Assembly of viral particles in Xenopus oocytes: pre-surface-antigens regulate secretion of the hepatitis B viral surface envelope particle.

Abstract

Infection with hepatitis B virus (HBV) is associated with the production of a viral envelope particle that contains membrane lipids, surface antigen (S), and two presurface-antigens (pre-S) comprised of the entire S moiety with approximately 55 (pre-S2) and 174 (pre-Sl) additional NH2-terminal amino acids. We show here that Xenopus oocytes hUjected with synthetic S mRNA assemble and secrete characteristic 22-nm viral envelope particles. In contrast, pre-Sl and pre-S2 antigens are synthesized but not secreted. By coinjecting mRNAs, we found that synthesis of high levels of pre-S proteins specifically inhibits S antigen secretion. On the other hand, high levels of S synthesis can drive the secretion of small amounts of either pre-S antigen. These observations are consistent with a model for viral envelope assembly in which both S and pre-S proteins are incorporated into a multimeric particle, presumably via interactions between the S protein domains, while the pre-S amino-terminal moieties regulate the secretion of this structure. Our results indicate that Xenopus oocytes will provide a powerful system for studying the morphogenesis of simple structures of viral or cellular origin.Hepatitis B virus (HBV), on infection, produces two types of particles (1): the 42-nm virus, with a core containing HBV DNA and viral proteins surrounded by an envelope containing three related surface antigen (S) molecules, and 22-nm particles and filaments comprised of the elements of the viral envelope. The latter particles, either isolated from the sera of HBV carriers or produced in heterologous cells by recombinant DNA methods, form the basis of an effective HBV vaccine (2, 3). Whereas knowledge of the structure, transcription, and replication cycle of HBV has increased dramatically in recent years (4, 5), the details ofthe assembly and secretion of these viral particles are less clear. Recent studies (6-8) suggest that S particles bud into the lumen of the endoplasmic reticulum prior to secretion. Although the different classes of viral particles contain different ratios of pre-surface-antigens (pre-S) to S (9), the mechanism by which this is achieved and the role of pre-S in the assembly and secretion of viral particles is obscure.To dissect the complex problem of assembly, we have developed an approach involving the microinjection of individual and combinations of synthetic viral mRNAs into Xenopus oocytes, which allows the analysis of the contribution of specific viral proteins to viral structures. These studies have revealed that the pre-S antigens can regulate the secretion of the S particle.MATERIALS AND METHODS Individual stage 5 or 6 oocytes were isolated manually, rinsed well, and stored overnight at 17-19'C in MBSH medium. Healthy oocytes were injected in the vegetal pole with 20-30 nl of the appropriate synthetic mRNA (0.4 mg/ml in distilled H20). For studies on the translation of synthetic mRNAs in oocytes, see refs. 12 and 13. Batches of 20 healthy oocytes were transferred next day to 0.5 ml of MBSH medium con...

Novel N-terminal amino acid sequence required for retention of a hepatitis B virus glycoprotein in the endoplasmic reticulum.

“…First, despite the presence of the entire S domain at its C terminus, when the preSl protein is expressed alone in cultured cells (or Xenopus oocytes), it is not secreted from the cell but is retained in the ER as an integral membrane protein (3, 28, 37; K. Simon, V. Lingappa, and D. Ganem, unpublished data). Second, when the preSl protein is overexpressed in the presence of the S protein, the export of the latter is profoundly and specifically inhibited (3,4,28,37). These results imply that some feature of the preSl-specific domain is capable of overriding the secretory information in S and causing the ER retention of the polypeptide.…”

Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes.

“…In eucaryotes, on the other hand, initiation at a downstream ATG codon is completely suppressed when it is overlapped by an upstream cistron (8,35,40,54,65). (This is true only under conditions that preclude leaky scanning, and at present it can be shown most reliably in vivo.…”

Viruses, Immunity, and Cancer: Lessons from Hepatitis B

“…The polymerase protein contains a viral RNA packaging signal and performs multiple enzymatic functions including reverse transcriptase, DNA polymerase, and RNase H activity that are essential for viral replication. 15 The core protein, known as hepatitis B core antigen (HBcAg), rapidly forms homodimers that self-assemble into capsid particles 16 in the cytoplasm and the nucleus of the hepatocyte. The intranuclear capsid particles appear to be empty, and their role in the viral life cycle is not understood.…”