The pre S surface proteins of hepatitis B virus: Identification, characterization, and membrane insertion

Abstract

Hepatitis B virus (HBV) is the prototype of the hepadna viruses and is the cause of a medically significant disease. The S open reading frame (ORF S) which encodes the major surface protein, HBsAg, has the capacity to encode an additional 174 amino acids (pre-S) at the amino-terminus of HBsAg. However definitive identification of pre-S:HBsAg has been difficult because of the inability to propagate HBV in vitro until recently; consequently, it was unclear if a protein encoded by the entire ORF S, pre-S:HBsAg, was expressed during infection. To identify the existence of such a polypeptide, pre-S determinants were synthesized in bacteria as fusion proteins with chloramphenicol acetyltransferase (CAT) and with {dollar}\beta{dollar}-galactosidase. Polyclonal antisera were generated to these antigens and used to identify in HBV particles HBsAg containing polypeptides having a Mr consistent with their being translation products of the entire pre-S open reading frame. Immunoprecipitation studies with Dane particles using anti-pre-S antibodies demonstrated the existence of pre-S on the surface of the virion coat, a finding which is consistent with a proposed role in recognition of host cell receptors. To achieve such a surface topology, these data predict that the nascent pre-S region of pre-S:HBsAg would be inserted into the ER membrane during its synthesis. Based on these observations and computer predictions, a transmembrane model for pre-S:HBsAg was proposed.;To test the model and determine the mechanism of membrane insertion, HBsAg and pre-S:HBsAg were synthesized in a cell-free translation system in the presence of microsomal membranes. Analysis of the translation products confirmed the assumption that HBsAg and pre-S:HBsAg were integral membrane proteins. However, protease protection studies showed that the pre-S region was translocated inefficiently. To identify protein domains involved in pre-S insertion, a series of truncated pre-S polypeptides were synthesized and their in vitro membrane insertion analyzed. Rather surprisingly, pre-S was found to possess "weak" membrane insertion signals and was able to translocate into membranes even in the absence of hydrophobic domains. Furthermore, the pre-S region was demonstrated to be capable of post-translational translocation without the requirements for on-going protein synthesis, a ribosome-membrane junction, or the signal recognition particle (SRP). The membrane insertion of pre-S:HBsAg was proposed to occur by a two-step mechanism. (Abstract shortened with permission of author.).