| Background: Human endogenous retroviruses(HERVs)became infected and integrated into the human genome tens of millions of years ago,making up 8% of the total human genome,and can be passed on to future generations.The Human Genome Project revealed that HERV-K is the most biologically specific form of endogenous retroviruses,but most HERV-K members are genetically defective because mutations impair the ability to form infectious virus particles.And those HERV-K members that have the ability to replicate can cause disease if activated.In recent years,abnormal expression of HERV-K protein and reverse transcription factors may lead to the development of pathological states.HML-2 is the most active member of the HERV-K family and has the shortest time to integrate with the human genome.RNA expression and protein products of HERV-K(HML-2)have been identified in a variety of autoimmune diseases and malignancies,and the cytotoxic response of T cells to HERV-K antigens has been identified in malignancies such as melanoma and seminoma.Although the number of these reports is increasing,no cause-and-effect relationship has been established.Some pathological conditions may be related to the up-regulation of RNA transcription of HERV-K(HML-2),the irregular expression of viral proteins and virus particles,or the production of HERV-K autoantibodies.It has been shown that HERV-K(HML-2)can increase its copy number in and out of cells by infection with other viruses,which also confirms the existence of some replicable HERV-K proviruses in the human genome.HIV-1 virus is an exogenous retrovirus.It has been reported in many pieces of literature that the reverse E transcriptional element of HIV-1 is common in HERV-K.Clinical trials have also shown that anti-HERV-K antibodies are detected in 70% of HIV-1 patients and that HERV-K transcription levels are elevated in patients diagnosed with HIV-1 encephalopathy.Related studies have found that the expression of some RNA fragments of HERV-K(HML-2)in the plasma of HIV-1 infected patients is up-regulated.Through wild Human Immunodeficiency Virus type 1(Human Immunodeficiency Virus comes,HIV-1)strain infected people different cells in vitro,study different cells after infection with HIV-1 and endogenous retroviruses K race(HERV-K)expression of transcription,thus carry out to the HERV-K(HML-2)in HIV infection in the body to activate or inhibit the related problems of medical science research.Objectives: The transcriptional level of HERV-K(HML-2)in peripheral blood lymphocytes of different HIV-1 subtypes in China was detected.Then,cell models infected with different subtypes of HIV-1 were used to observe the changes of HERV-K(HML-2)transcription level after HIV-1 infection,and the transcriptional expression level of HERV-K(HML-2)in each cell infection model was compared,objective to construct monoclonal cell lines stably expressing Tat proteins of different HIV-1subtypes and explore the role of Tat proteins of different HIV-1 subtypes in activating HERV-K transcription.Finally,we summarized the role of HIV-1 infection in activating HERV-K(HML-2)in vivo and in vitro,and explored the possible mechanism.Methods: First,peripheral lymphocyte(PBMCs)samples from 106 HIV-1 infected patients without antiretroviral therapy and 64 healthy blood donors were collected.The viral subtypes of HIV-1 infected patients were identified by RT-PCR amplification and sequencing.The transcriptional expression of HERV-K(HML-2)in PBMCs was detected by real-time quantitative PCR,and the differences of HERV-K(HML-2)transcriptional levels among different HIV-1 subtypes and between HIV infected patients and healthy people were analyzed Different.Then,different HIV-1 subtypes(subtype B,CRF01＿AE)were infected with different cell lines(MT2,H9,TZM-bl,293T)in vitro＿The optimal infection does MOI was 0.1.The cells and supernatant were collected at 6,12,24,and 48 hours after infection.HIV-1 p24 in culture supernatant and HIV-1 RNA in cells were detected to confirm HIV-1 infection.The transcriptional levels of HERV-K(HML-2)were detected by real-time quantitative PCR and RNAscope.Finally,MT2,the most suitable cell line for infection,was selected to construct the stable expression of different HIV-1 subtypes(subtype B,CRF01)based on pLVX-Teton system＿Objective to explore the effect of Tat protein of different HIV-1 subtypes on the transcriptional activation of HERV-K(HML-2)and compare the differences of different subtypes.Results:(1)106 pol gene sequences,28 approximately full-length gene sequences,fifteen5 ’ hemimolecular sequences,and two 3’-end hemimolecular sequences were obtained from HIV-1 infected patients.Virus genotypes were identified in 95 HIV-1 infected patients,of which subtype B accounted for 41%(39/95),CRF01＿AE 25.3%(24/95),CRF07＿BC 24.2%(23/95),CRF01＿AE/CRF07＿BC recombinant subtype 9.5%(9/95),and 11 subtypes were unknown.