FS cell and HeLa pcDNA3.1 cell were monitored by flow cytometry and the DNA fragmentation was analyzed by agarose gel electrophoresis. Results BamH Ⅰ and Hind Ⅲ cleavaged products of pcDNA3.1-vpr and pcDNA3.1-vpr-Fincluded 342 bp length fragments suggesting that the length of DNA sequence containing HIV vpr (HIV vpr-FS) within pcDNA3.1 was the same as theoretical length. The HeLa cells transfected by pcDNA3.1-vpr or pcDNA3, l-vpr-FS and selected with G418 could express HIV vpr or HIV vpr-FS by RT-PCR, and express HIV Vpr or HIV Vpr-FS protein by Western blot. The results of flow cytometry and DNA fragmentation showed that there was significant different in the number of apoptotic cells between HeLa HIV vpr cell and HeLa HIV vpr-FS cell, but the difference between HeLa HIV vpr-FS cell and control group was not obvious. Conclusion Recombinant plasmids pcDNA3.1-vpr and pcDNA3. 1-vpr-FS were constructed successfully, and the cell strain expressing HIV Vpr and HIV Vpr-FS proteins was established. The HIV Vpr could induce host cell apoptosis, while the mutant of Vpr did not or weakened this ability. This study provides foundation for further study on HIV vpr gene.