HBVx could be a drug target for HBV induced hepatocellular carcinoma.

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

Hepatitis B virus (HBV) infection is the main risk factor for hepatocellular carcinoma (HCC) worldwide. Epidemiologic studies have shown that individuals who are chronic HBV carriers have a greater than 100-fold increase in the risk of developing liver cancer. The HBV genome is a partially double-stranded, circular DNA containing four overlapping genes: S/preS, C/preC, P, and X. The X gene encodes a 17-kd HBV X protein (HBx), which is a multifunctional transactivator of both viral and cellular genes. It is widely accepted that HBx plays crucial roles in the pathogenesis of HBV-induced HCC. HBV belongs to the family hepadnaviridae. It is a small, enveloped DNA virus that replicates via reverse transcription of an RNA intermediate. HBV virions, also called Dane particles, are spherical lipid-containing structures with a diameter of ~42 nm. The inner shell of the virus consists of an icosahedral capsid, which is assembled from 180 or 240 subunits of the core protein. The capsid is covered by a lipid bilayer membrane densely packed with the three envelope proteins, large (L), middle (M), and predominantly small (S) protein, and is acquired by budding into the endoplasmic reticulum. They are translated from individual start codons but share the open reading frame and the same C-terminal amino acids, called the S domain. As a consequence, the M protein shares the S and has an extra N-terminal domain called preS2, and the L protein encompasses the S and two extra domains: preS2 and preS1. Capsids contain a single copy of the HBV genome consisting of a 3.2-kb partially double-stranded relaxed circular (rc) DNA molecule. The viral polymerase serves as a protein primer and remains covalently linked to the 5’ end of the complete strand, also called viral (–) strand DNA of the rcDNA after reverse transcription. Besides virions, HBV infection leads to secretion of huge amounts of subviral particles, which consist of empty viral envelopes with filamentous or spherical shapes containing mainly S and little L protein. Subviral particles are the most abundant HBV structures released into the blood stream, are commonly defined as hepatitis B surface (HBs) antigen and are thought to facilitate virus spread and persistence in the host by adsorbing virus-neutralizing antibodies and tolerizing T cell responses. In addition to polymerase and the structural proteins, the HBV genome also encodes for two non-structural proteins, which have less well-defined functions.

Life cycle of HBV

HBV infection is restricted to hepatocytes. HBV entry into these cells is thought to be a multistep process. Virions are first trapped at the surface of the cell by heparan sulfate proteoglycans and then bind to a receptor allowing uptake into the cells via an endocytosis process. So far, this cellular receptor has not been identified. Proteolytic cleavage of the surface protein occurs within the endosomal compartment, probably resulting in a conformational change that exposes some translocation motifs at the surface of the viral particle allowing fusion of viral and cellular membranes and release of the capsid into the cytosol. The naked capsid is then directed towards the nucleus, and the HBV genome is translocated to the nucleus. In the nucleus, the rcDNA genome is converted by cellular enzymes into a covalently closed circular DNA (cccDNA), the episomal persistance form of the virus serving as transcription template. The 3.5 kb RNA species serves as pregenomic RNA (pgRNA) and as messenger RNAs for the synthesis of polymerase and core proteins as well as HBeAg. The 2.1 and 2.4 kb subgenomic RNAs encode for the three viral envelope proteins, a small 0.7 kb RNA for the HBx. The pgRNA is exported in an unspliced form, encapsidated together with the viral polymerase and used as a template for reverse transcription. The capsid spontaneously self-assembles from core dimers present in the cytoplasm due to the nucleic acid-binding domain of the core protein. Specific packaging of pgRNA into the capsid is mediated by binding of the primer region of the viral polymerase to the stem-loop in the 5’ region of pgRNA. The pgRNA is then reverse transcribed by the reverse transcriptase domain of the polymerase within the capsid in the cytoplasm of the infected cell. Upon minus and then plus strand DNA synthesis the capsid matures and can be enveloped or reimported into the nucleus to fill up a cccDNA pool.

General features and functions of HBVx

HBx is translated from a small subgenomic RNA controlled by the HBx promoter. Alternatively, HBx may be produced form a very long RNA (3.9 kb) containing all the HBV open reading frames (ORF). The ORF was originally designated X because of the lack of homology with known sequences. HBx is a protein composed of 154 amino acid residues with a molecular mass of around 17.5 kDa.

