Hepatitis B Surface Proteins Diagrammed below are the predicted folding patterns of the various hepatitis B virus (HBV) surface proteins. Within the HBV genome, the region encoding the HBV surface proteins contains three in-frame start sites which share a common termination codon. Because of this, the various HBV surface proteins are all related to each other by a shared region known as the S-domain. Small HBsAg | Subtypes | Middle HBsAg | Large HBsAg Small Hepatitis B Surface Antigen (HBsAg or SHBsAg) This protein is the smallest of the hepatitis B surface proteins, containing solely the S-domain. Historically, it also has been referred to as the Australia antigen (Au antigen). It is highly hydrophobic, containing four-transmembrane spanning regions. The HBsAg contains a high number of cysteines,14 all together, each of which is cross-linked to one another. It also may be glycosylated at Asp146. The two forms of this protein are commonly seen on gels run on HBsAg particles purified from carrier serum. This protein is the prime constituent of all hepatitis B particle forms. As such, this protein appears to be manufactured by the virus in high quantities. It also contains a highly antigenic epitope. Analysis of this epitope allows for the subtyping of HBV carriers. Computer modelling implicates helices 3 and 4 as transmembrane spanning regions inserted postranslationally into the ER membrane. These two helices are thought to be the site of multimerization. This has been supported by observations which show truncated versions of the HBsAg missing in these helices. These helices are unable to form particles and remain in the ER. Infected cells in the early stages produce this protein in the greatest quantities. The titre of the resulting non-infectious HBsAg particles found in a carrier's serum can be as high as 200ug/mL. Expression of the HBsAg appears to be inducible by stress in the endoplasmic reticulum typically due to the presence of high amount of LHBsAg. Despite the high antigenicity and prevalence of these particles, the immune system appears basically oblivious to their presence. Studies, on T-lymphocyte-derived soluble factors in the maintenance of HBV infection, have shown that HBsAg of T-cell origin appears to suppress HBsAg-antibody production in other T-cells. Suppression is antigen specific for HBsAg. Immune suppression of antibodies against the various components of HBV favours persistence found in the chronic HBV carrier state. The S promoter lies within the preS region. Mutations in this region result in lowered HBsAg production. Reduced production of the HBsAg appears to lead to intracellular retention of the virus. It also causes viral misassembly. Subtypes Subtypes of SHBsAg were originally defined by antibody recognition. Antigenic domains present on all known HBs isolates were classified as determinant a. The four other major subtypes are d or y and w or r. These two sets are paired and the members of each pair are mutually exclusive. Determinant d has a lysine at residue 122 while y has an arginine. Similarly, determinant w has a lysine at residue 160 while r has an arginine. Recently, other determinants have been found which contain antigenic epitopes unrecognizable by antibodies against the above-mentioned subtypes. Because some antibodies are sub-type specific, it leads to the question: Does vaccination using HBs particles immunize a person against all HBV strains? The answer is "Yes", so far. However, in the more recent years, escape mutants have been found, showing a need for an improved vaccine or treatment. Middle Hepatitis B Surface Antigen (MHBsAg) This intermediate or middle-sized HBV surface protein contains an additional 55 amino-acid domain known as Pre-S2. This domain is hydrophilic and appears to reside extracellularly. The Pre-S2 domain also contains an additional glycosylation site at Asp4. It appears that this site is always glycosylated, but the glycosylation site on the S-domain is only glycosylated at times, resulting in either a fully or partially glycosylated form of this protein. Some have proposed that this protein is involved in HBV attachment and entry into the liver. However, in a study involving genetic analysis of HBV in patients with fulminant hepatitis, the pre-S2 start codon carried a double mutation, preventing expression of the corresponding protein. As such, it appears pre-S2 is not required for HBV infectivity nor viral particle morphogenesis. This likely excludes the middle HBsAg from being the HBV binding protein, though it may contribute to viral attachment as a secondary mechanism. Large Hepatitis B Surface Antigen (LHBsAg) This protein is the largest of the HBV surface proteins, containing the Pre-S1 domain as well as the Pre-S2 and S domains. The Pre-S1 domain's sequence appears to be highly variable among infected patients, suggesting that this may be the HBV protein involved in liver attachment. The Pre-S1 domain contains no additional glycosylation sites, but contains a myristylation signal at its N-terminus, anchoring the N-terminus to the membrane. There are two proposed different folding patterns for this protein: one found on the cell surface and in mature virions, the other found on the surface of the endoplasmic reticulum (ER). The predicted folding patterns are based on protease and antibody studies. In the related duck hepatitis B virus, the LHBsAg has also been shown to have dual topology. It appears that both the Pre-S1 and Pre-S2 domains remain cytoplasmic when the LHBsAg is in the ER. As such, the Pre-S2 domain remains unglycosylated whereas the Pre-S1 domains becomes myristylated. When, where, and how the PreS domains are translocated across the membrane are still under debate. However, a model has been proposed for the duck hepatitis B viral model. The model predicts that the PreS domains are translocated through an aqueous pore in the virus envelope. This pore is likely formed by the oligomerization of the transmembrane spanning regions in the S-domain Overexpression of the LHBsAg alone results in ER retention of the protein. However, it was first suggested that ER retention was due to a cytosolic factor binding the LHBsAg as a transmembrane protein. However, more recent evidence shows formation of intracellular particles of LHBsAg in the lumen of the ER. Retention appears to be due to the binding of LHBsAg to calnexin. This protein is believed by most to be the one responsible for mediating viral attachment onto its host cells. However, the receptor for HBV has not been isolated.

