Assembly of hepatitis B surface antigen

Abstract

Hepatitis B virus infected cells produce, in addition to v1nons, non-infectous particles composed of the major viral coat protein, hepatitis B surface antigen (HBsAg), and lipid. These particles are stabilized by extensive disulphide crosslinking. Cellular expression of HBsAg leads to its secretion as particles indistinguishable from those found in sera of infected patients. The assembly of these particles was classically believed to occur entirely in the endoplasmic reticulum (ER). This was paradoxical since the ER contains a high concentration of protein disulphide isomerase (PDI) which should resolve such extensive linkages. HBsAg biosynthesis was studied using a stably transfected cell line. Kinetic studies showed that disulphide linked dimers begin to form during or immediately after the polypeptide synthesis. Their concentration increases rapidly until a peak at one hour after the initial synthesis, and then decreases monotonically as the dimers are crosslinked to higher oligomers. Several immunocytochemical techniques in combination with biochemical studies demonstrate that HBsAg is sorted rapidly after its dimerization in the ER to a morphologically and functionally distinct post-ER, pre-Golgi compartment in which the assembly is completed. This compartment excludes lumenal ER proteins and Golgi proteins. It was shown to be discontinuous with the ER. Two established intermediate compartment proteins, rab2 and the 72 kD KDEL-binding protein, colocalize to this compartment. The exit from the ER was shown to be a requirement for the assembly, and the incubation of purified HBsAg particles with recombinant PDI led to the disassembly of highly crosslinked mature HBsAg multimers to dimers. This indicates that the PDI exclusion may be a condition for particle assembly. Stereological analysis of electron microscopy data provided the first quantitation of an intermediate compartment, demonstrating that this compartment is a major and dynamic element of the secretory pathway.