Preferential ribosomal scanning is involved in the differential synthesis of the hepatitis B viral surface antigens from subgenomic transcripts

Sheu, Shih Yi; Lo, Szecheng J.

doi:10.1016/0042-6822(92)90764-g

Cited by 22 publications

(17 citation statements)

References 17 publications

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“…The plasmid pMTMS was derived from a partial deletion of pMH3/3097 plasmid (Junker et al, 1987) as described previously (Sheu & Lo, 1992). Briefly, the 1.6 kb HindlIIMstII fragment of pMH3/3097 was deleted followed by Klenow fragment fill-in and blunt-end ligation.…”

Section: Methodsmentioning

confidence: 99%

“…After transfection into human hepatoma cells, the pMTMS plasmid under the human metallothionine (hMT) promoter control can be transcribed into a 2.1 kb mRNA responsible for the synthesis of M and S proteins. The flanking sequence to the initiation codon (5' GCCATGC 3') of the pre-S2 region in pMTMS was substituted by the sequence 5' ACCATGG 3' to give the pMTMMS plasmid (Sheu & Lo, 1992) (see Fig. 1 …”

Section: Methodsmentioning

confidence: 99%

“…Our previous study has also revealed that the secreted M proteins gp33 and gp36 are more complex than those of intracellular counterparts, resulting in their slower migration in SDS-PAGE (Sheu & Lo, 1992). In this study, the superscript' *' is used to designate the complex form of glycosylation, i.e.…”

Section: Glycosylation Patterns Of M Protein In Secreted and Nonsecrementioning

confidence: 99%

“…Our previous experiments have demonstrated that the HUH-7 human hepatoma cells transfected by the pMTMMS plasmid which contains an HBV sequence with an optimal translational initiation context of pre-S2 region can produce a higher amount of M protein, and that the ratio of M to S protein differs inside and outside of cells (Sheu & Lo, 1992). We then examined whether the surface antigens in the pMTMMS-transfected cells, which consist of more M than S proteins in contrast to more S than M proteins in the wild-type, could form particles.…”

Section: Assembly and Secretion Of 22-nm-like Surface Antigen Particlmentioning

confidence: 99%

“…Studies of expression and secretion of the M protein have already been reported in various transfected cell systems (Cheng & Moss, 1987;McLachlan et al, 1987;MolnarKimber et al, 1988) but full information on glycosylation and secretion pathway is lacking. In this study, we used transfected human hepatoma cells that produce higher amount of M proteins (Sheu & Lo, 1992) to address the biogenesis of M protein in more detail.…”

Section: Introductionmentioning

confidence: 99%

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Biogenesis of the Hepatitis B Viral Middle (M) Surface Protein in a Human Hepatoma Cell Line: Demonstration of an Alternative Secretion Pathway

Sheu

1994

Journal of General Virology

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In the serum of hepatitis B virus (HBV)-infected patients, two different types of particles, a 42 nm virion and a 22 nm subviral particle, were identified. The envelope of both particles is composed of three proteins, the large (L), middle (M), and major/small (S) surface proteins but the ratio between these components varies in each.The M protein appears in a lesser amount than the S protein in both virion and subviral particles, although it is translated from the same subgenomic RNA, and this is due to its poor initiation context of translation. In addition, only the glycosylated form of M protein is secreted in contrast to both glycosylated and unglycosylated forms of L and S proteins that are secreted. To investigate the biogenesis of M protein, human hepatoma cells transfected with plasmids containing a mutated HBV DNA were used to produce a high amount of M protein. Electron microscopic observation revealed that despite a higher proportion of the M protein being found in the transfected cells, the secreted surface antigen particles possess similar size and density to 22 nm subviral particles. Detailed biochemical analyses showed the following. (1) The unglycosylated M protein was predominantly present in the microsomal fraction but not present in any other subcellular fractions. (2) The M protein formed 22-nm-like particles in the endoplasmic reticulum (ER) and was retained in the post-ER or preGolgi regions. (3) In addition to the complex glycosylated form of M protein, a high-mannose form of M protein could be secreted. (4) Normally, no unglycosylated M protein was secreted. However, glycosylation was not essential for M protein secretion since M protein deprived of glycosylation by tunicamycin treatment was detected in the medium. These findings suggest that (i) the M protein was probably translated and co-translocated into the ER and at least one site was glycosylated before leaving the ER resulting in no secretion of unglycosylated M protein, and (ii) the M protein had two secretion pathways, one through the conventional pathway and the other probably directly through the ER.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Glycosylation Patterns Of M Protein In Secreted and Nonsecrementioning

confidence: 99%

Section: Assembly and Secretion Of 22-nm-like Surface Antigen Particlmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Biogenesis of the Hepatitis B Viral Middle (M) Surface Protein in a Human Hepatoma Cell Line: Demonstration of an Alternative Secretion Pathway

