Replication of hepatitis B virus in differentiated adult rat hepatocytes transfected with cloned viral DNA

119

The hepatitis B virus (HBV) X protein (HBx) is a multifunctional protein that regulates numerous cellular signal transduction pathways, including those that modulate apoptosis. However, different HBx-dependent effects on apoptosis have been reported; these differences are likely the consequence of the exact conditions and cell types used in a study. Many of the previously reported studies that analyzed HBx regulation of apoptosis were conducted in immortalized or transformed cells, and the alterations that have transformed or immortalized these cells likely impact apoptotic pathways. We examined the effects of HBx on apoptotic pathways in cultured primary rat hepatocytes, a biologically relevant system that mimics normal hepatocytes in the liver. We analyzed the effects of HBx on apoptosis both when HBx was expressed in the absence of other HBV proteins and in the context of HBV replication. HBx stimulation of NF-B inhibited the activation of apoptotic pathways in cultured primary rat hepatocytes. However, when HBx-induced activation of NF-B was blocked, HBx stimulated apoptosis; blocking the activity of the mitochondrial permeability transition pore inhibited HBx activation of apoptosis. These results suggest that HBx can be either proapoptotic or antiapoptotic in hepatocytes, depending on the status of NF-B, and confirm previous studies that link some HBx activities to modulation of the mitochondrial permeability transition pore. Overall, our studies define apoptotic pathways that are regulated by HBx in cultured primary hepatocytes and provide potential mechanisms for the development of HBV-associated liver cancer.

“…1). These results are consistent with other published studies that have used similar isolation and culture techniques to maintain differentiated rat hepatocytes (7,22,23,29,36,69).…”

Section: Confirmation Of Hepatocyte Differentiation In Culturesupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

See 1 more Smart Citation

Hepatitis B Virus X Protein Modulates Apoptosis in Primary Rat Hepatocytes by Regulating both NF-κB and the Mitochondrial Permeability Transition Pore

Clippinger¹,

Gearhart²,

Bouchard

2009

119

“…Primary rat hepatocytes are a biologically relevant model system used to study normal hepatocyte physiology and liver diseases (7,18,21,30,52). Our studies addressed the effect of HBx on the levels and activities of cell cycle-regulatory proteins and on overall cell cycle progression and linked HBxinduced modulation of cell proliferation pathways to cytosolic calcium signaling.…”

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confidence: 99%

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

Gearhart¹,

Bouchard

2010

102

121

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...

“…Indeed, hepatocytes from refractory species are able to support viral replication only if the normal pathway of viral entry is artificially bypassed. Thus, it has been shown that primary rat hepatocytes or a rat hepatoma cell line could support HBV replication upon transient transfection of cloned HBV DNA (8,39). Complete virions are also produced in hepatocytes of HBV transgenic mice (10,17,43), while there is no evidence for reinfection by the circulating virus.…”

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confidence: 99%

A Short N-Proximal Region in the Large Envelope Protein Harbors a Determinant That Contributes to the Species Specificity of Human Hepatitis B Virus

Chouteau

Seyec

Cannie

et al. 2001

Infection by hepatitis B virus (HBV) is mainly restricted to humans. This species specificity is likely determined at the early phase of the viral life cycle. Since the envelope proteins are the first viral factors to interact with the cell, they represent attractive candidates for controlling the HBV host range. To investigate this assumption, we took advantage of the recent discovery of a second virus belonging to the primate Orthohepadnavirus genus, the woolly monkey HBV (WMHBV). A recombinant plasmid was constructed for the expression of all WMHBV envelope proteins. In additional constructs, N-terminal sequences of the WMHBV large envelope protein were substituted for their homologous HBV counterparts. All wild-type and chimeric WMHBV surface proteins were properly synthesized by transfected human hepatoma cells, and they were competent to replace the original HBV proteins for the production of complete viral particles. The resulting pseudotyped virions were evaluated for their infectious capacity on human hepatocytes in primary culture. Virions pseudotyped with wild-type WMHBV envelope proteins showed a significant loss of infectivity. By contrast, infectivity was completely restored when the first 30 residues of the large protein originated from HBV. Analysis of smaller substitutions within this domain limited the most important region to a stretch of only nine amino acids. Reciprocally, replacement of this motif by WMHBV residues in the context of the HBV L protein significantly reduced infectivity of HBV. Hence this short region of the L protein contributes to the host range of HBV.