Hepatitis B Virus X Protein Transactivates the Inducible Nitric Oxide Synthase Promoter

Abstract

The capability of hepatitis B virus (HBV) to increase the transcription of the human hepatic inducible nitric oxide synthase (iNOS) by transactivating its promoter has been studied. We have observed by reverse-transcription polymerase chain reaction (RT-PCR) that although the mRNA for the iNOS was almost undetectable in the human hepatoblastoma cell line, HepG2, it was constitutively expressed in the 2. The hepatitis B virus (HBV), the prototype member of the Hepadnaviridae family of animal viruses, is a noncytopathic enveloped virus that causes acute and chronic liver disease and is related to the development of hepatocellular carcinoma in chronically infected patients. 1 The HBV genome is a 3.2-kb-long circular partially double-stranded DNA molecule. 2 The HBV DNA contains four overlapping open reading frames (ORFs) that encode the structural and nonstructural viral proteins. Among them, the X protein (pX) encoded by the ORFX activates the transcription of HBV and other viral and cellular promoters in vitro. 3 Furthermore, pX transactivates gene promoters containing binding motifs for nuclear factor-B (NF-B) among other transcription factors. 4 Nitric oxide (NO) is produced during the conversion of L-arginine to L-cytruline, a process that is catalyzed by NO synthase. 5 There are three different isoforms of this enzyme. 6 Two of them (cNOS) are constitutively expressed in neuronal and endothelial cells, while the third, inducible isoform (iNOS) is expressed in macrophages and hepatocytes in response to several cytokines, tumor necrosis factor ␣ (TNF-␣), interleukin-1␤ (IL-1␤), interferon gamma (IFN-␥), and endotoxins. [7][8][9] It has been reported that woodchucks chronically infected by the woodchuck hepatitis virus (another member of the Hepadnaviridae family) show an increase in NO production and endogenous formation of the DNA damaging agent, N-nitrosodimethylamine in vivo. 10,11 However, the mechanism responsible for the increase in NO production remains unclear. Recently, an increase in iNOS mRNA levels in hepatoma cells transfected with a pX expression plasmid has been reported. 12 Because the promoter of the human hepatic iNOS contains functional binding sites for NF-B, 13 it may be hypothesized that HBV pX protein can transactivate this promoter.In the present report, we have studied if HBV can transactivate the hepatic iNOS gene promoter and whether pX or other viral-encoded proteins are responsible for this transactivation. MATERIALS AND METHODSCell Lines. The Chang liver cell line, which is derived from nonmalignant human liver tissue, the human hepatoblastoma cell line, HepG2, and the 2.2.15 cell line (kindly provided by Dr. Manuel Ló pez-Cabrera, Molecular Biology Unit, Hospital de la Princesa, Madrid, Spain), which is derived from the HepG2 cell line and which is stably transfected with a dimer of the complete HBV genome, 14 were used. To check whether 2.2.15 cells produce HBV proteins, hepatitis B surface (HBsAg) and e (HBeAg) antigens were tested in culture supernatants by commercial enzyme im...

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“…In addition, NOS2 is also induced directly by the HBV and HCV. In vitro studies from Elmore et al (1997); Amaro et al (1999); Majano et al (1998) demonstrate that the HBx protein is capable of transcriptional transactivation of NOS2. An induction of the NOS2 expression has also been seen after hepatocytes are exposed to woodchuck hepatitis virus surface antigen (Liu et al, 1994).…”

Hepatitis B virus X protein: a multifunctional viral regulator

“…Furthermore, several kinase pathways were reported to be modulated by HBx, either directly through HBx-associated factors, or indirectly. 20,[48][49][50][51] Recently, several host genes have been reported to be indirectly affected by HBx, such as the upregulation of IL6, 52 the induction of nitric oxide synthetase, 53 and Fas ligand, 54 phenomena which probably occur via NF-kB activation. [52][53][54] Lara-Pezzi et al 55 reported that the tumor necrosis factor (TNF)α signaling pathway was Fig.…”

Hepatitis B virus X protein transactivates inducible nitric oxide synthase gene promoter through the proximal nuclear factor κB-binding site: Evidence that cytoplasmic location of X protein is essential for gene transactivation

“…Furthermore, 2 copies of activator protein 1 (AP-1) binding sites have also been found. 6 Although it has been suggested that HBV X protein (HBx) transactivates the iNOS gene promoter through the induction of NF-B, 7 the mechanism of this activity remains only partially understood. It is conceivable, therefore, that understanding how HBx activates the iNOS gene transcription can provide insights into the pathogenic role of the enhanced intrahepatic iNOS expression that we observed in patients with CHB.…”