(2)Three cases of CRF01＿AE/CRF07＿BC were identified as a novel circulating second-generation Recombinant virus(CRF)by the analysis of approximately full-length gene sequence and epidemiological information,and the genomic chimerism was identified.CRF102＿0107 was published in HIV Databases,btf@lanl.gov a(3)The transcriptional level of HERV-K(HML-2)gag region in HIV-1 subtype B infected patients was significantly up-regulated compared with healthy controls(P<0.05),while the transcription level of HERV-K(HML-2)pol region in non-B subtype strains(CRF01＿AE,CRF07＿BC,etc.)was significantly up-regulated(P <0.05).There was no significant correlation between viral load and transcriptional levels of HERV-K(HML-2)regions in infected patients(P >0.05).(4)Compared with uninfected cells,HIV-1 Ⅲ B virus infection in different cell lines in vitro transcription for 48 hours,after infection MT2 cell line HERV-K(HML-2)transcription by about 5 times(P <0.05),H9 and TZM-bl cells were upregulated by about 1.5 times(P <0.05).(5)Compared with uninfected cells,the transcription level of HERV-K(HML-2)in HIV-1 GX002 strain(CRF01＿AE recombinant subtype)was upregulated 15 times after 48 h infection with MT2 cells(P<0.05),GX002 strain infection in the genome HERV-K(HML-2)transcription increases significantly higher levels of Ⅲ B strain and NL4-3 strian(P <0.05).RNAscope experiment results show that the infection after 24 hours Ⅲ strain,NL4-3 strian and GX002 strain genome HERV-K(HML-2)transcription are slightly raised,48 hours after infection in the three groups were raised obviously and GX002 group raised the level of higher,consistent with experimental results in real-time quantitative PCR.(6)MT2 cell models that could regulate the stable expression of Tat protein,pLVX-Teton-NL4-3-Tat(subtype B),pLVX-Teton-GX002-Tat(subtype CRF01＿AE and pLVX-Teton-puro(no-load control),were successfully constructed.(7)Different HIV-1 Tat proteins had different effects on HERV-K(HML-2)transcription level in MT2 cells(P<0.05),and the increase in subtype B and CRF01＿AE groups was about 4-fold and 2-fold,respectively.Conclusion:(1)A novel epidemic recombinant strain(CRF)of the second generation was found and identified in the United States HIV Databases,btf@lanl.gov,and named CRF102＿0107.(2)The transcription level of the HERV-K(HML-2)gag region in HIV-1 subtype B infected patients without antiviral treatment was up-regulated compared with that in healthy people.The transcription level of the HERV-K(HML-2)pol region in HIV-1patients with non-B subtype(CRF01＿AE,CRF07＿BC)was higher than that in healthy people.The transcriptional expression of HERV-K(HML-2)in HIV-1 infected patients with different genetic subtypes is different,and further studies are needed to explain the possible mechanism.There was no significant correlation between viral load and transcriptional levels of HERV-K(HML-2)regions in infected patients.(3)Different cell lines have different susceptibility to HIV-1,mainly reflected in different viral replication capacities at the same time,and the MT2 cell line is the most susceptible.Different subtypes of HIV-1 strains resulted in significant differences in the upregulation of HERV-K(HML-2)transcriptional levels in MT2 cells.CRF01＿AE strain in regulating genome HERV-K(HML-2)raises stronger than subtype B strain.(4)Tat protein can activate the transcription of HERV-K(HML-2)in MT2 cells,and there are differences in the ability of Tat protein of different HIV-1 subtype strains to up-regulate HERV-K(HML-2).Compared with the Tat protein of CRF01＿AE strain,subtype B strain has a stronger regulation of HERV-K(HML-2)up-regulation. |
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