Bancroft, W.H., Mundon, F.K. and Russell, P.K. 1972. Detection of Additional Antigenic Determinants of Hepatitis B Antigen. J Immuno; 109: 985-992.

Bock, C.T., Tillmann, H.L., Maschek, H.J., Manns, M.P., and Trautwein, C. A PreS Mutation Isolated from a Patient with Chronic Hepatitis B Infection Leads to Virus Retention and Misassembly. Gastroenterology; 113(6): 1976-1982.

Bruss, V. and Ganem, D. 1991. Mutational Analysis of Hepatitis B Surface Antigen Particle Assembly and Secretion. J Virol; 65: 3813-3820.

Bruss, V. and Vieluf, K. 1995. Functions of the Internal pre-S Domain of the Large Surface Protein in Hepatitis B Virus Particle Morphogenesis. J Virol; 69: 6652-6657.

Budkowska, A., Bedossa, P., Groh, F., Louise, A. and Pillot, J. 1995. Fibronectin of Human Liver Sinusoids Binds Hepatitis B Virus: Identification by an Anti-Idiotypic Antibody Bearing the Internal Image of the Pre-S2 Domain. J Virol; 69: 840-848.

Cheng, K.C., Smith, L. and Moss, B. 1986. Hepatitis B Virus Large Surface Protein Is Not Secreted But Is Immunogenic When Selectively Expressed by Recombinant Vaccinia Virus. J Virol; 60: 337-344.

Eble, B.E., Lingappa, V.R. and Ganem, D. 1986. Hepatitis B Surface Antigen: An Unusual Secreted Protein Initially Synthesized as a Transmembrane Polypeptide. Mol and Cell Biology; 6: 1454-1463.

Franco, A., Paroli, M., Testa, U., Benvenuto, R., Peschle, C., Balsano, F. and Barnaba, V. 1992. Tranferrin Receptor Mediates Uptake and Presentation of Hepatitis B Envelope Antigen by T Lymphocytes. J Exper Med; 175: 1195-1205.

Guo, J-T and Pugh, J.C. 1996. Topology of the Large Envelope Protein of Duck Hepatitis B Virus Suggests a Mechanism for Membrane Translocation During Particle Morphogenesis. J Virol; 71: 1107-1114.

Heermann, K.H., Goldmann, U., Schwartz, W. Seyffarth, T., Baumgarten, H. and Gerlich, W.H. 1984. Large Surface Proteins of Hepatitis B Virus Containing the Pre-S Sequence. J Virol; 52: 396-402.

Heermann, K.H. and Gerlich, W.S. 1991. Surface Proteins of Hepatitis B Viruses. In: McLachlan, A. (ed.) Molecular Biology of the Hepatitis B Viruses. CRC Press, Boca Raton, pp. 109-144.

Le Bouvier, G.L., McCollum, R.W., Hierholzer, W.J.J., Irwin, G.R., Krugman, S. and Giles, J.P. 1972. Subtypes of Australia Antigen and Hepatitis B Virus. J American Medical Association; 222: 928-930.

Mehdi, H., Kaplan, M.J., Anlar, F.Y., Yang, X., Bayer, R., Sutherland, K. and Peeples, M.E. 1994. Hepatitis B Virus Surface Antigen Binds to Apolipoprotein H. J Virol; 68: 2415-2424.

Mehdi, H., Yang, X. and Peeples, M.E. 1996. An Altered Form of Apolipoprotein H Binds Hepatitis B Virus Surface Antigen Most Efficiently. Virology; 217: 58-66.

