| Background:The rapid development of the high-throughput sequencing,chromosome conformation capture and synthetic biology technology brings about unprecedented opportunities to understand the biological characteristics of the diseases caused by viral infection.Using three-dimensional genome technology combined with genomics,transcriptomics and epigenomics data analysis,and bacterial artificial chromosome(BAC)biological system,we studied the life features of two RNA viruses called human immunodeficiency virus type 1(HIV-1)and severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)in host cell in various aspects,including the integration preference and transcription landscape of viral genome,and virus-host genome interaction,as well as their regulatory effects on virus or host gene function,which provided insightful views for further elucidating the latent or pathogenic mechanism and improving the clinical detection,prediction and treatment measures of viral infectious diseases.Objective:To comprehensively delineate the precise positioning of HIV-1 integration sites within host nucleus space and virus-host interaction at the genome-wide level,and systematically uncover the regulatory mechanism of host genome structure and function by viral integration,and to establish a virus simulation system allowing basic researches and applications on SARS-Co V-2 being widely carried out under the condition of non-biosafety level 3,thus provided potential targets and useful tools for exploring the infection mechanism,biomarkers,antiviral drugs and vaccines of HIV-1 and SARS-Co V-2.Methods:The wild type and HIV-1 infected A3.01 cell line and CD4~+T lymphocytes from patients as study objects were collected for multi-omics data synthetic analysis,including high-throughput chromosome conformation capture(Hi-C),integration site sequencing,assay for transposase accessible chromatin using sequencing(ATAC-seq)and RNA-seq,to reconstruct the three-dimensional genomic structure of host nucleus,characterize the HIV-1 integration landscape in a hierarchical view of host genome organization,and compare the differences of chromatin accessibilities and transcription profiles between pre-and post-integrated host genome.We also designed and constructed a non-infectious and DNA-based SARS-Co V-2 replicon system in BAC backbone,and the vectors containing wild type or mutant nucleocapsid(N)gene for Vero E6 cell transfection assays,together with the improved quantitative reverse transcription PCR(RT-q PCR)methods,to mimic the authentic replication circle of SARS-Co V-2,represent different stages among replication and transcription processes of replicon RNA,the regulatory activities of N protein in viral transcription process,and the sensitivity of BAC system to antiviral drugs.Results:HIV-1 genome did not randomly integrated into host chromatin but preferred to occur within the regions including the interior of host nucleus,gene-dense chromosomal segments,chromatin compartment A,topologically associated domains(TADs),introns of actively transcribed genes and active regulatory elements in open chromatin regions,which not only attenuated the segregation of compartment A and B,the consolidation of TADs,and the insulation of TADs boundaries,but also enhanced the switches and contacts across compartment A and B,TADs splitting and fusion,the interactions between certain chromatin regions or genomic loci,and the activities of cis-acting elements(like promoter and enhancer)along with their contact frequencies and chromatin accessibilities.These changes finally led to local chromatin structure being more relaxed and gene regulatory networks being reset,which further supplied molecular basis for integration,interaction and expression of the genes that are conducive to viral life cycle.The SARS-Co V-2 replicon system was not merely able to express normally in Vero E6 cells.We also found that the wild type N plasmid being co-transfected with replicon significantly enhanced the signal of reporter gene enhanced green fluorescent protein(EGFP),the abundance and ratio of plus-and minus-strand subgenomic RNAs(sg RNAs)of EFGP and N.Moreover,the mutant N vectors N1(R203K)and N4(S194L)could improved the levels both plus-and minus-strand sg RNAs of EFGP and N obviously as well,while N5(S188L)compromised the transcription process of them.The two known drugs against SARS-Co V-2 infection which called remdesivir and chloroquine was introduced to act in different stages of the transcription process of replicon and consequently effectively suppress the expression levels of EGFP and N both in a dose-dependent manner.Conclusion:The results based on multi-omics data analysis about HIV-1 integration revealed high selectivity of this process in multiple folding space of host genome and complicated influence of the conservative integration pattern on host chromatin structure and gene activity,which shed light on our complete and profound understanding on the formation and latent mechanism of HIV-1 proviral reservoir and the relationship between virus and host genome.The changes appeared in single gene loci was observed and described according to above conclusions,which provided important evidences for the research and development of targeted medicine or other specific treatment against HIV-1 infection.In addition,our designed and constructed replicon system could perform autonomous,efficient and stable amplification,and nearly had no toxicity to Vero E6 cells,no capacity to produce virion,and no difficulty to transfection operation.The trans-acting N protein can not only promote the overall transcription of replicon RNA,but also facilitate the transformation efficiency from template minus-strand sg RNAs to newly synthesized plus-strand sg RNAs.Furthermore,the two kinds of anti-SARS-Co V-2 drugs showed significant inhibitory effect on the transcription process of replicon,though the work mode were not quite the same.The above results indicated that the BAC system has potential application value both in the basic research and new antiviral drug screening of SARS-Co V-2. |
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