Clathrin-mediated endocytosis and lysosomal cleavage of hepatitis B virus capsid-like core particles

The hepatitis B virus (HBV) core particle serves as a protective capsid shell for the viral genome and is highly immunogenic. Recombinant capsid-like core particles are used as effective carriers of foreign T and B cell epitopes and as delivery vehicles for oligonucleotides. The core monomer contains an arginine-rich C terminus that directs core particle attachment to cells via membrane heparan sulfate proteoglycans. Here we investigated the mechanism of recombinant core particle uptake and its intracellular fate following heparan sulfate binding. We found that the core particles are internalized in an energy-dependent manner. Core particle uptake is inhibited by chlorpromazine and by cytosol acidification known to block clathrin-mediated endocytosis but not by nystatin, which blocks lipid raft endocytosis. Particle uptake is abolished by expression of dominant negative forms of eps15 and Rab5, adaptors involved in clathrin-mediated endocytosis and early endosome transport, respectively. Endocytosed particles are transported to lysosomes where the core monomer is endoproteolytically cleaved into its distinct domains. Using protease inhibitors, cathepsin B was identified as the enzyme responsible for core monomer cleavage. Finally we found that monomer cleavage promotes particle dissociation within cells. Together, our results show that HBV capsid-like core particles are internalized through clathrin-mediated endocytosis, leading to lysosomal cleavage of the core monomer and particle dissociation.     

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge.

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.     

乙肝病毒核心蛋白钉突部位基因工程改造对其功能的影响

通过在乙肝病毒核心蛋白钉突部位插入标签蛋白EGFP及小片段多肽,研究各种改造对HBc功能的影响。采用RLIC方法,构建野生型HBc、HBc钉突部位带不同接头的EGFP融合重组体、缩短的EGFP融合重组体,并构建与HBc功能互补的质粒HBV1.1c－,将不同重组体与HBV1.1c－共转染HEK293细胞,通过观察荧光及Southern blotting检测病毒复制中间体,判断相应基因工程改造对重组蛋白中不同结构域功能的影响。RLIC方法可有效地用来进行片段缺失,且缺失片段大小及位置无明显限制。带柔性或刚性接头的重组HBc-EGFP均可产生绿色荧光,但荧光在细胞内分布形态不同,两种重组HBc-EGFP均不能支持正常的HBV复制,各种截短的插入片段以及aa79-80单独缺失体亦不能支持HBV复制。结果表明RLIC方法是一种基因工程改造的有力工具,不同类型接头对重组蛋白的结构和功能有不同影响,aa79-80对维持HBc的主要功能之一——支持HBV复制有重要作用。     

HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein

Core particles of the hepatitis B virus (HBV) potentiate the immune response against foreign epitopes presented on their surface. Potential insertion sites in the monomeric subunit of the HBV core protein were previously identified at the N- and C-terminus and in the immunodominant c/e1 region. In a C-terminally truncated core protein these sites were used to introduce the entire 120 amino acid (aa)-long potentially immunoprotective region of the hantavirus (serotype Puumala) nucleocapsid protein. The N- and C-terminal fusion products were unable to form core-like particles in detectable amounts. However, a suppressable stop codon located between the HBV core and the C-terminally fused hantavirus sequence restored the ability to form particles ('mosaic particles'); in contrast to the C-terminal fusion product the mosaic construct allowed the formation of particles built up by the core protein itself and the HBV core-Puumala nucleocapsid-readthrough protein. The mosaic particles exposed the 120 aa region of the PUU nucleocapsid protein on their surface as demonstrated by ELISA and immuno electron microscopy applying different monoclonal antibodies. Insertion of the hantaviral sequence into the c/e1 region not only allowed the formation of chimeric particles, but again the surface accessibility of the sequence. HBV core antigenicity itself was, however, reduced in the particles carrying insertions in the c/e1 region, probably due to a masking effect of the 120 aa long insert.     

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.     

Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein.

