Hepatitis B virus (HBV) DNA synthesis occurs by reverse transcription of viral 3.5kb pregenomic RNA (pgRNA) synthesized from the nucleocapsid promoter (Nassal M, 2008; Ondracek C R, et al., 2011; Ondracek C R, et al., 2009; Ondracek C R, et al., 2009). Therefore, it is apparent that controlling the pgRNA level is a key regulatory step both in viral gene expression and in viral replication (Li L, et al., 2009; Tang H, et al., 2001). Liverenriched nuclear receptors (NRs) have been demonstrated to be essential for HBV RNA synthesis (Ondracek C R, et al., 2009; Ondracek C R, et al., 2009; Reese V C, et al., 2013; Shlomai A, et al., 2008). Complementation studies utilizing the human embryonic kidney cell line 293T, which support neither viral 3.5kb pgRNA synthesis nor HBV replication, revealed that only the NR hepatocyte nuclear factor 4α (HNF4α) and RXRα/PPARα (retinoid X receptor α plus peroxisome proliferator-activated receptor α) are capable of rescuing viral biosynthesis (Reese V, et al., 2011; Tang H, et al., 2001). These observations indicate that nuclear receptors may have a unique capacity to regulate HBV transcription and replication during natural infection. Indeed, it has been reported that HBV biosynthesis is likely to be completely dependent on HNF4α in vivo using a transgenic mouse model of chronic HBV infection (Ondracek C R, et al., 2011; Reese V, et al., 2011).
HNF4α and its coactivator are important regulators of energy homeostasis within the liver (Rodgers J T, et al., 2005; Yoon J C, et al., 2001). Peroxisome proliferatoractivated receptor-γ coactivator 1α (PGC1α), specifically and robustly coactivates key gluconeogenesis genes (Yoon J C, et al., 2001). Similarly, the amounts of HBV transcripts driven by the nucleocapsid promoter have also been shown to increase due to the coactivation effect of PGC1α and HNF4α (Shlomai A, et al., 2006). Tandem mass spectrometry analysis has shown that PGC1α is acetylated at 13 lysine sites. Mutation of these 13 lysines to arginines abolished the acetylation of PGC1α. If only some of the sites were mutated, PGC1α could still be still acetylated (Rodgers J T, et al., 2005). General Control Non-repressed Protein 5 (GCN5) acetylates PGC1α, resulting in a transcriptionally inactive state that relocalizes from promoter regions to nuclear foci. This process inhibits gluconeogenesis and is particularly important for the maintenance of glucose homeostasis. Adenovirus-mediated expression of GCN5 in cultured hepatocytes and in mouse liver significantly represses activation of gluconeogenic enzymes and decreases hepatic glucose levels (Doitsh G, et al., 2004; Lerin C, et al., 2006). However, there is still no direct study on the effect of GCN5 on HBV transcription and replication enhanced by PGC1α and HNF4α.
On the basis of these considerations, it was of interest to determine whether the acetyltransferase GCN5 might modulate HBV pgRNA synthesis and viral replication by regulating PGC1α activity. To address this issue, the expression vectors bearing PGC1α, acetylation site mutant PGC1αR13, GCN5 and acetyltransferase inactive mutant GCN5m were transferred into human hepatoma cell line Huh-7 and BALB/C mouse to evaluate the effects in vitro and in vivo respectively. Our results clearly show that GCN5, depending on the acetyltransferase activity, inhibits the coactivation activity of PGC1α in HBV transcription and replication.
GCN5 Acetyltransferase Inhibits PGC1α-induced Hepatitis B Virus Biosynthesis
- Received Date: 14 May 2013
- Accepted Date: 05 June 2013
- Published Date: 02 July 2013
Abstract: Hepatitis B virus (HBV) biosynthesis is primarily restricted to hepatocytes due to the governing of liver-enriched nuclear receptors (NRs) on viral RNA synthesis. The liver-enriched NR hepatocyte nuclear factor 4α (HNF4α), the key regulator of genes implicated in hepatic glucose metabolism, is also a primary determinant of HBV pregenomic RNA synthesis and HBV replication. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) coactivates and further enhances the effect of HNF4α on HBV biosynthesis. Here, we showed that the acetyltransferase General Control Non-repressed Protein 5 (GCN5) acetylated PGC1α, leading to alteration of PGC1α from a transcriptionally active state into an inactive state. As a result, the coactivation activity of PGC1α on HBV transcription and replication was suppressed. Apparently, an acetylation site mutant of PGC1α (PGC1αR13) still had coactivation activity as GCN5 could not suppress the coactivation activity of the mutant. Moreover, a catalytically inactive acetyltransferase mutant GCN5m, due to the loss of acetylation activity, failed to inhibit the coactivation function of PGC1α in HBV biosynthesis. Our results demonstrate that GCN5, through its acetyltransferase activity, inhibits PGC1α-induced enhancement of HBV transcription and replication both in vitro and in vivo.