Mehta, A., Lu, X., Block, T.M., Blumberg, B.S. and Dwek, R. 1997. Hepatitis B Virus Envelope Glycoproteins Vary Drastically in their Sensitivity to Glycan Processing: Evidence that Alteration of a Single N-Linked Glycosylation Site Can Regulate HBV Secretion. Proc Natl Acad Sci USA; 94: 1822-1827.

Melegari, M., Scaglioni, P.P. and Wands, J.R. 1997. The Small Envelope Protein Is Required for Secretion of a Naturally Occuring Hepatitis B Virus Mutant with Pre-S1 Deleted. J Virol; 71: 5449-5454.

Nagaraju, K., Naik, S.R. and Naik, S. 1997. Functional Implications of Hepatitis B Surface Antigen (HBsAg) in the T Cells of Chronic HBV Carriers. J Viral Hepat; 4(4): 221-230.

Neurath, A.R., Strick, N. and Sproul, P. 1992. Search for Hepatitis B Virus Cell Receptors Reveals Binding Sites for Interleukin 6 on the Virus Envelope Protein. J Experimental Medicine; 175: 461-469.

Norder, H., Hammas, B., Lofdahl, S., Courouse, A.M. and Magnius, L.O. 1992. Comparison of the Amino Acid Sequences of Nine Different Serotypes of Hepatitis B Surface Antigen and Genomic Classification of the Corresponding Hepatitis B Virus Strains. J Gen Virol; 73: 1201-1208.

Okamoto, H., Tsuda, F., Sakugawa, H., Sastrosoewignjo, R.I., Imai, M., Miyakawa, Y. and Mayumi, M. 1988. Typing Hepatitis B Virus by Homology in Nucleotide Sequence: Comparison of Surface Antigen Subtypes. J Gen Virol; 69: 2575-2583.

Persing, D.H., Varmus, H.E. and Ganem, D. 1987. The preS1 Protein of Hepatitis B Virus Is Acylated at Its Amino Terminus with Myristic Acid. J Virol; 61: 1672-1677.

Peterson, D.L. 1981. Isolation and Characterization of the Major Protein and Glycoprotein of Hepatitis B Surface Antigen. J Biological Chem; 256: 6975-6983.

Peterson, D.L., Paul, D.A., Lam, J., Tribby, I.I. and Achord, D.T. 1984. Antigenic Structure of Hepatitis B Surface Antigen: Identification of the "d" Subtype Determinant by Chemical Modification and Use of Monoclonal Antibodies. J Immuno; 132: 920-927.

Poisson, F., Severac, A., Hourious, C., Goudeau, A. and Roingeard, P. 1997. Both Pre-S1 and S Domains of Hepatitis B Virus Envelope Proteins Interact with the Core Particle. Virology; 228: 115-120.

Pollicino, T, Zanetti, A.R., Cacciola, I, Petit, M.A. Smedile, A., Campo, S., Sagliocca, L., Pasquiali, M., Tanzi, E., Longo, G. and Raimondo, G. 1997. Pre-S2 Defective Hepatitis B Virus Infection in Patients with Fulminant Hepatitis. Hepatology; 26(2): 495-499.

Pontisso, P, Ruvoletto, M.G., Gerlich, W.H., Heermann, K-H, Bardini, R. and Alberti, A. 1989. Identification of an Attachment Site for Human Liver Plasma Membranes on Hepatitis B Virus Particles. Virology; 173: 522-530.

Stibbe, W. and Gerlich, W.H. 1983. Structural Relationships Between Minor and Major Proteins of Hepatitis B Surface Antigen. J Virol; 46: 626-629.

Stirk, H.J., Thorton, J.M. and Howard, C.R. 1992. A Topological Model for Hepatitis B Surface Antigen. Intervirology; 33: 148-158.

Swameye, I. and Schaller, H. 1997. Dual Topology of the Large Envelope Protein of Duck Hepatitis B Virus: Determinants Preventing Pre-S Translocation and Glycosylation. J Virol; 71: 9434-9441.

Werr, M. and Prange, R. 1997. Role for Calnexin and N-Linked Glycosylation in the Assembly and Secretion of Hepatitis B Virus Middle Envelope Protein Particles. J Virol; 72: 778-782.

Xu, Z., Jensen, G. and Yen, T.S. 1997. Activation of Hepatitis B Virus S Promoter by the Viral Large Surface Protein via Induction of Stress in the Endoplasmic Reticulum. J Virol; 71(10): 7387-7392.

Xu, Z., Bruss, V. and Yen, T.S.B. 1997. Formation of Intracellular Particles by Hepatitis B Virus Large Surface Protein. J Virol; 71: 5487-5494.