As a step toward understanding the assembly of the hepatitis B virus (HBV) nucleocapsid at a molecular level, we sought to define the primary sequence requirements for assembly of the HBV core protein. This protein can self assemble upon expression in Escherichia coli. Applying this system to a series of C-terminally truncated core protein variants, we mapped the C-terminal limit for assembly to the region between amino acid residues 139 and 144. The size of this domain agrees well with the minimum length of RNA virus capsid proteins that fold into an eight-stranded beta-barrel structure. The entire Arg-rich C-terminal domain of the HBV core protein is not necessary for assembly. However, the nucleic acid content of particles formed by assembly-competent core protein variants correlates with the presence or absence of this region, as does particle stability. The nucleic acid found in the particles is RNA, between about 100 to some 3,000 nucleotides in length. In particles formed by the full-length protein, the core protein mRNA appears to be enriched over other, cellular RNAs. These data indicate that protein-protein interactions provided by the core protein domain from the N terminus to the region around amino acid 144 are the major factor in HBV capsid assembly, which proceeds without the need for substantial amounts of nucleic acid. The presence of the basic C terminus, however, greatly enhances encapsidation of nucleic acid and appears to make an important contribution to capsid stability via protein-nucleic acid interactions. The observation of low but detectable levels of nucleic acid in particles formed by core protein variants lacking the Arg-rich C terminus suggests the presence of a second nucleic acid-binding motif in the first 144 amino acids of the core protein. Based on these findings, the potential importance of the C-terminal core protein region during assembly in vivo into authentic, replication-competent nucleocapsids is discussed.     

Conformational equilibria and rates of localized motion within hepatitis B virus capsids

Functional analysis of hepatitis B virus (HBV) core particles has associated a number of biological roles with the C terminus of the capsid protein. One set of functions require the C terminus to be on the exterior of the capsid, while others place this domain on the interior. According to the crystal structure of the capsid, this segment is strictly internal to the capsid shell and buried at a protein-protein interface. Using kinetic hydrolysis, a form of protease digestion assayed by SDS-PAGE and mass spectrometry, the structurally and biologically important C-terminal region of HBV capsid protein assembly domain (Cp149, residues 1-149) has been shown to be dynamic in both dimer and capsid forms. HBV is an enveloped virus with a T = 4 icosahedral core that is composed of 120 copies of a homodimer capsid protein. Free dimer and assembled capsid forms of the protein are readily hydrolyzed by trypsin and thermolysin, around residues 127-128, indicating that this region is dynamic and exposed to the capsid surface. The measured conformational equilibria have an opposite temperature dependence between free dimer and assembled capsid. This work helps to explain the previously described allosteric regulation of assembly and functional properties of a buried domain. These observations make a critical connection between structure, dynamics, and function: made possible by the first quantitative measurements of conformational equilibria and rates of conversion between protein conformers for a megaDalton complex.     

Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner

Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV-replicating cells release non-enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non-vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional protein with key roles in membrane biology, as a regulator of capsid budding. Ectopic overexpression of Alix enhanced capsid egress, while its depletion inhibited capsid release. Notably, the loss of Alix did not impair HBV production, furthermore indicating that virions and capsids use diverse export routes. By mapping of Alix domains responsible for its capsid release-mediating activity, its Bro1 domain was found to be required and sufficient. Alix binds to core via its Bro1 domain and retained its activity even if its ESCRT-III binding site is disrupted. Together, the boomerang-shaped Bro1 domain of Alix appears to escort capsids without ESCRT.     

Phosphorylation of hepatitis B virus core C-terminally truncated protein (Cp149) by PKC increases capsid assembly and stability

The HBV (hepatitis B virus) core is a phosphoprotein whose assembly, replication, encapsidation and localization are regulated by phosphorylation. It is known that PKC (protein kinase C) regulates pgRNA (pregenomic RNA) encapsidation by phosphorylation of the C-terminus of core, which is a component packaged into capsid. Neither the N-terminal residue phosphorylated by PKC nor the role of the C-terminal phosphorylation have been cleary defined. In the present study we found that HBV Cp149 (core protein C-terminally truncated at amino acid 149) expressed in Escherichia coli was phosphorylated by PKC at Ser(106). PKC-mediated phosphorylation increased core affinity, as well as assembly and capsid stability. In vitro phosphorylation with core mutants (S26A, T70A, S106A and T114A) revealed that the Ser(106) mutation inhibited phosphorylation of core by PKC. CD analysis also revealed that PKC-mediated phosphorylation stabilized the secondary structure of capsid. When either pCMV/FLAG-Cp149[WT (wild-type)] or pCMV/FLAG-S106A Cp149 was transfected into Huh7 human hepatoma cells, mutant capsid level was decreased by 2.06-fold with the S106A mutant when compared with WT, although the same level of total protein was expressed in both cases. In addition, when pUC1.2x and pUC1.2x/S106A were transfected, mutant virus titre was decreased 2.31-fold compared with WT virus titre. In conclusion, PKC-mediated phosphorylation increased capsid assembly, stability and structural stability.     

Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.     

Conformational changes in adeno-associated virus type 1 induced by genome packaging

Adeno-associated virus (AAV) is frequently used as a vector for gene therapy. The viral capsid consists of three structural proteins (VP1, VP2, and VP3) that have a common C-terminal core (VP3), with N-terminal extensions of increasing length in VP2 and VP1. The capsid encloses a single-stranded genome of up to 4.7 kb, which is packaged into empty capsids. The N-terminal extension of VP1 carries a phospholipase domain that becomes accessible during infection in the endosomal pathway. We have used cryo-electron microscopy and image reconstruction to determine subnanometer-resolution structures of recombinant AAV1 that has packaged different amounts of a 3. 6-kb recombinant genome. The maps show that the AAV1 capsid undergoes continuous conformational changes upon packaging of the genome. The rearrangements occur at the inner capsid surface and lead to constrictions of the pores at the 5-fold symmetry axes and to subtle movements of the β-sheet regions of the capsid proteins. In fully packaged particles, the genome forms stem-like features that contact the inner capsid surface at the 3-fold symmetry axes. We think that the reorganization of the inner surface has an impact on the viral life cycle during infection, preparing the externalization of phospholipase domains through the pores at the 5-fold symmetry axes and possibly genome release.     

Core protein domains involved in hepatitis C virus-like particle assembly and budding at the endoplasmic reticulum membrane

Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LPs) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV particle morphogenesis by electron microscopy. Various mutated HCV core proteins with engineered internal deletions were expressed with this system, to identify core domains required or dispensable for HCV-LP assembly. The HCV core protein sequence was compared with its counterpart in GB virus B (GBV-B), the virus most closely related to HCV, to identify conserved domains. GBV-B and HCV display similar tropism for liver hepatocytes and their core proteins are organized similarly into three main domains (I, II and III), although GBV-B core is smaller and lacks approximately 35 amino acids (aa) in domain I. The deletion of short hydrophobic domains (aa 133-152 and 153-167 in HCV core) that appear highly conserved in domain II of both GBV-B and HCV core proteins resulted in loss of HCV core ER anchoring and self-assembly into HCV-LPs. The deletion of short domains found within domain I of HCV core protein but not in the corresponding domain of GBV-B core according to sequence alignment had contrasting effects. Amino acids 15-28 and 60-66 were shown to be dispensable for HCV-LP assembly and morphogenesis, whereas aa 88-106 were required for this process. The production of GBV-B core protein from a recombinant SFV vector was associated with specific ER ultrastructural changes, but did not lead to the morphogenesis of GBV-B-LPs, suggesting that different budding mechanisms occur in members of the Flaviviridae family.     

The herpes simplex virus type 1 DNA packaging protein UL17 is a virion protein that is present in both the capsid and the tegument compartments

The UL17 protein of herpes simplex virus type 1 is essential for packaging the viral genome into the procapsid, a spherical assembly intermediate, and is present in the mature virus particle. We have examined the distribution of UL17 in various assembly products and virions to determine which component of the virus particle UL17 is associated with and at what stage in capsid assembly UL17 is required. UL17 was present in the procapsid, in the DNA-containing angularized C capsid, and in two other angularized capsid forms, A and B, that lack DNA and are thought to be dead-end products. The results suggest that UL17 is a minor capsid protein which is incorporated into the procapsid during assembly of the particle. UL17 was also found in virions and in noninfectious structures known as light (L) particles, which possess a tegument and envelope but lack a capsid. The level of UL17 in these particles was much greater than the amount that could be attributed to capsid contamination of the purified L-particle preparation, suggesting that UL17 is also a tegument protein. The finding that virions contain approximately twofold more UL17 than do C capsids provided further support for the idea that UL17 is present in two different structural components within the mature virion. The UL25 packaging protein, which is also present in virions, was not found in significant amounts in L particles, indicating that it is associated only with the capsid. UL6, the third virion-associated packaging protein, was present in slightly increased levels in L particles.     

