# Peroxisome Proliferator-Activated Receptors in HBV-Related Infection

Authors: Laurent Dubuquoy; Alexandre Louvet; Antoine Hollebecque; Philippe Mathurin; Sébastien Dharancy
Journal: PPAR Research
 (2009)

---

## Abstract

Thirty years after its discovery, the hepatitis B virus (HBV) still remains a major global public health problem. Worldwide, two billion subjects have been infected, 350 million have a chronic infection and more than 600 000 die annually of HBV-related liver disease or hepatocellular carcinoma; new infections occur because of the presence of a large reservoir of chronic carriers of the virus. Since a decade several studies describe the interrelations between HBV and nuclear receptors and more particularly the peroxisome proliferator-activated receptors (PPARs). After a brief introduction, this review will make a rapid description of HBV incidence and biology. Then a report of the literature on the role of PPARs on viral transcription and replication will be developed. Finally, the role of HBV on PPARγ expression and activity will be discussed. Concluding remarks and perspectives will close this review.


---

## Body

## 1. Introduction

Hepatitis B  virus
(HBV) infection  is a major public health problem with approximately 350 million
people chronically infected but the prevalence of HBV infection and patterns of
transmission vary greatly throughout the world. Fifteen percent to 40% of
HBV-infected patients will develop cirrhosis, liver failure, and hepatocellular
carcinoma (HCC) [1]. Hepatitis B
virus was considered to be not directly cytopathic, and the development of
HCC in individuals with chronic HBV infection is a multistage, multifactorial
process including the interaction between  host and environmental factors. However,
a recent study suggested that elevated serum HBV DNA level (≥10 000 copies/mL) was a risk predictor of HCC
independent of hepatitis B e antigen (HBeAg), serum alanine aminotransferase
level, and liver cirrhosis suggesting that HVB proteins themselves may have
direct effect on cellular functions [2].Recent data
suggested the implication of nuclear hormone receptor and especially of the
retinoid X receptors (RXRs) and peroxisome
proliferator-activated receptors (PPARs) in the transcription and the replication of
the HBV. The peroxisome proliferator-activated receptors (PPARs)α, β/δ, and γare members of the nuclear receptor
superfamily activated by fatty acids and involved in the transduction of
metabolic and nutritional signals into transcriptional responses [3, 4]. Among these transcription factors, PPARα/γ together with their
obligate partner the RXR are three main nuclear receptors expressed in the
liver [5–7]. However, despite strong
expression in the liver, proof of an eventual role of PPARs in hepatic disease
remains limited to the link between hepatic tumorigenesis and chronic
administration of PPARα activators in rodents [8], the development of extensive hepatic steatosis in
response to fasting and delayed liver regeneration in PPARα knock-out mice [9, 10], impaired expression of PPARα in a murine model of
alcoholic liver diseases [11], and
impaired liver expression of PPARα influenced by the HCV
core protein during chronic hepatitis C virus infection [12].This review
will first describe the importance of HBV infection worldwide and the biology
of the virus. Then the interactions between PPARs and HBV will be developed to
provide a precise picture of the potential role of PPARs in HBV pathophysiology.


## 2. Hepatitis B Virus: Incidence and Prevalence

Approximately 2
billion people have been exposed to the HBV and 350 million people are
chronically infected with the virus. Each year over 1 million people die from HBV-related
liver disease. The chapter below will expose the incidence and prevalence of
this huge public health problem worldwide.The prevalence of
HBV infection varies depending on the geographical area. In the Far East, the
Middle East, Africa, and parts of South America,
the prevalence is high, with hepatitis B surface antigens (HBsAgs) rates ranging
from 8% to 15% [13]. In regions of high
HBsAg endemicity, serologic evidence of prior HBV infection (anti-HBc and/or
anti-HBs Ag) is almost universal in subjects without active infection. As a general
rule, in these areas with high HBV endemicity the source of infection is mainly
through perinatal transmission from the chronically infected mother or through
infection during early childhood.Areas of intermediate
prevalence (2–7%) include Japan, parts of South America, Eastern and
Southern Europe, and parts of central Asia. Areas
with low HBV endemicity (prevalence of chronic infection <2%) include
Northwestern Europe, North America, and Australia
[14].
The source of infection in these areas is mainly through sexual contacts and
needle sharing among injecting drug users, with a peak incidence in the 15–25-year-old age group.Globally
the incidence of acute HBV infection has been falling in the last decade, due
to changes in behavior (e.g., increase in safe sexual practices related to HIV
education efforts) and, to a lesser extent, to the introduction of effective
vaccination programs [15]. Transmission of
HBV via transfusion of blood and plasma-derived products has been eliminated in
most countries through donor screening for HBsAg and viral inactivation
procedures.