HBx selectively promotes degradation of Smc5/6 via an E3-ubiquitin ligase pathwaw by hijacking the DDB1–E3 ligase to target Smc5/6 for degradation. Smc5/6 is a complex that directly binds DNA and is required for chromosome dynamics and stability. Smc5/6 play a role in homologous recombination as well as in resolving replication-induced DNA supercoiling. In addition, a recent study demonstrated that Smc5/6 binds and topologically entraps plasmid DNA in an ATP-dependent manner. Smc5/6 binds episomes (including cccDNA) and blocks episome transcription.

REFERENCES

Michielsen P, Ho E. Viral hepatitis B and hepatocellular carcinoma. Acta Gastroenterol Belg 2011;74:4–8.

Henkler FF, Koshy R. Hepatitis B virus transcriptional activators: mechanisms and possible role in oncogenesis. J Viral Hepat 1996;3:109–21.

 Tang H, Oishi N, Kaneko S, Murakami S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci 2006;97: 977–83.

Motavaf M, Safari S, Saffari JM, Alavian SM. Hepatitis B virus-induced hepatocellular carcinoma: the role of the virus x protein. Acta Virol 2013;57:389–96

Bertoletti, A. & Gehring, A. J. (2006). The immune response during hepatitis B virus infection. J Gen Virol 87, 1439-1449.

Chen, M., Sallberg, M., Hughes, J., Jones, J., Guidotti, L. G., Chisari, F. V., Billaud, J. N. & Milich, D. R. (2005). Immune tolerance split between hepatitis B virus precore and core proteins. J Virol 79, 3016-3027.

Chen, M. T., Billaud, J. N., Sallberg, M., Guidotti, L. G., Chisari, F. V., Jones, J., Hughes, J. & Milich, D. R. (2004). A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci U S A 101, 14913-14918.

Visvanathan, K., Skinner, N. A., Thompson, A. J., Riordan, S. M., Sozzi, V., Edwards, R., Rodgers, S., Kurtovic, J., Chang, J., Lewin, S., Desmond, P. & Locarnini, S. (2007). Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology 45, 102-110.

Schulze, A., Gripon, P. & Urban, S. (2007). Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans. Hepatology 46, 1759-1768.

Stoeckl, L., Funk, A., Kopitzki, A., Brandenburg, B., Oess, S., Will, H., Sirma, H. & Hildt, E. (2006). Identification of a structural motif crucial for infectivity of hepatitis B viruses. Proc Natl Acad Sci U S A 103, 6730-6734.

Kott, N., König, A., Glebe, D. (2010). Hepatitis B virus (HBV) bypasses classical endocytic pathways to infect primary hepatocytes in vitro. Journal of Hepatology 52, S48-S49.

Leistner, C. M., Gruen-Bernhard, S. & Glebe, D. (2008). Role of glycosaminoglycans for binding and infection of hepatitis B virus. Cell Microbiol 10, 122-133.

Rabe, B., Glebe, D. & Kann, M. (2006). Lipid-mediated introduction of hepatitis B virus capsids into nonsusceptible cells allows highly efficient replication and facilitates the study of early infection events. J Virol 80, 5465-5473.

Zlotnick, A., Johnson, J. M., Wingfield, P. W., Stahl, S. J. & Endres, D. (1999). A theoretical model successfully identifies features of hepatitis B virus capsid assembly. Biochemistry 38, 14644-14652.

Hirsch, R. C., Lavine, J. E., Chang, L. J., Varmus, H. E. & Ganem, D. (1990). Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription. Nature 344, 552-555.

Junker-Niepmann, M., Bartenschlager, R. & Schaller, H. (1990). A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA. EMBO J 9, 3389-3396.

Knaus, T. & Nassal, M. (1993). The encapsidation signal on the hepatitis B virus RNA pregenome forms a stem-loop structure that is critical for its function. Nucleic Acids Res 21, 3967-3975.

Nassal, M. (1992). The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol 66, 4107-4116.

Porterfield, J. Z., Dhason, M. S., Loeb, D. D., Nassal, M., Stray, S. J. & Zlotnick, A. (2010). Full-length hepatitis B virus core protein packages viral and heterologous RNA with similarly high levels of cooperativity. J Virol 84, 7174-7184.

Doitsh, G. & Shaul, Y. (2003). A long HBV transcript encoding pX is inefficiently exported from the nucleus. Virology 309, 339-349.

Guo, W. T., Wang, J., Tam, G., Yen, T. S. & Ou, J. S. (1991). Leaky transcription termination produces larger and smaller than genome size hepatitis B virus X gene transcripts. Virology 181, 630-636.