Underrepresentation of the 3' region of the capsid pregenomic RNA of duck hepatitis B virus

The pregenomic RNA (pgRNA) of hepadnaviruses is packaged into capsids where it is reverse transcribed to yield mature DNA genomes. This report describes differences between the 3' region and other regions of the pgRNA isolated from capsids. Analysis of capsid pgRNA isolated by using an established method involving micrococcal nuclease treatment demonstrated reduced levels of the 3' region of the pgRNA compared to the 5' region. This underrepresentation of the 3' region was partly a result of microccocal nuclease digestion of the 3' region because isolation of capsid pgRNA by an alternative method that did not involve nuclease treatment led to a greater, but not complete, recovery of the 3' region. These results indicate that the 3' region of the capsid pgRNA is susceptible to micrococcal nuclease digestion during its isolation and that the 3' region can still be underrepresented when capsid pgRNA is isolated without nuclease digestion. Additional experiments show that the 3' ends of capsid pgRNA isolated by micrococcal nuclease treatment are heterogeneously dispersed from nucleotide 2577 to the poly(A) tail. These data provide evidence that the 3' region of the capsid pgRNA has biochemical properties different from those of its 5' region. Possibly, the 3' region of the pgRNA is not packaged into the interior of the capsid but rather is associated with a part of the capsid where it is susceptible to microccocal nuclease digestion.     

Norovirus P particle, a novel platform for vaccine development and antibody production

The norovirus P particle is an octahedral nanoparticle formed by 24 copies of the protrusion (P) domain of the norovirus capsid protein. This P particle is easily produced in Escherichia coli, extremely stable, and highly immunogenic. There are three surface loops per P domain, making a total of 72 loops per particle, and these are potential sites for foreign antigen presentation for immune enhancement. To prove this concept, a small peptide (His tag, 7 amino acids [aa]) and a large antigen (rotavirus VP8, 159 aa) were inserted into one of the loops. Neither insertion affects P particle formation, while both antigens were presented well on the P particle surface. The immune-enhancement effect of the P particle was demonstrated by significantly increased antibody titers induced by the P particle-presented antigens compared to the titers induced by free antigens. In addition, the measured neutralization antibody titers and levels of protection against rotavirus shedding in mice immunized with the VP8 chimeric P particles were significantly higher than those of mice immunized with the free VP8 antigen. Sera from P particle-VP8 chimera-vaccinated animals also blocked norovirus virus-like particle (VLP) binding to the histo-blood group antigen (HBGA) receptors. From these data, the P particle appears to be an excellent vaccine platform for antigen presentation. The readily available three surface loops and the great capacity for foreign antigen insertion make this platform attractive for wide application in vaccine development and antibody production. The P particle-VP8 chimeras may serve as a dual vaccine against both rotavirus and norovirus.     

Generation of filamentous instead of icosahedral particles by repression of African swine fever virus structural protein pB438L

The mechanisms involved in the construction of the icosahedral capsid of the African swine fever virus (ASFV) particle are not well understood at present. Capsid formation requires protein p72, the major capsid component, but other viral proteins are likely to play also a role in this process. We have examined the function of the ASFV structural protein pB438L, encoded by gene B438L, in virus morphogenesis. We show that protein pB438L associates with membranes during the infection, behaving as an integral membrane protein. Using a recombinant ASFV that inducibly expresses protein pB438L, we have determined that this structural protein is essential for the formation of infectious virus particles. In the absence of the protein, the virus assembly sites contain, instead of icosahedral particles, large aberrant tubular structures of viral origin as well as bilobulate forms that present morphological similarities with the tubules. The filamentous particles, which possess an aberrant core shell domain and an inner envelope, are covered by a capsid-like layer that, although containing the major capsid protein p72, does not acquire icosahedral morphology. This capsid, however, is to some extent functional, as the filamentous particles can move from the virus assembly sites to the plasma membrane and exit the cell by budding. The finding that, in the absence of protein pB438L, the viral particles formed have a tubular structure in which the icosahedral symmetry is lost supports a role for this protein in the construction or stabilization of the icosahedral vertices of the virus particle.