## 3. Viral Structure, Genomic Organization and Replication

HBV
is a member of the family of the hepadnaviridae, hepatotropic DNA viruses.
Characteristics of these viruses are as follows: a partially
double-stranded DNA, with an outer lipoprotein envelope and an inner
nucleocapsid or core bearing the viral genome; a polymerase with reverse
transcription activity; the massive overproduction of viral envelope proteins
(e.g., HBsAg), and a relative but not absolute hepatotropism. The following
chapter will briefly describe the viral structure, genomic organization and
replication mode of the HBV.HBV
virions are 42 nm double-shelled particles. The genome contains four open
reading frames (ORFs) (S, P, C, and X) that encode four major proteins (surface,
polymerase, core, and X protein, resp.) (Figure1). The major abundant
protein on the virus surface is the HBsAg or S protein, 24 kDa in size. In the
viral envelope there are two other proteins, the L—involved in binding the virus to a
receptor on the hepatocyte surface—and the M protein, whose function is unknown.Figure 1
HBV
virion and genomic organization. The HBV virion is
composed of a viral envelop that contains the surface proteins, which are of
different lengths (L, M, and S). The nucleocapsid or core wraps the viral DNA.
The viral genome contains four open reading frames, the S that encodes for the
surface protein (red), P that encodes for the viral polymerase (blue), preC and
C that encode for the core (yellow) and X that encodes for the X protein
(green).The
27 nm nucleocapsid  is an icosahedral symmetric structure containing  180 or 240
copies of the viral core (C) protein [16, 17], known as
hepatitis B core antigen (HBcAg). The nucleocapsid contains the viral genome
(Figure 1), a relaxed circular molecule that consists of a 3.2 kB minus strand
and a smaller, complementary DNA (plus strand) of variable length. Circularity
of HBV is maintained by hydrogen bonds between 250 bp at the two 50 ends of the
plus and minus strands. The 50 ends of the DNA strands are each linked
covalently to additional structures, essential for the initiation of DNA
synthesis, that is, the polymerase and an oligo RNA. The viral polymerase is
encoded by the P gene of the virus and is implicated in the synthesis of both
strands of viral DNA through a reverse transcriptase (protein P) enzyme (RT).
This RT shares sequence similarities with retroviral RT; the latter has been
used in the development of antiviral drugs against HBV.In
addition to complete virions, HBV-infected hepatocytes produce in great excess
two distinct subviral lipoprotein particles: the spheres, containing primarily
the S protein, and the filaments, less numerous, rich in L protein. As these
subviral particles contain only envelope glycoproteins and host-derived lipids,
but not viral DNA; they are not infectious; nevertheless, they strongly
stimulate the production of neutralizing anti-HBs antibodies. The
overproduction of these particles makes it easy to diagnose HBV infection by
the detection of the surface antigen in the blood.Little
is known about the earliest steps in the HBV life cycle. Virion binding to hepatocytes
is mediated by a 180 kDa host protein identified as a member of the
carboxypeptidase family [18]; antibodies against
this protein block viral infection [19]. After
direct membrane fusion uncoating of the virus allows the presentation of the
nucleocapside to the cytosol. The naked viral core migrates to the nucleus
where the viral genome is repaired to a covalently closed circular form
(cccDNA). This cccDNA is transcribed by host RNA polymerase II to generate
genomic and subgenomics stable RNAs. All viral RNAs are transported to the
cytoplasm for translation yielding the viral envelope, core and preC, viral DNA
polymerase, and X proteins. Finally, nucleocapsids are assembled in the cytosol;
assembly requires the binding of viral polymerase (P) to a selective structure
located at the 5'end of the genomic RNA. Once the P-RNA complex is formed, RNA
packaging and reverse transcription begin. The replication of HBV requires an
RNA intermediate followed by the synthesis of viral DNA by RT [20]. After
replication is completed, viral cores are transported back into the nucleus,
where they are either converted to cccDNA to maintain a stable intranuclear
pool of transcriptional templates or more frequently, bud into the endoplasmic
reticulum or Golgi apparatus; in this site nucleocapsidic particles are wrapped
in the envelope proteins (surface, L, and M) and finally exported from the cell
as full virions by vesicular transport [21].


