Synthesis of hepatitis B surface antigen in mammalian cells: expression of the entire gene and the coding region

Laub, Orgad; Rall, Leslie B.; Truett, Martha; Shaul, Yosef; Standring, David N.; Valenzuela, Pablo; Rutter, William J.

doi:10.1128/jvi.48.1.271-280.1983

Cited by 108 publications

(43 citation statements)

References 32 publications

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“…7 B). We and others have shown that HBsAg secreted from SV24 and other transfected cells is particulate, as judged by EM and CsCI: centrifugation (Huovila, A.-P. J., and S. D. Fuller, manuscript in preparation; Dubois et al, 1980;Moriarty et al, 1981;Liu et al, 1982;Laub et al, 1983). This is consistent with the fact that none of the secreted HBsAg entered the gel without reduction (data not shown), indicating all of it was crosslinked in the particles.…”

Section: Time Course Of Particle Formation and Secretionsupporting

confidence: 80%

Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment.

Huovila

Eder

Fuller

1992

The Journal of Cell Biology

261

221

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AbstracL Expression of hepatitis B surface antigen (HBsAg), the major envelope protein of the virus, in the absence of other viral proteins leads to its secretion as oligomers in the form of disk-like or tubular lipoprotein particles. The observation that these lipoprotein particles are heavily disulphide crosslinked is paradoxical since HBsAg assembly is classically believed to occur in the ER, and hence in the presence of high levels of protein disulphide isomerase (PDI) which should resolve these higher intermolecular crosslinks. Indeed, incubation of mature, highly disulphide crosslinked HBsAg with recombinant PDI causes the disassembly of HBsAg to dimers. We have used antibodies against resident ER proteins in double immunofluorescence studies to study the stages of the conversion of the HBsAg from individual protein subunits to the secreted, crosslinked, oligomer. We show that HBsAg is rapidly sorted to a post-ER, preGolgi compartment which excludes PDI and other major soluble resident ER proteins although it overlaps with the distribution of rab2, an established marker of an intermediate compartment. Kinetic studies showed that disulphide-linked HBsAg dimers began to form during a short (2 rain) pulse, increased in concentration to peak at 60 rain, and then decreased as the dimers were crosslinked to form higher oligomers. These higher oligomers are the latest identifiable intracellular form of HBsAg before its secretion (h/2 = 2 h). Brefeldin A treatment does not alter the localization of HBsAg in this PDI excluding compartment, however, it blocks the formation of new oligomers causing the accumulation of dimeric HBsAg. Hence this oligomerization must occur in a pre-Golgi compartment. These data support a model in which rapid dimer formation, catalyzed by PDI, occurs in the ER, and is followed by transport of dimers to a pre-Golgi compartment where the absence of PDI and a different lumenal environment allow the assembly process to be completed.NVELOPED animal viruses have been major tools in the characterization of the secretory pathway and in advancing our understanding of the mechanisms of protein sorting. Frequently, particular features of the pathways of protein traffic are exploited in the maturation pathways of different viruses so that a study of virus assembly leads to unexpected rewards in terms of an understanding of intracellular transport. This paper presents a study of the assembly of the lipoprotein particle formed by the transmembrane glycoprotein of hepatitis B virus. This particle has the unusual feature of being stabilized by extensive intermolecutax crosslinks and here we show that this reflects its assembly in an intermediate compartment between the ER and the Golgi.Hepatitis B virus (HBV) ~ is a small (440/~) DNA virus which is the type virus of the hepadnaviridae (hepatotropic DNA viruses). HBV consists of a nucleoprotein core containing a relatively small DNA genome (3,200 bp) surrounded by a protein capsid composed of the core protein, hepatitis B core antigen. The nucleocapsid i...

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Section: Time Course Of Particle Formation and Secretionsupporting

confidence: 80%

Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment.

