Ultrastructural evidences of HCV infection in hepatocytes of chronically HCV-infected patients

In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied by transmission electron microscopy (EM) and immunoelectron microscopy (IEM). The 13 biopsies showed ultrastructural cell damage typical of acute viral hepatitis. In four of the 13 liver biopsies enveloped virus-like particles (VLPs) inside cytoplasmic vesicles and in the cytoplasm of hepatocytes were observed. We also detected the presence of unenveloped VLPs mainly in the cytoplasm and in the endoplasmic reticulum. IEM using anti-core, E1 and E2 monoclonal antibodies (mAbs) confirmed the specific localization of these proteins, in vivo, inside cytoplasm and endoplasmic reticulum. Thus, this work provided evidence for hepatocellular injury related to HCV infection. It also suggested the presence of HCV-related replicating structures in the cytoplasm of hepatocytes and raised the possibility of hepatitis C virion morphogenesis in intracellular vesicles.     

HCV core protein localizes in the nuclei of nonparenchymal liver cells from chronically HCV-infected patients

Understanding the mechanism of hepatitis C virus (HCV) pathogenesis is an important part of HCV research. Recent experimental evidence suggests that the HCV core protein (HCcAg) has numerous functional activities. These properties suggest that HCcAg, in concert with cellular factors, may contribute to pathogenesis during persistent HCV infection. HCV is capable of infecting cells other than hepatocytes. Although the extrahepatic cellular tropism of HCV may play a role in the pathophysiology of this infection, the precise biological significance of the presence of HCV components in different liver cell types presently remains to be established. In this study, HCcAg was detected in nonparenchymal liver cells of six patients out of eight positive for serum HCV RNA. Immunostaining with anti-HCcAg mAbs revealed the presence of this protein in different liver cell types such as lymphocytes, Kupffer, polymorphonuclear, pit, endothelial, stellate, and fibroblast-like cells. Interestingly, HCcAg was immunolabeled not only in the cytoplasm but also in the nucleus of these cells. Remarkably, HCcAg co-localized with large lipid droplets present in stellate cells and with collagen fibers in the extracellular matrix. Moreover, HCcAg was immunolabeled in bile canaliculus suggesting the involvement of the biliary system in the pathobiology of HCV. Data suggest that nonparenchymal liver cells may constitute a reservoir for HCV replication. Besides, HCcAg may contribute to modulate immune function and fibrosis in the liver as well as steatosis.     

Innate form of HCV core protein plays an important role in the localization and the function of HCV core protein

Hepatitis C Virus (HCV) has been identified as the major causative agent of non-A, non-B hepatitis. Core protein is not only a capsid protein of HCV but also a regulator of cellular functions, and plays an important role in the pathogenesis of HCV. Core protein is produced as an innate form (amino acids [a.a.] 1-191), and following processing produces a mature form (a.a. 1-173). This study demonstrates that the innate form regulates subcellular localization of the mature form, and that the innate form in the cytoplasm enhances p21 expression; on the other hand, the mature form in the nucleus suppresses p21 expression. These observations suggest that the innate form is not only a precursor of the mature form but also a regulator of the localization and functions of core protein.     

In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein

Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.     

Affinity analysis of differentially expressed genes in hepatocytes expressing HCV core genotype 1b or 3a

Chronic hepatitis C patients display many genotype-specific clinical features of HCV infection. The core proteins encoded by different genotypes dysregulate numerous sets of distinct host genes. In this study we tested the hypothesis that HCV core proteins 1b and 3a would actually act on a limited number of independent cellular players, as well as on several functionally linked gene products. Structural and functional tests identified a core set of host genes dysregulated by HCV core genotypes 1b and 3a. The core proteins of HCV genotypes 1b and 3a target specifically limited sets of functionally related gene products, which may be responsible for the variations in the clinical spectra associated with HCV infection.     

Nucleic acid binding properties and intermediates of HCV core protein multimerization in Pichia pastoris

Little is known about the in vivo assembly pathway or structure of the hepatitis C virus nucleocapsid. In this work the intermediates of HCcAg multimerization in Pichia pastoris cells and the nucleic acid binding properties of structured nucleocapsid-like particles (NLPs) were studied. Extensive cross-linking was observed for HCcAg after glutaraldehyde treatment. Data suggest that HCcAg exists in dimeric forms probably representing P21-P21, P21-P23, and P23-P23 dimers. In addition, the presence of HCcAg species that might represent trimers and multimers was observed. After sucrose equilibrium density gradient purification and nuclease digestion, NLPs were shown to contain both RNA and DNA molecules. Finally, the analysis by electron microscopy indicated that native NLPs were resistant to nuclease treatment. These results indicated that HCcAg assembles through dimers, trimers, and multimers' intermediates into capsids in P. pastoris cells. Assembly of NLPs in its natural environment might confer stability to these particles by adopting a compact structure.     