## 4. Impact of PPAR on Viral Transcription and Replication

Studies
in hepatoma cell line HepG2 and studies on a transgenic mouse model for HBV
have provided evidence for a role of PPARS in controlling viral transcription
and replication.HBV
has a partially double-stranded DNA genome and replicates through an RNA
intermediate. After infecting host liver cells, there are four HBV transcripts
from four different viral promoters: Core, SPI, SPII, and X promoter. The first
studies that have linked PPAR and HBV have shown the presence of hormone
response elements (HREs) in the promoters of HBV genome (Figure2). In the
dedifferentiated hepatoma cell line, HepG2, it was found that the nucleocapsid
and large surface antigen promoters were transactivated in the presence of hepatocyte
nuclear factor 4 (HNF4) whereas the enhancer I/X gene, nucleocapsid, and large
surface antigen promoters were transactivated in the presence of RXR and PPAR [22].
Characterization of the nucleocapsid promoter region demonstrated that HNF4 is
the primary transcription factor binding to the regulatory region spanning
nucleotides −127 to −102 whereas HNF4, RXR-PPAR heterodimers, and chicken ovalbumin
upstream promoter transcription factor 1 (COUP-TF1) bind the regulatory
region spanning nucleotides −34 to −7 [22]. Modulation
of the level of transcription from the nucleocapsid promoter by RXR-PPAR
appears to be regulated by the regulatory sequence element spanning nucleotides
−34 to −7 and the HBV enhancer I region (Figure 2). Another study demonstrated
that HNF4 and testicular receptor 2 (TR2) repressed synthesis of the pre-C RNA,
whereas PPAR-RXR activated synthesis of the pregenomic RNA and COUP-TF1
repressed synthesis of both the pre-C and pregenomic RNAs [23].Figure 2
NR
regulatory region in HBV genome. Schematic diagram of
the HBV genome. The viral polymerase (P), surface proteins (S), precore (preC),
core (C), and X protein (X) open reading frames are indicated by open
rectangular boxes. Enhancers (ENHs) I and II are indicated by grey rectangular
boxes. The hormone response elements (HREs) are indicated by small black
rectangular boxes. Nuclear receptors that can bind these HREs are indicated
into brackets.The
regulation of HBV transcription and regulation were then explored in vivo.
Using an HBV transgenic mouse model, Guidotti et al. demonstrated that
activation of PPARα increased
transcription and replication of HBV and suggested that even a modest
alteration in transcription could have big impact on virus replication [24]. To point
out the importance of nuclear receptors and specially PPARα on the HBV
replication, Tang and McLachlan have shown that ectopic expression of HNF4 and PPARα was
necessary and sufficient to allow HBV replication in nonhepatic cells, which
is normally impossible due to the virus tropism [25].Two studies
performed in the team of McLachlan in La Jolla specified the sequences of interaction between the HBV and PPARα [26, 27]. Indeed, this team has developed a transgenic mouse for
a natural hepatitis B virus (HBV) variant associated with seroconversion from
HBeAg to anti-HBe antibody that contains two nucleotide substitutions (A1764T
and G1766A) in the proximal nuclear hormone receptor binding site in the
nucleocapsid promoter. This model suggested that peroxisome proliferators may
enhance viral transcription directly in a PPARα-dependent manner through the nuclear hormone receptor
recognition site in the enhancer I region of the HBV genome. Moreover, those
mice transcribe very little precore RNA and secrete extremely low levels of HBe
antigen compared with the wild-type HBV transgenic mice [26].
Analysis of HBV transcription and replication in nonhepatoma cells indicates
that PPARα/RXRα heterodimers support
higher levels of pregenomic RNA transcription from the wild-type than from the
variant nucleocapsid promoter, producing higher levels of wild-type than of
variant replication intermediates [27]. These
observations indicate that the replication of wild-type and variant viruses can
be differentially regulated by the liver-specific transcription factors that bind
to the proximal nuclear hormone receptor binding site of the nucleocapsid
promoter.More recent
data concern approaches to counteract this nuclear receptor-induced HBV
transcription and replication. Oropeza et al. showed that the nuclear
receptor short heterodimer partner (SHP) inhibits the nuclear receptor-mediated
HBV replication [28]. HBV replication
that is dependent on HNF4 seemed considerably more sensitive to SHP-mediated
inhibition than PPARα/RXRα-directed viral
biosynthesis. A nonnucleosidic compound, Helioxanthin (HE-145), was found to
suppress HBV gene expression and replication in HCC cells. It was found that
HE-145 selectively suppresses surface antigen promoter II (SPII) and core
promoter (CP) but has no effect on surface antigen promoter I (SPI) or promoter
for X gene (XP). Tseng et al. showed that HE-145 acted by decreasing the
DNA-binding activity of PPAR to specific cis element of HBV promoter for core
antigen [29]. Taken together, all these
data provide an interesting rationale for modulating the PPARα/RXRα heterodimer to control
the HBV infection.


