Chimeric hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies

Patient, Romuald; Hourioux, Christophe; Vaudin, Pascal; Pagès, Jean‐Christophe; Roingeard, Philippe

doi:10.1016/j.nbt.2009.01.001

Cited by 42 publications

(51 citation statements)

References 33 publications

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“…Similar results have been reported recently for HBsAg chimeras containing the ectodomains and TM domains of envelope proteins E1 and E2 of hepatitis C virus (11,33). These authors replaced TM1 of HBsAg with the TM domains of the HCV envelope proteins E1 and E2.…”

Section: Discussionsupporting

confidence: 84%

“…Prior studies, based on the deletion of membrane-spanning domains, showed that transmembrane domains TM1 and TM2 were required for particle formation (5,6). However, subsequent studies have shown that, at least for TM1, the replacement of amino acids is permitted (21,33). In this paper, we have substituted an entire TM domain of another transmembrane protein (HIV gp41) for TM1 or TM3 of HBsAg and probed the requirements for particle assembly.…”

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confidence: 99%

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Hepatitis B Virus Surface Antigen Assembly Function Persists when Entire Transmembrane Domains 1 and 3 Are Replaced by a Heterologous Transmembrane Sequence

Berkower

Spadaccini

Chen

et al. 2011

J Virol

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Native hepatitis B surface antigen (HBsAg) spontaneously assembles into 22-nm subviral particles. The particles are lipoprotein micelles, in which HBsAg is believed to span the lipid layer four times. The first two transmembrane domains, TM1 and TM2, are required for particle assembly. We have probed the requirements for particle assembly by replacing the entire first or third TM domain of HBsAg with the transmembrane domain of HIV gp41. We found that either TM domain of HBsAg could be replaced, resulting in HBsAg-gp41 chimeras that formed particles efficiently. HBsAg formed particles even when both TM1 and TM3 were replaced with the gp41 domain. The results indicate remarkable flexibility in HBsAg particle formation and provide a novel way to express heterologous membrane proteins that are anchored to a lipid surface by their own membrane-spanning domain. The membrane-proximal exposed region (MPER) of gp41 is an important target of broadly reactive neutralizing antibodies against HIV-1, and HBsAg-MPER particles may provide a good platform for future vaccine development.HBsAg is the surface antigen of hepatitis B virus. In the virus, translation of the same open reading frame can begin at one of three in-frame start codons, resulting in proteins of large (42 kDa), medium (33 kDa), and small size (27 kDa), which share the identical 226-amino-acid sequence at the carboxyl end. The 27-kDa form of HBsAg is fully capable of spontaneous assembly into virus-like particles, so we have focused on this protein to study the requirements for particle assembly. In nature, small HBsAg participates in forming at least four different kinds of particles: 42-nm infectious hepatitis B virions (Dane particles), 22-nm spherical subviral particles, 22-nm-diameter rods, or as the borrowed envelope protein in 36-nm hepatitis D virus (39, 45).Recombinant-derived small HBsAg forms subviral particles when expressed in a number of different cell types, including mammalian (39), yeast (25), and insect cells (19), but not Escherichia coli (25,26). The particles are lipoprotein micelles, which contain 25% lipid. In these particles, HBsAg is believed to span the lipid four times, with four transmembrane domains (TM1 to TM4). This conformation exposes three external domains on the lipid surface at the amino end, middle, and carboxyl end of the protein (7, 42). Prior studies, based on the deletion of membrane-spanning domains, showed that transmembrane domains TM1 and TM2 were required for particle formation (5, 6). However, subsequent studies have shown that, at least for TM1, the replacement of amino acids is permitted (21,33). In this paper, we have substituted an entire TM domain of another transmembrane protein (HIV gp41) for TM1 or TM3 of HBsAg and probed the requirements for particle assembly.When other proteins were expressed in tandem with HBsAg, the surface antigen acted as a carrier protein and incorporated proteins as large as gp120 into the particles (2). This method allowed us to express membrane proteins and transmembrane proteins...

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Section: Discussionsupporting

confidence: 84%

mentioning

confidence: 99%

Hepatitis B Virus Surface Antigen Assembly Function Persists when Entire Transmembrane Domains 1 and 3 Are Replaced by a Heterologous Transmembrane Sequence

Berkower

Spadaccini

Chen

et al. 2011

J Virol

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“…However, mutagenesis of three hydrophobic clusters of amino acids in TM1 demonstrated that these residues were critical for S protein expression as well as for infectivity in the background of the L protein (17). On the other hand, a chimera consisting of an N-terminal hepatitis C virus E1 domain, including its transmembrane region, fused to the portion of the HBV S protein downstream of TM1 also was able to be cosecreted with wild-type S protein as SVP (18). In this construct the hepatitis C virus-derived TM replaced TM1.…”

Section: Discussionmentioning

confidence: 99%

Role of Transmembrane Domains of Hepatitis B Virus Small Surface Proteins in Subviral-Particle Biogenesis

