| Backgrounds: According to the latest data released by the Joint United Nations Programme on HIV-1/AIDS(UNAIDS),as of June 30,2021,the AIDS pandemic has claimed 40.1 million lives [33.6 million – 48.6 million],which is one of the public health challenges facing human society.Human Immunodeficiency Virus(HIV)is the causative agent of AIDS,including HIV-1 and HIV-2,among which HIV-1 is the main cause of the AIDS pandemic.The HIV 1 provirus is mainly composed of gag,pol,and env,with a pair of identical Long Terminal Repeat(LTR)distributed at both ends.Although LTR only accounts for 13.05%(1268/9719)of the total length of the provirus,its role is crucial.Take 5’ LTR as an example,it plays a key role in the reverse transcription,integration,and transcriptional regulation of HIV-1,and is essential for HIV-1 to maintain its normal life cycle.Therefore,an in-depth study of HIV-1 5’ LTR gene polymorphism and transcriptional regulation function is of great significance to the transmission and epidemic,prevention and treatment of HIV-1.However,there are relatively few studies on HIV-15’ LTR in the current field,mainly reflected in the following three aspects.First,LTR sequences in the HIV Database database are rarely included,accounting for only 0.086% of all sequences(722/835940).Secondly,the research on HIV-1 gene polymorphism and gene function is mainly concentrated in the internal region,and the related research on the LTR region is few.Thirdly,the subtle differences in HIV-1 LTR of different subtypes can significantly affect the activity of its promoter,enhancer,and transcription factor binding sites(TFBS),as well as the replication dynamics and toxicity of HIV-1.However,the research on the transcription function of LTR in the current international field mainly focuses on subtype B in Europe and the United States,and research on the function of LTR involving other subtypes and circulating recombinant forms(CRFs)is relatively rare.Few domestic documents elaborate on the LTR polymorphism and functional characteristics of the prevalent HIV-1 subtype in China.Therefore,this study focused on the 5’ LTR gene polymorphism and transcriptional activity of the HIV-1 strain.Methods:(1)A variety of quality control strategies,including jpHMM,recombination detection,and phylogenetic analysis,were used to screen the sequences in the HIV database.According to the four principles of determining the reference sequences recognized in the field,the LTR reference sequence system was constructed,and the reference sequence system was tested and applied by introducing sample sequences.(2)The virus RNA was extracted from the plasma samples of HIV-1-infected patients in China.Following the HIV-1 reverse transcription process,two incomplete LTR sequences at the 5 ’and 3’ ends were obtained through reverse transcription and two rounds of PCR,respectively,and the same R region were combined to form a complete LTR..(3)Based on the established typing system,the fluorescent report plasmids of different subtypes of LTR were obtained by sequence synthesis,enzyme digestion,connection,and transformation.The corresponding stable cell lines were obtained by transfection and screening.The 5’ LTR characteristics of different subtypes of HIV-1 virus strains prevalent in China were further studied by using the method of luciferase activity detection.(4)Through the prediction of transcription factor binding sites,the differential transcription factor binding sites that affect the transcription activity of LTR were analyzed and obtained.The influence of differential transcription factor binding sites on the transcription activity of LTR was analyzed using luciferase activity detection.Results:(1)First of all,after systematic typing analysis,we updated some 5’ LTR sequence typing information in the HIV Database sequence database and found that the LTR of CRF02＿AG was recombined from the G subtype and A1 subtype.In addition,based on the four principles of phylogenetic typing,76 individual 5’ LTR sequences and302 5’ LTR sequences with complete genomes were screened from the database,and the reference sequence system of LTR was successfully constructed.The results showed that a total of 83 sequences were selected as reference sequences,including 2 groups,6subtypes,6 subtypes,and 9 CRF.(2)Given the fact that there is a serious lack of 5’ LTR sequence in the HIV database,we simulated the reverse transcription process of HIV-1 under natural conditions to establish an LTR amplification method based on HIV-1 RNA and expanded the plasma samples of HIV-1 infected people in Hebei Province and Shenzhen City we collected.The results showed that 22 complete LTR sequences were amplified from the samples of Hebei Province,3 sequences were subtype B and 8 sequences were CRF01＿AE,3sequences are CRF07＿BC,2 sequences are CRF08＿BC,3 sequences are CRF55＿01B.The recombinants of CRF07＿BC and CRF01＿AE(HB010151 and HB010161),subtype B,and CRF08＿BC(HB030133)were also found.219 complete LTR sequences were amplified from the samples in Shenzhen,including 9 sequences of subtype B,146 sequences of subtype C,3 sequences of subtype G,and 61 sequences of CRF01＿AE.There are four LTR recombinants of subtype C and CRF01＿AE,LS13145,LS11614,LS14862,and LS14863,respectively.(3)Based on CRF01＿AE,CRF07＿BC,CRF08＿BC,and CRF55＿01B the LTR shared sequences of the four major epidemic strains in China,respectively constructed the LTR fluorescent report expression plasmids of the four CRF strains,and completed the transfection of HEK 293 T cell line and Hela cell line,and screened a total of 10 stable cell lines.Through the luciferase report experiment,the results showed that there was a significant difference in the transcription activity of the 5’ LTR of the four main CRF prevalent in China,of which the LTR transcription activity of CRF08＿BC was the highest,and the LTR transcription activity of CRF01＿AE was the lowest.(4)To explore the reasons for the difference in the transcription activity between CRF08＿BC and CRF01＿AE,the NF-AT2 transcription factor was found to exist independently in the LTR of CRF08＿BC and showed different distribution,and the location was single.The sequence at NF-AT2 of CRF08＿BC was exchanged with CRF01＿AE,and the fluorescent report expression plasmids of CRF01＿AE and CRF08＿BC after mutation were successfully constructed and transfected into HEK293 T cells.The stable cell lines containing the fluorescent report expression plasmids of CRF01＿AE and CRF08＿BC after the mutation was screened.Through the luciferase report experiment,we found that the fluorescence value of CRF01＿AE increased after CRF01＿AE obtained NF-AT2,and the fluorescence value of CRF08＿BC without NFAT2 decreased slightly,suggesting that the NF-AT2 transcription factor binding site may be one of the key factors affecting the transcription activity of CRF01＿AE and CRF08＿BC LTR region. |
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