Abstract
Here we identified PUF60, a splicing factor and a U2 small nuclear ribonucleoprotein auxiliary factor, as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) 3.5 kb, precore plus pregenomic RNA. We demonstrate that PUF60 is involved in: 1) up-regulation of core promoter activity through its interaction with transcription factor TCF7L2, 2) promotion of 3.5 kb RNA degradation and 3) suppression of 3.5 kb RNA splicing. When the 1.24-fold HBV genome was introduced into cells with the PUF60-expression plasmid, the 3.5 kb RNA level was higher at days 1–2 post-transfection but declined thereafter in PUF60-expressing cells compared to viral replication control cells. Deletion analyses showed that the second and first RNA recognition motifs (RRMs) within PUF60 are responsible for core promoter activation and RNA degradation, respectively. Expression of PUF60 mutant deleting the first RRM led to higher HBV production. To our knowledge, this is the first to identify a host factor involved in not only positively regulating viral gene expression but also negative regulation of the same viral life cycle. Functional linkage between transcriptional and post-transcriptional controls during viral replication might be involved in mechanisms for intracellular antiviral defense and viral persistence.
Highlights
Upon infection, the uncoated viral genome is transported to the nucleus and converted into cccDNA, which serves as the template for synthesis of viral transcripts
To address how PUF60 is involved in gene expression and HBV replication, viral RNAs in cells co-transfected with pUCHB-Ce carrying the 1.24-fold HBV genome derived from genotype C and the FLAG-tagged PUF60-expressing plasmid were analyzed by northern blotting
In semi-quantitative (Fig. 1b) and quantitative (Fig. 1c, Supplementary Fig. S1) RT-PCR analyses, both a marked increase and decrease in the 3.5 kb RNA level were observed in PUF60-expressing cells at days 2 and 4 pt, respectively, compared to data obtained from northern blotting
Summary
The uncoated viral genome is transported to the nucleus and converted into cccDNA, which serves as the template for synthesis of viral transcripts. Livers of hepatitis B patients as well as in cultured cells transfected with the viral genome[6,7,8] Their significance and regulatory mechanisms underlying post-transcriptional processing events in the HBV life cycle are essentially unclear. During the course of investigating involvement of host cell factors with dual DNA- and RNA-binding capacities in HBV replication in siRNA-mediated gene knockdown and over-expression experiments, we identified PUF60 as a versatile regulator of both transcriptional and post-transcriptional steps in expression of HBV 3.5 kb RNA. We found that PUF60 up-regulated core promoter activity through its interaction with transcription factor 7-like 2 (TCF7L2), which is necessary for direct binding with the ENII region. PUF60 contributed to 3.5 kb RNA degradation and suppression of 3.5 kb RNA splicing
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