Siegler

Bruss

2013

J Virol

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The hepatitis B virus (HBV) surface proteins not only are incorporated into the virion envelope but in addition form subviral particles (SVP) consisting solely of surface proteins and lipids. Heterologous expression of the small HBV envelope protein S produces secreted spherical SVP 20 nm in diameter, with approximately 100 S molecules per particle. The pathway leading from the initial S translation product as a multispanning transmembrane protein to the final SVP is largely unknown. To investigate the role of the four transmembrane domains (TM) of S in this process, we introduced mutations in these regions and characterized their effects on SVP formation in transfected Huh7 cells. We found that the insertion of one amino acid in the center of the ␣-helix of TM1 or the exchange of TM1 with a heterologous TM blocked SVP release and SVP formation by coexpressed wildtype S chains in a transdominant negative fashion. Surprisingly, this effect was partially neutralized when the mutations were expressed in the background of the HBV surface protein M, suggesting that mutations in TM1 could partially be complemented by the pre-S2 domain. The exchange of TM2 with heterologous TMs that form ␣-helices of the same lengths was also incompatible with SVP formation. However, these mutants no longer blocked SVP formation by coexpressed wild-type S. We conclude that TM2 is essential for the stable assembly of S chains by establishing intramembrane interactions. Hepatitis B virus (HBV) causes chronic infections of the liver in more than 350 million people worldwide and an increased risk for the development of hepatocellular carcinoma in these individuals (1). Today's options for the treatment of persistent HBV infections are unsatisfying. Therefore, the prevention of HBV infections is particularly important and can be achieved by active vaccination. The most common vaccine consists of the major HBV envelope protein S expressed in yeast. This protein is peculiar as it not only is incorporated into the envelope of virions which have a diameter of 42 nm but, together with host lipids, also assembles into spherical or filamentous lipoprotein particles of 20 nm in diameter (2). Such subviral particles (SVP) are secreted in great excess over virions from infected hepatocytes. Spherical SVP containing approximately 100 protein molecules are also formed by cells expressing the S protein in the absence of any other HBVencoded protein and represent the antigenic component of HBV vaccines.The gene encoding the S protein constitutes the 3= end of a longer continuous open reading frame that is divided into the regions pre-S1, pre-S2, and S. Two further HBV envelope proteins, M and L, are encoded by the pre-S2 plus S and by the pre-S1 plus pre-S2 plus S sequences, respectively. Therefore, all three proteins share the S domain at their C termini. Virion envelopes contain the three proteins S, M, and L at a ratio of approximately 4:1:1. Natural spherical SVP contain mainly S proteins and some M proteins but only small amounts of L, while filamen...

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“…In particular, its N-terminal ectodomain tolerates large heterologous insertions derived from, for example, HIV, globin, or green fluorescent protein [85,102,103]. While former studies demonstrated an essential role of the TM segments in SVP biogenesis [85,86], two recent reports showed that TM1 and TM3 of S could be replaced by foreign TM domains derived from human immunodeficiency virus (HIV) and hepatitis C virus envelope proteins [102,104]. Hence, SVP formation appears to depend on a functional TM domain rather than on S-specific TM sequences.…”

Section: Assembly and Release Of Empty Envelope Particlesmentioning

confidence: 99%

Host factors involved in hepatitis B virus maturation, assembly, and egress

Prange

2012

Med Microbiol Immunol

112

122

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Hepatitis B virus (HBV) is a major cause of liver disease. Due to the tiny size of its genome, HBV depends on the critical interplay between viral and host factors for the generation of new viral particles from infected cells. Recent work has illuminated a multiplicity of spatially and temporally coordinated virus-host interactions that accompany HBV particle genesis. These interactions include the requirement of cellular chaperones for the maturation of the three viral envelope proteins, the cellular factors involved in dynamic modification, maturation, and intracellular trafficking of the nucleocapsids, and the host components of the multivesicular body (MVB) pathway enabling virion budding at intracellular compartments. Beside infectious virions, HBV produces at least two other types of particles, subviral empty envelope particles and subviral naked capsid particles, likely as a result of the engagement of different host factors by the viral structural proteins. Accordingly, HBV exploits distinct cellular pathways to release its particle types. Here, I review recent progress in these areas of the cell biology of HBV genesis.

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Chimeric hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies

Cited by 42 publications

References 33 publications

Hepatitis B Virus Surface Antigen Assembly Function Persists when Entire Transmembrane Domains 1 and 3 Are Replaced by a Heterologous Transmembrane Sequence

Hepatitis B Virus Surface Antigen Assembly Function Persists when Entire Transmembrane Domains 1 and 3 Are Replaced by a Heterologous Transmembrane Sequence

Role of Transmembrane Domains of Hepatitis B Virus Small Surface Proteins in Subviral-Particle Biogenesis

Host factors involved in hepatitis B virus maturation, assembly, and egress

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