The Hepatitis B Virus X Protein Modulates Hepatocyte Proliferation Pathways To Stimulate Viral Replication

Abstract

Worldwide, there are over 350 million people who are chronically infected with the human hepatitis B virus (HBV); chronic HBV infections are associated with the development of hepatocellular carcinoma (HCC). The results of various studies suggest that the HBV X protein (HBx) has a role in the development of HBVassociated HCC. HBx can regulate numerous cellular signal transduction pathways, including those that modulate cell proliferation. Many previous studies that analyzed the impact of HBx on cell proliferation pathways were conducted using established or immortalized cell lines, and when HBx was expressed in the absence of HBV replication, and the precise effect of HBx on these pathways has often differed depending on experimental conditions. We have studied the effect of HBx on cell proliferation in cultured primary rat hepatocytes, a biologically relevant system. We demonstrate that HBx, both by itself and in the context of HBV replication, affected the levels and activities of various cell cycle-regulatory proteins to induce normally quiescent hepatocytes to enter the G 1 phase of the cell cycle but not to proceed to S phase. We linked HBx regulation of cell proliferation to cytosolic calcium signaling and HBx stimulation of HBV replication. Cumulatively, our studies suggest that HBx induces normally quiescent hepatocytes to enter the G 1 phase of the cell cycle and that this calcium-dependent HBx activity is required for HBV replication. These studies identify an essential function of HBx during HBV replication and a mechanism that may connect HBV infections to the development of HCC.Viruses often encode proteins that modulate normal cellular processes to create an environment that facilitates viral replication. A potential consequence of the activities of viral proteins that alter normal cellular signaling pathways is that they can stimulate cell transformation and cancer progression. Included among confirmed oncogenic viruses is the hepatitis B virus (HBV); HBV is the prototype member of the Hepadnaviridae, a family of hepatotropic viruses (57). The HBV genome is a partially double-stranded, circular DNA that contains four overlapping open reading frames (ORFs) that encode the viral envelope, capsid, reverse transcriptase, and X (HBx) proteins (57). Worldwide, there are over 350 million people who are chronically infected with HBV; approximately 25% of these chronically HBV-infected individuals develop hepatocellular carcinoma (HCC) (3, 57). While there is evidence of a strong link between chronic HBV infections and HCC development, exactly how chronic HBV infections can lead to the development of HCC has not been defined. The results of numerous studies suggest that the development of HBV-associated HCC involves a combination of the activities of HBV proteins that modulate cell signal transduction pathways, such as HBx, and the consequences of the host immune response to infection, including immune-mediated destruction of HBV-infected hepatocytes that induces repeated liver regeneration cycles.HBx is a...

Granzyme H of Cytotoxic Lymphocytes Is Required for Clearance of the Hepatitis B Virus through Cleavage of the Hepatitis B Virus X Protein

“…To determine whether granule exocytosis is involved in the clearance of HBV, we incubated HepG2.2.15 cells with LAK cells at the E:T ratio of 30. HepG2.2.15 cell line is a human hepatoblastoma cell line that contains an integrated HBV genome and can produce infectious HBV particles (23). Total genomic DNAs were isolated, and viral titers were measured by Southern blotting and real-time PCR at 0, 3, and 6 h after incubation.…”

The Hepatitis B Virus (HBV) HBx Protein Activates AKT To Simultaneously Regulate HBV Replication and Hepatocyte Survival

“…While studies conducted with various cell lines have provided important insights concerning the effect of HBx expression on cellular signal transduction pathways, the influence of HBx expression on cellular signal transduction pathways in normal hepatocytes, and in the context of an HBV infection, remains poorly understood. Recent studies with ex vivo-cultured primary hepatocyte model systems and in vivo models have begun to identify HBx effects on normal hepatocytes and how these HBx activities influence HBV replication and affect hepatocyte physiology (8,11,14,23,26,27).…”

“…HBx stimulates HBV replication in multiple experimental systems, including cultured primary rat and human hepatocytes, some liver cell lines, livers of normal mice, and chimeric mice with humanized livers (14)(15)(16)(17)(18)(19). HBx is also thought to play an important role in the development of HBV-associated HCC due to the regulatory effects of HBx on cellular signaling pathways, DNA damage repair mechanisms, the cell cycle, and apoptosis (14,(20)(21)(22)(23)(24). Some HBx activities, such as its role in regulating apoptosis, appear to be context dependent and have varied depending upon the model system that was used to assess this HBx activity (8,11,25).…”

“…transcriptase PCR (RT-PCR) was performed, as previously described, to assess the expression of known hepatocyte differentiation markers and to demonstrate that hepatocytes remain differentiated throughout the time course of our experiments (14,23,26).…”

Hepatitis B Virus X Protein: A Key Regulator of the Virus Life Cycle

“…For example, using primary rat hepatocytes, it was recently demonstrated that HBx induces normally quiescent hepatocytes to enter the G 1 phase of the cell cycle and that this calcium-dependent HBx activity is required for HBV replication (Gearhart & Bouchard, 2010). While this effect of HBx on cell cycle progression can probably lead to carcinogenesis and thus become deleterious for the host, it is believed that it might be important for the virus to induce expansion of available deoxynucleoside triphosphate pools within the cells which it needs for replication (Bouchard et al, 2003).…”