ABSTRACT
BACKGROUND AND AIMS: Direct elimination of cccDNA remains a formidable obstacle due to the persistent and stable presence of cccDNA in hepatocyte nuclei. The silencing of cccDNA transcription enduringly is one of alternative strategies in the treatment of hepatitis B. Protein binding to cccDNA plays an important role in its transcriptional regulation; thus, the identification of key factors involved in this process is of great importance. APPROACHES AND RESULTS: In the present study, high mobility group nucleosome binding domain 1 (HMGN1) was screened out based on our biotin-avidin enrichment system. First, chromatin immunoprecipitation and fluorescent in situ hybridization assays confirmed the binding of HMGN1 with cccDNA in the nucleus. Second, functional experiments in HBV-infected cells showed that the promoting effect of HMGN1 on HBV transcription and replication depended on the functional region of the nucleosomal binding domain, while transfection of the HMGN1 mutant showed no influence on HBV compared with the vector. Third, further mechanistic exploration revealed that the silencing of HMGN1 increased the level of phosphorylase CLK2 and promoted H3 phosphorylation causing the reduced accessibility of cccDNA. Moreover, silenced HMGN1 was mimicked in HBV (r) cccDNA mouse model of HBV infection in vivo. The results showed that silencing HMGN1 inhibited HBV replication in vivo. CONCLUSIONS: In summary, our study identified that a host protein can bind to cccDNA and promote its transcription, providing a candidate strategy for anti-HBV targeting to interfere with the transcriptional activity of cccDNA microchromosomes.
Subject(s)
HMGN1 Protein , Hepatitis B , Animals , Mice , Histones/metabolism , Hepatitis B virus/physiology , HMGN1 Protein/genetics , HMGN1 Protein/metabolism , Chromatin , Carrier Proteins/genetics , Phosphorylation , In Situ Hybridization, Fluorescence , Virus Replication/genetics , DNA, Circular/genetics , DNA, Circular/metabolism , Transcription Factors/genetics , Hepatitis B/metabolism , DNA, Viral/geneticsABSTRACT
A respiratory illness has been spreading rapidly in China, since its outbreak in Wuhan city, Hubei province in December 2019. The illness was caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical manifestations related to SARS-CoV-2 infection ranged from no symptom to fatal pneumonia. World Health Organization (WHO) named the diseases associated with SARS-CoV-2 infection as COVID-19. Real time RT-PCR is the only laboratory test available till now to confirm the infection. However, the accuracy of real time RT-PCR depends on many factors, including sampling location and of methods, quality of RNA extraction and training of operators etc.. Variations in these factors might significantly lower the sensitivity of the detection. We developed a peptide-based luminescent immunoassay to detect IgG and IgM. Cut-off value of this assay was determined by the detection of 200 healthy sera and 167 sera from patients infected with other pathogens than SARS-CoV-2. To evaluate the performance of this assay, we detected IgG and IgM in the 276 sera from confirmed patients. The positive rate of IgG and IgM were 71.4% (197/276) and 57.2% (158/276) respectively. By combining with real time RT-PCR detection, this assay might help to enhance the accuracy of diagnosis of SARS-CoV-2 infection.
ABSTRACT
AIM:To investigate the role of heat shock protein 70(HSP70)in hepatitis B virus (HBV) replica-tion.METHODS:The effect of HBV replication on the expression of HSP 70 was analyzed by RT-qPCR.The overexpres-sion efficiency of HSP70 was confirmed by Western blot .The effect of HSP70 overexpression on HBV DNA replicative in-termediates was analyzed by RT-qPCR and Southern blot .The effects of HSP70 overexpression on the expression level of HBV 3.5 kb mRNA and HBV core protein were measured by RT-qPCR and Western blot, respectively.The Effect of HSP70 overexpression on HBV promoter activity was detected by dual luciferase reporter system .RESULTS: The mRNA levels of HSP70 were inhibited by HBV replication .Overexpression of HSP70 repressed the expression of HBV DNA repli-cative intermediates, 3.5 kb mRNA and core protein, as well as HBV core promoter activity .CONCLUSION:HBV rep-lication inhibits the expression of HSP70.Overexpression of HSP70 represses HBV replication.These data suggest that HSP70 repressed HBV replication by inhibiting HBV core promoter activity .