Abstract
The core promoter of hepatitis B virus (HBV) genome is a critical region for transcriptional initiation of 3.5 kb, pregenome and precore RNAs and for the viral replication. Although a number of host-cell factors that potentially regulate the viral promoter activities have been identified, the molecular mechanisms of the viral gene expression, in particular, regulatory mechanisms of the transcriptional repression remain elusive. In this study, we identified LUC7 like 3 pre-mRNA splicing factor (LUC7L3, also known as hLuc7A or CROP) as a novel interacting partner of HBV enhancer II and basal core promoter (ENII/BCP), key elements within the core promoter, through the proteomic screening and found that LUC7L3 functions as a negative regulator of ENII/BCP. Gene silencing of LUC7L3 significantly increased expression of the viral genes and antigens as well as the activities of ENII/BCP and core promoter. In contrast, overexpression of LUC7L3 inhibited their activities and HBV replication. In addition, LUC7L3 possibly contributes to promotion of the splicing of 3.5 kb RNA, which may also be involved in negative regulation of the pregenome RNA level. This is the first to demonstrate the involvement of LUC7L3 in regulation of gene transcription and in viral replication.
Highlights
The core promoter and ENII are located in the region that overlaps with the X gene
To identify novel host factors which are involved in regulating the transcription of 3.5-kb pregenome/precore RNA of Hepatitis B virus (HBV), we first carried out proteomics screening in which nuclear proteins binding to biotinylated capture DNA of HBV sequence covering enhancer II and basal core promoter (ENII/BCP)(nt 1627–1817) were isolated using the magnetic microbeads separation system (Supplementary Table S1)
We found that LUC7L3 knock-down led to marked increase in the promoter activities derived from both genotypes A and B (Fig. 1A)
Summary
The core promoter can be divided into two elements; basal core promoter (BCP)(nucleotide [nt] 1743–1849) sufficient to initiate transcription and an upper regulatory region (nt 1643–1742). The latter region is composed of the core upstream regulatory sequence (CURS) that stimulates the BCP and of negative regulatory element (NRE). It is assumed that ENII/BCP mutations potentially affect binding between the region and cellular factors involved in the transcriptional regulation, leading to less production of the 3.5-kb mRNA and HBe antigen. One of the identified proteins, LUC7 like 3 pre-mRNA splicing factor (LUC7L3, known as hLuc7A or CROP) was found to be a negative regulator of ENII/BCP activity. Ectopic expression of the siRNA-resistant LUC7L3 apparently restored down-regulation of the core promoter activity mediated by the gene silencing. Our data suggested a role of LUC7L3 as a novel negative regulator in HBV replication process
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