Sheu

1994

Journal of General Virology

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Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein.

et al. 1994

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The preS domain at the N‐terminus of the large envelope protein (LHBs) of the hepatitis B virus is involved in (i) envelopment of viral nucleocapsids and (ii) binding to the host cell. While the first function suggests a cytosolic location of the preS domain during virion assembly, the function as an attachment site requires its translocation across the lipid bilayer and final exposure on the virion surface. We compared the transmembrane topology of newly synthesized LHBs in the endoplasmic reticulum (ER) membrane with its topology in the envelope of secreted virions. Protease sensitivity and the absence of glycosylation suggest that the entire preS domain of newly synthesized LHBs remains at the cytosolic side of ER vesicles. However, virions secreted from transfected cell cultures or isolated from the blood of persistent virus carriers expose antibody binding sites and proteolytic cleavage sites of the preS domain at their surface in approximately half of the LHBs molecules. Thus, preS domains appear to be transported across the viral lipid barrier by a novel post‐translational translocation mechanism to fulfil a dual function in virion assembly and attachment to the host cell.

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Assembly of Hepatitis Delta Virus Particles: Package of Multimeric Hepatitis Delta Virus Genomic RNA and Role of Phosphorylation

Yeh¹,

Lee²

1998

Virology

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We previously demonstrated that both casein kinase II (CKII) and protein kinase C (PKC) positively modulate the hepatitis delta virus (HDV) RNA replication but not the assembly of the empty hepatitis delta antigen (HDAg) particle. In this study, we investigated whether phosphorylation of HDAg by these two kinases plays a role in assembly of the HDV virion. As demonstrated by in vivo labeling and kinase inhibitor experiments, the phosphorylation level of large HDAg but not small HDAg in HDAg-expressing HuH-7 cells was diminished by CKII inhibitor (DRB), whereas no effect was observed for the phosphorylation level of two HDAgs when treated with protein kinase A (PKA) inhibitor (HA1004) or PKC inhibitor (H7). Cotransfection experiment also demonstrated that packaging of HDV genomic RNA was not affected by the kinase inhibitor DRB or H7 and mutation at the putative CKII phosphorylation sites (serine-2, serine-123, or both), and the putative PKC site (serine-210) of HDAg did not elicit any significant effect on the HDV virion assembly. Therefore, based on the previous work and the present study, it seems that the status and biological significance of phosphorylation of HDAg vary depending on the HDV life cycle. Although in the HDV RNA replication cycle, phosphorylation of small HDAg by CKII or PKC plays important role in HDV replication, phosphorylation of the same HDAg by these two kinases does not occur during the HDV RNA virion assembly, and phosphorylation of the large HDAg by CKII does not confer any regulatory role in the assembly of HDV virion and empty viral particles. Our study also showed that the large HDAg without the small HDAg could efficiently assemble both monomeric and dimeric HDV genomic RNAs into secreted HBV-enveloped virus-like particles. Increasing the transfected small HDAg-expressing plasmid led to an enhancement of the packaging efficiency for the monomeric HDV genomic RNA with little effect on the packaging of dimeric HDV RNA. Similarly, HDAgs could package the trimeric HDV genomic RNA, albeit less efficiently. CsCl density gradient centrifugation confirmed that HDAgs and the monomeric and multimeric (dimer and trimer) HDV genomic RNAs formed an HBV-enveloped virus-like particle at a density of 1.23-1.25 g/ml. Thus, the assembly of the HDV virion seems to not impose much restriction on the size of HDV RNA for packaging.

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Preferential ribosomal scanning is involved in the differential synthesis of the hepatitis B viral surface antigens from subgenomic transcripts

Cited by 22 publications

References 17 publications

Biogenesis of the Hepatitis B Viral Middle (M) Surface Protein in a Human Hepatoma Cell Line: Demonstration of an Alternative Secretion Pathway

Biogenesis of the Hepatitis B Viral Middle (M) Surface Protein in a Human Hepatoma Cell Line: Demonstration of an Alternative Secretion Pathway

Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein.

Assembly of Hepatitis Delta Virus Particles: Package of Multimeric Hepatitis Delta Virus Genomic RNA and Role of Phosphorylation

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