## 5. HBV Modulates PPARγ Expression: Role in Steatosis

Until
now, two studies described a role of HBx protein on the regulation of PPARγ
expression and
activation and one of which suggests a role in steatosis.In the
below paragraph, we have described the role of PPAR on HBV transcription and
replication. Conversely, the HBx protein of HBV modulated PPARγ by protein-protein interaction. Indeed, ligand
activation of PPARγ has been reported to
induce growth inhibition and apoptosis in various cancers including HCC. Choi
and coll demonstrated that HBx counteracted growth inhibition caused by PPARγ ligand in HBx-associated HCC cells [30]. They found that HBx bound to DNA binding domain
of PPARγ and this interaction blocked
nuclear localization and binding to PPRE. HBx significantly suppressed the PPARγ mediated transactivation.More recent report described
a positive effect of HBx protein on PPARγ expression and transcriptional activity [31]. Some observations suggest that chronic
HBV infection is associated with hepatic steatosis, which is a common
histological feature of chronic infection with hepatitis C virus [32]. Even if other report described lower
frequency of steatosis in hepatitis B [33, 34],
evidence indicates that hepatic steatosis is a more vulnerable factor
that leads to liver inflammation, fibrosis, and cancer. Based on these
observations, Kim et al. demonstrated that overexpression of HBx induced
hepatic lipid accumulation [31]. This
phenomenon was accompanied by increased expression of sterol regulatory element
binding protein 1 (SREBP1) and PPARγ. The authors proposed that HBx could participate to hepatic
steatosis during HBV infection by regulating SREBP1 and PPARγ expression and
activation (Figure 3) but a direct proof remains to be obtained.Figure 3
HBx
protein could influence liver steatosis through SREBP1 and PPARγ. Protein X of the HBV
(HBx) increases the kinase AKT phosphorylation and inhibits PTEN expression
that leads to increased expression and activation of SREBP1 in the liver. In
another way HBx enhances C/EBPα that in turn induces PPARγ expression and activation.
Both pathways lead to an increased expression of adipogenic and lipogenic
genes, which finally could contribute to liver steatosis.


## 6. Conclusion

HBV infection is a
global health problem and recent data indicate that the HBV DNA level is a strong
risk predictor of liver cirrhosis and HCC. Studies indicate the presence
of hormone response elements in the promoters of HBV genome. Peroxisome
proliferators may enhance HBV viral transcription directly in a PPARα-dependent manner.
Conversely, HBx protein of HBV is able to induce the gene expression and
transcriptional activity of SREBP1 and PPARγ, thereby causing hepatic
lipid accumulation by increasing adipogenic and lipogenic gene expression. This
regulation loop between PPAR and HBV may contribute to the progression of
HBV-induced pathogenesis and the development  of PPAR antagonist could represent
a new therapeutic strategy.



---

*Source: 145124-2009-04-09.xml*