PGC1α Transcriptional Adaptor Function Governs Hepatitis B Virus Replication by Controlling HBcAg/p21 Protein-Mediated Capsid Formation

Abstract

In the human hepatoma cell line Huh7, the coexpression of the coactivators peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), cyclic AMP-responsive element binding protein binding protein (CBP), steroid receptor coactivator 1 (SRC1), and protein arginine methyltransferase 1 (PRMT1) only modestly increase hepatitis B virus (HBV) biosynthesis. However, by utilizing the human embryonic kidney cell line HEK293T, it was possible to demonstrate that PGC1α alone can support viral biosynthesis independently of the expression of additional coactivators or transcription factors. In contrast, additional coactivators failed to support robust HBV replication in the absence of PGC1α. These observations indicate that PGC1α represents a novel adaptor molecule capable of recruiting the necessary transcriptional machinery to the HBV nucleocapsid promoter to modestly enhance viral pregenomic 3.5-kb RNA synthesis. Although this change in transcription is associated with a similar modest change in hepatitis B virus core antigen polypeptide (HBcAg/p21) synthesis, it mediates a dramatic increase in viral capsid production and robust viral replication. Therefore, it is apparent that the synthesis of cytoplasmic HBcAg/p21 above a critical threshold level is required for the efficient assembly of HBV replication-competent viral capsids. Hepatitis B virus (HBV) is a major human pathogen, and novel targets for the development of additional therapeutic agents are urgently needed. Here we demonstrate that the coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) serves as a unique adaptor molecule for the recruitment of additional coactivator proteins, which can finely regulate HBV transcription. The consequence of this precise regulation of viral RNA levels by PGC1α is a subtle increase in cytoplasmic HBcAg/p21 polypeptide translation, which shifts the equilibrium from dimer formation dramatically in favor of viral capsid assembly. These findings suggest that both PGC1α and capsid assembly may represent attractive targets for the development of antiviral agents against chronic HBV infection.

Modulation of hepatitis B virus pregenomic RNA stability and splicing by histone deacetylase 5 enhances viral biosynthesis

“…However, HBV capsids were increased approximately 50-fold with HDAC5 expression but were undetectable in the presence of the other HDACs ( Fig 1B). The observation that an HDAC5-mediated 5-fold increase in HBV core polypeptide (p21) levels is associated with a 50-fold enhancement in HBV capsid abundance is consistent with previous reports indicating that the conversion of HBcAg dimers into capsids occurs by a highly cooperative process [35][36][37]. Moreover, the limited expression of HDAC5 protein in HepG2 cells compared with HEK293T cells (S1C Fig) may explain the modest effect of HDAC5 expression on HBV biosynthesis in HepG2 cells compared with the robust effect in HEK293T cells.…”

“…This might be reflected as a modest decrease in HBV 3.5 and 2.2 kb RNA levels, which potentially could be missed without the appropriate analysis to distinguish between the HBV spliced 2.2 kb and unspliced 2.1 kb RNAs. However, this change in viral transcript profile might result in a modest reduction in core/p21 polypeptide with an associated dramatic loss of capsid formation due to the concentration-dependent cooperative process involved in capsid assembly [ 35 – 37 ]. In fact, studies utilizing proinflammatory signals typically report a post-transcriptional inhibitory effect on HBV biosynthesis associated with limited changes in viral RNA levels but with a loss of cytoplasmic capsids [ 58 , 59 ].…”

“…Transfected cells from a single plate were divided equally and used for the preparation of total cellular RNA, cytoplasmic protein, and viral DNA replication intermediates as described [37,73]. RNA (Northern) and DNA (Southern) filter hybridization analyses were performed using 10 μg of total cellular RNA and viral DNA replication intermediates derived from one third of the cells per 10 cm plate.…”

“…For western blot analysis of viral capsids, 50 μg of cytoplasmic lysate was loaded onto an 0.8% agarose gel in TAE and electrophoresed at 75V for 2 hours. The gel was transferred to PVDF membrane by capillary transfer in TNE buffer (10 mM Tris pH 7.5, 150 mM NaCl, and 1 mM EDTA) overnight as described previously [76]. For detection of HBcAg/ p21 polypeptide, 50 μg of cytoplasmic lysate was loaded onto 4-12% Bis-Tris SDS-PAGE (Thermo Fisher) and electrophoresed for 2 hours at 100V.…”

“…HDAC5 was detected with 1:200 mouse antibody C-11 (Santa Cruz) in 5% milk in TBST. Western blot analysis was conducted as described [76,77]. For detection of viral capsids and HDAC5, horseradish peroxidase-labeled goat anti-mouse IgG (Cell Signaling Technology, 1:5000 dilution) was used and imaged using enhanced chemiluminescence substrate (Thermo Fisher) and the ChemiDoc MP Imaging System (BioRad).…”