Subcellular localization of HCV core protein regulates its ability for p53 activation and p21 suppression

An internal RNA standard proved less suitable in bacterial gene expression experiments. We therefore developed a method for simultaneous RNA and gDNA (genomic DNA) isolation from in vitro and in vivo samples containing staphylococci and combined it with quantitative PCR. The reliability of gDNA for bacterial quantification and for standardisation in gene expression experiments was evaluated. Quantitative PCR proves equivalent to quantitative culture for in vitro samples, and superior for in vivo samples. In gene expression experiments, gDNA permits a good standardisation for the initial amount of bacteria. The average interassay variability of the in vitro expression is 20.1%. The in vivo intersample variability was 73.3%. This higher variability can be attributed to the biological variation of gene expression in vivo. This method permits exact quantification of the number of bacteria and the expression of genes in staphylococci in vivo (e.g., in biofilms, evolution in time) and in vitro.     

Immunomorphological localization of Vasa protein and pre-mRNA splicing factors in Panorpa communis trophocytes and oocytes

The distribution of Vasa protein and splicing factors of pre-mRNA has been studied in oogenesis of Panorpa communis. This distribution was analyzed relative to three types of perinuclear bodies (PBs) in trophocyte cytoplasm, PBs and polar granules (PGs) in oocyte. Immunoelectron labeling using antibody against Vasa protein revealed PBs of the 2nd type of P. communis trophocytes as well as oocyte PBs and PGs to contain Vasa protein. From this evidence emerged proposal that PBs of the 2nd type are homologues to the "nuage" of Drosophila, a marker of germ line cells. Besides, we suggest that in P. communis, both trophocytes and oocytes take part in formation of PGs. Using immunoelectron microscopy, we also show small nuclear RNPs both in trophocyte PBs of the 2nd type and in oocyte PBs. The functional significance of coupling in the same structure of Vasa protein and snRNPs is discussed.     

Spectroscopic study of conformational changes accompanying self-assembly of HCV core protein

Electron microscopy and infrared and Raman spectroscopy have been used here to study the morphology, size distribution, secondary and tertiary structures of protein particles assembled from a truncated hepatitis C virus (HCV) core protein covering the first 120 aa. Particles of pure protein, having similar morphology and size distribution of those of nucleocapsids found in sera from HCV-infected patients, have been visualized for the first time. The secondary structure of these protein particles involve beta-sheet enrichment in relation to its protein monomer. Tertiary/quaternary structure has also been studied using the dynamics of H/D exchange. With this aim infrared spectra were measured as a function of H/D exchange time and subsequently analyzed by principal component analysis and two-dimensional correlation spectroscopy. Temporal dynamics of exchange for these protein particles were as follows: arginine residues exchanged first, followed by turn and unordered structures, followed by beta-sheets which may act as linkers of protein monomers.     

A C-terminal truncated hepatitis C virus core protein variant assembles in vitro into virus-like particles in the absence of structured nucleic acids

Little is known about the assembly pathway or structure of the hepatitis C virus (HCV). In this work a truncated HCcAg variant covering the first 120 aa (HCcAg.120) with a 32 aa N-terminal fusion peptide (6x Histag-Xpress epitope) was purified as a monomer under strong denaturing conditions. In addition, minor HCcAg.120 peaks exhibiting little different molecular mass by SDS-PAGE which possibly represents alternative forms harboring the N-termini of HCcAg.120 were detected. Analysis using gel filtration chromatography showed that HCcAg.120 assembled into high molecular weight structures in vitro in the absence of structured nucleic acids. The negative-stain electron microscopy analysis revealed that these structures correspond with spherical VLPs of uniform morphology and size distribution. The diameters of these particles ranged from 20 to 43nm with an average diameter of approximately 30 nm and were specifically immunolabelled with a mouse monoclonal antibody against the residues 5-35 of HCcAg. Results presented in this work showed that HCcAg.120 assembled in vitro into VLPs in the absence of structured nucleic acids with similar morphology and size distribution to those found in sera and hepatocytes from HCV-infected patients. Therefore, these VLPs would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure.     

Ultrastructural characterisation of a nuclear domain highly enriched in survival of motor neuron (SMN) protein

Mutations in the survival of motor neuron (SMN) gene are the major cause of spinal muscular atrophy (SMA). The SMN gene encodes a 38-kDa protein that localises in the cytoplasm and in nuclear bodies termed Gemini of coiled bodies (gems). When visualised by immunofluorescence microscopy, gems often appeared either in close proximity to, or entirely overlapping with coiled (Cajal) bodies (CBs) implying a possible functional relationship between these nuclear domains. With the aim of identifying subnuclear compartments corresponding to gems, we have investigated the intranuclear localisation of SMN and of its interacting protein Gemin2 by immunoelectron microscopy in cultured cells and in liver cells of hibernating dormouse. These antigens are highly enriched in round-shaped electron-dense fibro-granular clusters (EFGCs), which also display a biochemical composition similar to gems visualised by immunofluorescence microscopy. Our data reveal a novel SMN/Gemin2 containing nuclear domain and support the idea that it represents the structural counterpart of gems seen in the light microscope.     

Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles

Although sharing a T=1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence correlation spectroscopy showed that an average of nine EGFP domains were associated with these virus-like structures. Atomic force microscopy and immunoprecipitation studies showed that EGFP was displayed on the surface of these fVLPs. Confocal imaging indicated that these chimeric complexes were targeted to late endosomes when expressed in insect cells. The fVLPs were able to efficiently enter cancer cells and traffic to the nucleus via the microtubulus network. Finally, immunoglobulins present in human parvovirus B19 acute and past-immunity serum samples were able to detect antigenic epitopes present in these fVLPs. In summary, we have developed fluorescent virus-like nanoparticles displaying a large heterologous entity that should be of help to elucidate the mechanisms of infection and pathogenesis of human parvovirus B19. In addition, these B19 nanoparticles serve as a model in the development of targetable vehicles designed for delivery of biomolecules.     

Development and characterization of a recombinant cDNA-based hepatitis C virus system

Invention of subgenomic HCV replicon a few years ago and recent success of in vitro production of infectious HCV have improved our knowledge of the HCV life cycle, replication, pathogenesis, and screening of anti-HCV therapeutics. However, the highly genotype-dependent nature of the in vitro HCV production system has limited its potential for HCV research. In this study, we constructed a recombinant DNA-based HCV system that contained EF-1alpha promoter-driven HCV genotype 1b with HCV E1/E2 deleted and replaced by GFP. We co-transfected this recombinant cDNA with HCV E1/E2 or VSV-G expression plasmid into 293T cells, and we showed HCV protein expression and processing and demonstrated production of HCV-like particles in culture supernatant of co-transfected cells. These results support potential use of this system for studies on expression and processing of the HCV polyprotein and assembly and release of HCV-like particles.     

Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast

The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.     

Two types of virus-like particles in the bean leaf beetle,Cerotoma trifurcata

Two types of virus-like particles were observed in the cytoplasm of hemocytes of the bean leaf beetle, Cerotoma trifurcata. The polyhedral particles were 37–40 nm in diameter and were usually in a crystalline array. They were often associated with granular and laminated structures. The enveloped, spherical particles were 70–75 nm in diameter and were composed of three parts: an outer envelope, a central electron-dense core, and an electron-lucent space between the envelope and the central core. The envelope was similar in structure to the membranes of the cell organelles. These particles were also associated with granular and filamentous structures which were distinct from those associated with the nonenveloped, smaller, polyhedral particles. The nonenveloped particles were recovered in large amounts from partially purified preparations from beetles that contained the particles in thin sections and from soybean loopers, Pseudoplusia includens, which were injected with partially purified preparations from beetles.     

Enhancement of sapovirus recombinant capsid protein expression in insect cells

Human sapovirus (SaV) is uncultivable, but expression of the recombinant capsid protein (rVP1) in insect cells results in the formation of virus-like particles (VLPs) that are morphologically similar to the native viruses. However, the SaV rVP1 expression levels are considerably low. We have found that inclusions of short foreign nucleotide sequences inserted directly upstream from the predicted rVP1 AUG start codon lead to increased yield of VLPs. This method allowed us to express a SaV rVP1, which could not have been expressed to measurable or practical levels otherwise.     

Immuno-electron-microscopic localization of a centriole-related antigen in ciliated cells

Summary An antigen common to purported centriolar and basal body regions of a variety of cell types was previously visualized by immuno-fluorescence microscopy. The present study demonstrates the localization of the antigen relative to the defined basal body structures of ciliated tracheal cells at the electron-microscopic level. After ethyldimethylaminopropyl carbodiimide-glutaraldehyde-saponin (EGS) fixation and permeabilization, immunoferritin labeling is consistently found associated with amorphous electron-opaque material in proximity to basal bodies and their ciliary rootlets, but not with basal body microtubules themselves. This distribution pattern is distinct from that of other proteins found in the apical region of ciliated cells, such as calmodulin. It is proposed that the dense material may be analogous to pericentriolar material of centrosomes.     

HCV core protein immunization with Montanide/CpG elicits strong Th1/Th2 and long-lived CTL responses

An efficient vaccine against Hepatitis C virus (HCV) infection requires induction of strong humoral and cellular responses against viral proteins. We evaluated the immunogenicity of HCV core protein (HCVcp), a prime vaccine candidate, formulated in various human compatible adjuvants. An Escherichia coli-expressed HCVcp, purified in native conditions was used for murine immunization in separate groups of: free HCVcp (Ag), Ag+C/IFA (Freunds), Ag+CpG, Ag+M720 (Montanide ISA 720), Ag+F127 (Pluronic acid) and cocktails of Ag+F127+CpG and Ag+M720+CpG. Mice immunized with M720(+CpG) developed the highest HCVcp-specific titers of total IgG, IgG1, 2a, 2b, and that of IFN-gamma and IL-4 cytokines compared to all other groups. HCVcp-specific-CTLs against relevant MHC class I peptides were detected only for Ag+M720+CpG, Ag+M720, and Ag+CpG groups and could be blocked by antimouse-CD8 antibodies. While CTLs were stable, only F127 formulated groups demonstrated detectable IgG antibodies one year post-immunization. Hence, HCVcp formulated in M720 (with a synergistic effect by inclusion of CpG) could induce balanced and strong Th1/Th2 responses with long-lived CD4(-)CD8(+) CTLs.