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Biomedical subjects

Volker Bruss

Publications and source records attributed to Volker Bruss.

9 recordsLinked to original sources
PubMed2007

Assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes.

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells.

Antigens, Viral↗
PubMed2007

Hepatitis B virus morphogenesis.

The hepatitis B virus (HBV) particle consists of an envelope containing three related surface proteins and probably lipid and an icosahedral nucleocapsid of approximately 30 nm diameter enclosing the viral DNA genome and DNA polymerase. The capsid is formed in the cytosol of the infected cell during packaging of an RNA pregenome replication complex by multiple copies of a 21-kDa C protein. The capsid gains the ability to bud during synthesis of the viral DNA genome by reverse transcription of the pregenome in the lumen of the particle. The three envelope proteins S, M, and L shape a complex transmembrane fold at the endoplasmic reticulum, and form disulfide-linked homo- and heterodimers. The transmembrane topology of a fraction of the large envelope protein L changes post-translationally, therefore, the N terminal domain of L (preS) finally appears on both sides of the membrane. During budding at an intracellular membrane, a short linear domain in the cytosolic preS region interacts with binding sites on the capsid surface. The virions are subsequently secreted into the blood. In addition, the surface proteins can bud in the absence of capsids and form subviral lipoprotein particles of 20 nm diameter which are also secreted.

Capsid↗
PubMed2006

Hepatitis B virus particle formation in the absence of pregenomic RNA and reverse transcriptase.

Cytoplasmic hepatitis B virus (HBV) capsids are not enveloped and secreted unless the packaged RNA pregenome is reverse transcribed. The expression of the capsid protein C, together with envelope proteins in the absence of pregenomic RNA, produced normal amounts of intracellular capsids, but the secretion of virion-like particles was greatly reduced. The I97L C protein mutant, allowing immature nucleocapsid envelopment in the background of an HBV genome, did not promote the envelopment of capsids lacking a pregenome, suggesting that this mutation is not sufficient to induce secretion competence independently of the pregenome.

Capsid Proteins↗
PubMed2005

Determination of the minimal distance between the matrix and transmembrane domains of the large hepatitis B virus envelope protein.

The cytosolic matrix domain (MD) located between amino acids (aa) 103 and 124 of the large hepatitis B virus envelope protein L is essential for virion formation. We reduced the distance between MD and the transmembrane domain (TD; aa 254 to 272) by deletions starting at aa 132. Six mutants with deletions of up to aa 234 were wild type, and four mutants with slightly larger deletions were blocked with respect to virion morphogenesis. Thus, the minimal distance between MD and TD was around 26 aa. This spacer might be required by MD to reach contact sites on the capsid.

Animals↗
PubMed2004

Envelopment of the hepatitis B virus nucleocapsid.

The hepatitis B virus (HBV) is an enveloped DNA virus with an icosahedral capsid replicating via reverse transcription. The crystal structure of the capsid is known. It has a diameter of 36 nm and is formed by one protein species (C protein). The viral envelope contains three different coterminal proteins (S, M, and L proteins) spanning the membrane several times. These proteins are not only released from infected cells as components of the viral envelope but in 10,000-fold excess as subviral lipoprotein particles with a diameter of 22 nm containing no capsid. Assembly of the capsid occurs in the cytosol and results in packaging of a 3.5 kb RNA molecule together with viral and cellular factors. This newly formed capsid cannot be enveloped. Rather, synthesis of the viral DNA genome in the lumen of the capsid by reverse transcription is required to induce a budding competent state. Envelopment then takes place at an intracellular membrane of the pre-Golgi compartment. The S and the L protein, but not the M protein, is required for this process. The L protein forms two different transmembrane topologies. The isoform exposing the N-terminal part at the cytosolic side of the membrane is essential for budding. In this domain, a 22 amino acid (aa) long linear stretch has been mapped genetically to play a vital role in the morphogenetic process. This domain probably mediates the contact to the capsid. A second matrix domain was mapped to the cytosolic loop of the S protein. A similar genetic approach identified two small areas on the capsid surface, which might interact with the envelope proteins during envelopment.

Animals↗
PubMed2003

Mapping of amino acid side chains on the surface of hepatitis B virus capsids required for envelopment and virion formation.

The crystal structure of recombinant hepatitis B virus (HBV) capsids formed by 240 core proteins has recently been published. We wanted to map sites on the surface of the icosahedral 35-nm particle that are important for nucleocapsid envelopment by HBV surface proteins during virion morphogenesis. For this purpose, we individually mutated 52 amino acids (aa) within the N-terminal 140 aa of the 185-aa long core protein displaying their side chains to the external surface of the capsid to alanine residues. The phenotype of the mutations with respect to virion formation was tested by transcomplementation of a core gene-negative HBV genome in transiently cotransfected cells, immunoprecipitation of nucleocapsids from cells and secreted virions from culture media, and detection of the particles by radioactive endogenous polymerase reactions. Thirteen point mutations impeded nucleocapsid detection by endogenous polymerase reactions. Twenty-seven mutations were compatible with virion formation. Among these were all capsid-forming mutations in the upper half of the spike protruding from the particle shell and two additional triple mutations at tip of the spike. Eleven mutations (S17, F18, L60, L95, K96, F122, I126, R127, N136, A137, and I139) allowed nucleocapsid formation but blocked particle envelopment and virion formation to undetectable levels. These mutations map to a ring-like groove around the base of the spike and to a small area at the capsid surface close to the pores in the capsid shell. These residues are candidate sites for the interaction with envelope proteins during virion morphogenesis.

Capsid↗
PubMed2002

In vitro expression of human hepatitis B virus genomes carrying woodchuck hepatitis virus pre-S1 sequences.

Many hepatitis B virus (HBV) in vivo experiments are unfeasible because this virus infects only humans. Former studies demonstrated that the pre-S1 domain of the viral envelope protein L determines host range. Therefore, we tried to generate HBV recombinants which might be able to infect woodchucks by exchanging different portions of the pre-S1 encoding gene with homologous parts from the related woodchuck hepatitis virus (WHV) in a cloned HBV genome. In 6 mutants, 11-92 N-terminal HBV pre-S1 codons were replaced by 20-120 codons from WHV. Four mutants carried C-terminal substitutions. The pre-S1 region overlaps with the viral polymerase gene, which is therefore also affected. After transfection of Huh7 cells, the DNA polymerase activity in cytoplasmic nucleocapsids was found to be only slightly affected. All mutants except for the largest C-terminal substitution allowed virion formation. Only the smallest N- and C-terminal substitutions had a wild-type phenotype. The remaining 7 variants allowed virion yields between 5 and 50% of that of the wild type. This demonstrated that substitution of up to 92 pre-S1 codons in an HBV genome with up to 120 codons from WHV pre- S1 was compatible with DNA replication and virion formation. Some recombinant viruses might be able to grow in woodchucks.

Amino Acid Sequence↗