Dataset Information

Involvement of PUF60 in Transcriptional and Post-transcriptional Regulation of Hepatitis B Virus Pregenomic RNA Expression.

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.

SUBMITTER: Sun S

PROVIDER: S-EPMC5634508 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Involvement of PUF60 in Transcriptional and Post-transcriptional Regulation of Hepatitis B Virus Pregenomic RNA Expression.

Sun Suofeng S Nakashima Kenji K Ito Masahiko M Li Yuan Y Chida Takeshi T Takahashi Hirotaka H Watashi Koichi K Sawasaki Tatsuya T Wakita Takaji T Suzuki Tetsuro T

Scientific reports 20171009 1

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 ...[more]

PMID: 28993636

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Project description:BackgroundHepatitis B virus (HBV) pregenomic RNA (pgRNA) has gained increasing attention owing to its role in replication of covalently closed circular DNA (cccDNA) in HBV. This marker has the potential to be used in clinical programs aimed to manage HBV infections. However, several reports on HBV pgRNA levels in clinical cases have conflicting results. RNA is easily degraded when exposed to heat and other environmental stressors. However, the stability of HBV pgRNA, during blood sample collection before the standard automated quantification, has never been estimated. This study aimed to demonstrate the effect of two different temperature conditions and storage durations on the stability of HBV pgRNA.MethodBlood from forty patients with chronic hepatitis B infection, who also showed evidence of active HBV DNA replication, was collected and processed within 2 h of collection. Plasma from each patient was divided and stored at 4 °C and 25 °C (room temperature) for six different storage durations (0, 2, 6, 12, 24, and 48 h) and subsequently transferred to -80 °C for storage. The effect of multiple cycles of freezing and thawing of plasma at -20 °C or -80 °C was evaluated using samples from ten patients. Quantification of pgRNA from the samples was performed simultaneously, using the digital polymerase chain reaction (dPCR) method. The differences in pgRNA levels at baseline and each time point were compared using generalized estimating equation (GEE). A change greater than 0.5 log10 copies/mL of pgRNA is considered clinically significant. Statistical analyses were conducted using Stata 16.0.ResultsThe mean HBV pgRNA level in the initially collected plasma samples was 5.58 log10copies/mL (ranging from 3.08 to 8.04 log10 copies/mL). The mean pgRNA levels in samples stored for different time periods compared with the initial reference sample (time 0) significantly decreased. The levels of pgRNA for 6, 12, 24, and 48 h of storage reduced by -0.05 log10 copies/mL (95% confidence interval (CI) -0.095 to -0.005, p = 0.03), -0.075 log10 copies/mL (95% CI [-0.12 to -0.03], p = 0.001), -0.084 log10 copies/mL (95% CI [-0.13 to -0.039], p = < 0.001), and -0.120 log10 copies/mL (95% CI [-0.17 to -0.076], p = < 0.001), respectively. However, these changes were below 0.5 log10 copies/mL and thus were not clinically significant. Compared with the samples stored at 4 °C, there were no significant differences in pgRNA levels in samples stored at 25 °C for any of the storage durations (-0.01 log10 copies/mL; 95% CI [-0.708 to 0.689], p = 0.98). No significant difference in the levels of pgRNA was observed in the plasma samples, following four freeze-thaw cycles at -20 °C and -80 °C.ConclusionThe plasma HBV pgRNA level was stable at 4 °C and at room temperature for at least 48 h and under multiple freeze-thaw cycles. Our results suggest that pgRNA is stable during the process of blood collection, and therefore results of pgRNA quantification are reliable.

Project description:UnlabelledHepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication.ImportanceThe hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target. The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment.

Dataset Information

Involvement of PUF60 in Transcriptional and Post-transcriptional Regulation of Hepatitis B Virus Pregenomic RNA Expression.

Publications

Involvement of PUF60 in Transcriptional and Post-transcriptional Regulation of Hepatitis B Virus Pregenomic RNA Expression.

Similar Datasets

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