Huovila

Eder

Fuller

1992

The Journal of Cell Biology

261

221

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“…A similar proposal has also been made in the case of the alphaviruses based on the properties of capsid mutants (10), but alphaviruses, in contrast to flaviviruses, bud at the plasma membrane and apparently do not make subviral particles (11). However, secreted subviral particles produced by expression of envelope proteins alone have been reported for hepatitis B virus (27) and the coronavirus mouse hepatitis virus (45), but also in these cases, the exact role of the envelope proteins in the budding mechanism is still unknown.…”

Section: Discussionsupporting

confidence: 56%

Two Distinct Size Classes of Immature and Mature Subviral Particles from Tick-Borne Encephalitis Virus

Allison

Tao

O’Riordain

et al. 2003

J Virol

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Flaviviruses assemble in the endoplasmic reticulum by a mechanism that appears to be driven by lateral interactions between heterodimers of the envelope glycoproteins E and prM. Immature intracellular virus particles are then transported through the secretory pathway and converted to their mature form by cleavage of the prM protein by the cellular protease furin. Earlier studies showed that when the prM and E proteins of tick-borne encephalitis virus are expressed together in mammalian cells, they assemble into membranecontaining, icosahedrally symmetrical recombinant subviral particles (RSPs), which are smaller than whole virions but retain functional properties and undergo cleavage maturation, yielding a mature form in which the E proteins are arranged in a regular T ‫؍‬ 1 icosahedral lattice. In this study, we generated immature subviral particles by mutation of the furin recognition site in prM. The mutation resulted in the secretion of two distinct size classes of particles that could be separated by sucrose gradient centrifugation. Electron microscopy showed that the smaller particles were approximately the same size as the previously described mature RSPs, whereas the larger particles were approximately the same size as the virus. Particles of the larger size class were also detected with a wild-type construct that allowed prM cleavage, although in this case the smaller size class was far more prevalent. Subtle differences in endoglycosidase sensitivity patterns suggested that, in contrast to the small particles, the E glycoproteins in the large subviral particles and whole virions might be in nonequivalent structural environments during intracellular transport, with a portion of them inaccessible to cellular glycan processing enzymes. These proteins thus appear to have the intrinsic ability to form alternative assembly products that could provide important clues about the role of lateral envelope protein interactions in flavivirus assembly.Flaviviruses are small enveloped viruses that are assembled intracellularly, apparently by budding into the endoplasmic reticulum (ER) (28). The entire virion, consisting of a nucleocapsid, lipid membrane, and two envelope glycoproteins, prM and E, is then transported through the secretory pathway (32) and, after undergoing posttranslational modifications that include the cleavage of the prM protein, is exported from the cell by exocytosis. Newly synthesized prM and E proteins rapidly associate to form heterodimers (6, 30, 46), which then further associate to organize the envelope into a regular icosahedral structure (8,25,48). The idea that lateral envelope protein interactions play a dominant role in assembly is supported by the observations that ordered membrane-containing subviral particles can be obtained by coexpressing prM and E in the absence of other viral components (4, 9, 19-23, 35, 36, 41) and that subviral particles containing only the envelope proteins are also secreted as a by-product of natural infections (39).Shortly before being released from the cel...

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“…The results of this experiment show that >95% of the HBsAg synthesized is secreted within a 10 h time period, and that the half-time for secretion is '~3 h. Both p24s and gp27s are released into the medium with similar kinetics. In previous studies, we (18) and others (4,11,13,14) have shown that the material secreted from such transformed cell lines is particulate, as judged by electron microscopy and by equilibrium density centrifugation in CsC1.…”

Section: Resultsmentioning

confidence: 79%

Secreted hepatitis B surface antigen polypeptides are derived from a transmembrane precursor.

Simon

Lingappa

Ganem

1988

The Journal of Cell Biology

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Abstract. Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also independently secreted from infected cells as a lipoprotein particle. Secretion proceeds without signal sequence removal or cleavage of other segments of the polypeptide. We have examined the synthesis and transport of HBsAg in cultured cells expressing the cloned surface antigen gene. Our results show that HBsAg is initially synthesized as a integral membrane protein. This transmembrane form is slowly converted to a secreted lipoprotein complex in the lumen of the endoplasmic reticulum via a series of definable intermediates, after which it is secreted from the cell. This unusual export process shares many features with the assembly and budding reactions of conventional enveloped animal viruses. However, it differs importantly in its absence of a requirement for the participation of nucleocapsid or other viral proteins.

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Synthesis of hepatitis B surface antigen in mammalian cells: expression of the entire gene and the coding region

Cited by 108 publications

References 32 publications

Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment.

Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment.

Two Distinct Size Classes of Immature and Mature Subviral Particles from Tick-Borne Encephalitis Virus

Secreted hepatitis B surface antigen polypeptides are derived from a transmembrane precursor.

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