Distribution,Expression And Activation Factors Of Human Endogenous Retrovirus K Family(HERV-K) In HIV-1 Positive Individuals In China

Human Endogenous Retrovirus(HERV)exist as DNA remnants of retrovirus that has been infected and inherited germ lineage cells of our ancestors in millions of years ago.About 98,000 HERV elements constitute 8% of the human genome.HERVs plays an important role in fertilization,embryo implantation and development,and transcription.HERVs are associated with these diseases such as cancer,autoimmune diseases,mental disorder,neuropathy,inflammation and viral infections.To date,the study of HERV is still in its infancy.These questions such as that whether HERV had influence the replication and integration of HIV-1,the HIV-1 regulatory protein Tat may activate HERVs and which types of HERV integrate HIV positive individuals in China remain unclear.In this study,we illuminated the distribution,expression and activation factors of human endogenous retrovirus K(HERV-K)in HIV-infected patients in China.PartⅠ:Different distributions of HERV-K113 and K115 genes were found in HIV-1 infected individuals of different ethnics in ChinaHuman endogenous retroviruses,comprise 8 % of the human genome.HERV-K113 and K115 are full-length proviruses.Although generous previously published papers have documented their prevalence in the global population,to date,no data in China population has been reported.Objective: To demonstrate HERV-K113 and HERV-K115 genetic distributions among HIV infected individuals from different areas or ethnics in China.Methods: Peripherial blood was sampled and genomic DNA was extracted from HIV-1 positive individuals in China.The presence or absence of HERV-K113 and HERV-K115 was determined with PCR primers spanning the preintegration sites and sites within the viral sequences.Results: A total of 504 individuals were enrolled into the study.Among of them,238 belong to Han ethnic,175 belong to Yi minority ethnic,and 91 belong to Zhuang minority ethnic.The insertion frequencies of HERV-K113 and HERV-K115 were 30.0% and 4.8%.The distributions of HERV-K113 are significantly different among ethnics and regions.Highest frequency of K113 was identified in Zhuang minority ethnic(39.6%),followed by Han ethnic(30.3%)and Yi minority ethnic(24.6%)within HIV-1 positive individuals in China.HERV-K115 was found in 2.9% of Yi minority ethnic,5.0% of Han ethnic and7.7% of Zhuang minority ethnic.Conclusion: To date,there has been no report on HERV-K113 and HERV-K115 prevalence in HIV-1 infected individuals from China.This study will provide information for detecting the relationship between presence of the HERVs and disease procession of HIV-1 infected individuals.Part Ⅱ:The transcription levels of HERV-K RNA in PBMC from different populationsHERV-K is the most active and complete of HERV.They are capable of producing retroviral-like particles.HERV-K fragments were found in plasma and PBMC of HIV-infected individuals.Although HERV-K transcripts were detected in some HIV-1 viral positive plasma specimens,levels of HERV-K expression did not correlate with HIV-1 viral load.Objective: To examined whether levels of HERV-K expression are correlate with HIV-1 viral load,whether HERV-K RNA transcription is different among different populations,and whether HBV infection can up-regulate HERV-K RNA transcription.Methods: Blood samples were collected from untreated HIV-infected patients,HIV-infected patients,HBV positive individuals,and healthy populations.The total RNA was extracted from PBMC,and examined whether contaminated with DNA.The real-time quantitative PCR method was used to detect HERV-K RNA in PBMC specimens.Statistical analysis of one way anova and person-correlation test was used to analyze whether HERV-K RNA transcription was correlated with HIV load and whether HERV-K RNA transcription was different between different populations.Results: A total of 68 PBMC samples were collected,and 7 samples with genomic contamination of purified total RNA were excluded.HERV-K RNA was detected in all PBMC samples except for one healthy person.We found increased HERV-K RNA titers in HIV-1 patients with non-suppressive compared to suppressive regimens HAART,HBV-infected patients and healthy(p<0.05,there was no significant difference among HIV-infected patients receiving HAART,HBV-infected patients,and healthy people.Lack of Correlation between HERV-K expression in PBMC and HIV-1viral Load in Plasma Specimens(r=0.119,p=0.660>0.05).Conclusion: HIV infection significantly up-regulated the levels of HERV-K RNA.HIV-1 load was not associated with HERV-K RNA levels.Part Ⅲ : Establishment of stable expression HIV-1Tat cell line and the activation of HERVThe Tat protein is a regulatory protein encoded by HIV,it can bind to HERV-LTR and activate transcription of HERVs.However,it is not clear that which HERV LTR can be combined with Tat protein,and the function of these LTR.Objective: To construct a cell line that stably expressing Tat protein,and to explored the role of the HIV-1 Tat protein in inducing the expression of these endogenous retroviral genes.Methods:(1)Construction of a monoclonal cell line stably expression Tat proten.The two exon fragments of HIV-1 subtype B and HIV-1 subtype CRF01_AE tat gene were amplified by PCR,and purified the amplification product.And the tat gene fragments linked to vector p CDNA3.1(+).The successfully constructed Tat vector and empty vector were transfected into TZM-bl cells,and cultured in a medium containing G418 for two weeks.The surviving cells are infinitely diluted into 96-well cell culture plates,and the wells of individual cells are observed and labeled.After the cells are aggregated,the Tat protein is stably expressed by detecting the chemilumine scence value of the cells.To determine the tat gene integration in the selected monoclonal cells,extracted DNA and total RNA from the selected Monoclonal cell line,and amplified by PCR and RT-q PCR.(2)Transcriptome sequencing and analysis.Three biological replicates were set up for each monoclonal cell,the total RNA of these cells was extracted,a c DNA library was constructed,Paired-end sequencing of the library was performed on the Hi Seq XTen sequencers.Fast QC was used for evaluating the quality of sequenced data.Clean reads were mapped to the ensemble reference genome.DESeq2 was used to determin e differentially expressed genes(DEGs)between two samples.Functional enrichment analyses including Gene Ontology(GO)and KEGG was performed to identify which DEGs were significantly enriched in GO terms or metabolic pathways.The differentially expressed genes were aligned into the HERVd database to find the Tat-regulated HERV gene.Results: Monoclonal cell with good growth and high Tat expression was obtained in each subtype.The lumen escence value of the selected cell line was 100 times that of control.The selected monoclonal cell line has tat gene integration and transcription.Each cell transcriptome was sequenced with more than 6.5G data,Q30 was above 90%.subtype B Tat protein regulated 54 HERV transcriptions,30 up-regulation,24 down-regulation,most HERV are LTR,only 8 have an open reading frame.Subtype CRF01_AE regulates 58 HERV transcriptions,38 up-regulations,20 down-regulations,most are LTR,only 8 have an open reading frame.Both subtype B and CRF01_AE Tat have regulated 13 HERV.Tat protein related cell growth and apoptosis,signal transduction,cell communication,cancer,immune diseases,infectious diseases,chromosome structure,transcription,cell migration,cellular stress response and other functions.Transcriptome sequencing results suggest that Tat is associated with cell growth,through cells proliferation experiments demonstrated that Tat protein expression did not promote the growth of TZM-bl cells.Conclusion: The cell line stably expressing Tat protein was successfully constructed.The Tat-activated HERV is mainly LTR,which lays a foundation for studying how Tat activates HERV LTR.