Morphology of hepatitis C and hepatitis B virus particles as detected by immunogold electron microscopy

We performed indirect immunogold electron microscopy (EM) for immunological identification and characterization of hepatitis C virus (HCV). To clarify the morphology of HCV, an indirect immunogold EM of two plasma samples from patients with high HCV RNA titers was carried out using antibodies specific for the putative HCV envelope protein (E) 1. Spherical virus particles 55–65 nm in diameter with delicate spike projections were detected in the 1.14–1.16 g/ml fractions after sucrose density gradient centrifugation. Polyclonal and monoclonal antibodies to the putative HCV E1 specifically recognized these particles. In addition, immunogold EM of the samples was also performed to uncover the morphology of HCV core particles. Spherical particles 33–40 nm in diameter (average, 37 nm) were detected in the 1.22- to 1.25-g/ml fractions by conventional EM after sucrose density gradient centrifugation. Immunogold EM using rabbit polyclonal antibody (RR8) specific for the putative HCV core protein and colloidal gold-labeled goat antirabbit IgG showed binding of the gold particles with RR8. Some of the HCV core particles showed icosahedric morphology. Optical rotation technique showed that the HCV core particles exhibit sixfold symmetry and that the length of the regular hexagon side is approximately 20 nm, suggesting that they have an icosahedric structure. Further, the detection limit of the indirect immunogold EM was evaluated in 11 plasma samples from chronic hepatitis B patients with different degrees of hepatitis B virus (HBV) DNA titers using antihepatitis B surface antigen antibody. The study showed that the detection limit of virus using this method is 10⁷ virions/ml.     

Assembly of HCV E1 and E2 glycoproteins into coronavirus VLPs

Summary. Hepatitis C virus (HCV) is believed to assemble by budding into membranes of the early secretory pathway, consistent with the membrane location where the viral envelope glycoproteins E1 and E2 accumulate when expressed. Coronavirus assembly also takes place at pre-Golgi membranes. Here, we generated coronavirus-like particles carrying in their envelope chimeric HCV glycoproteins composed of the ectodomains of E1 and E2, each fused to the transmembrane plus endodomain of the mouse hepatitis coronavirus spike glycoprotein. The chimeric particle system will enable structural and functional studies of the HCV glycoproteins.     

Structural features of envelope proteins on hepatitis C virus-like particles as determined by anti-envelope monoclonal antibodies and CD81 binding

The envelope glycoprotein E2 of hepatitis C virus (HCV) is a major component of the viral envelope. Knowledge of its topologic features and antigenic determinants in virions is crucial in understanding the viral binding sites to cellular receptor(s) and the induction of neutralizing antibodies. The lack of a robust cell culture system for virus propagation has hampered the characterization of E2 presented on the virion. Here we report the structural features of hepatitis C virus-like particles (HCV-LPs) of the 1a and 1b genotypes as determined by various mouse and human monoclonal anti-envelope antibodies. Our results show that the E2 protein of HCV-LPs reacts with human monoclonal antibodies recognizing conformational determinants. Monoclonal antibodies (mAbs) specific for the hypervariable region 1 (HVR-1) sequence reacted strongly with HCV-LPs, suggesting that the HVR-1 is exposed on the viral surface. Several mAbs recognized both HCV-LPs with equally high affinity, indicating that the corresponding epitopes [amino acids (aa) 192-217 of E1 and aa 412-423, aa 522-531, and aa 640-653 of E2] are conserved in both genotypes and exposed on the surface of the HCV-LP. The E2 and E1/E2 dimers of 1a bound strongly to the recombinant large extracellular loop (LEL) of CD81 (CD81-LEL) of human and African green monkey, while the HCV-LP of 1a bound weakly to human CD81-LEL. E1/E2 dimers and the HCV-LPs of 1b did not bind CD81-LEL, consistent with the notion that CD81 recognition by E2 is strain-specific and does not correlate with permissiveness of infection. A model of the topology and exposed antigenic determinants of the envelope proteins of HCV is proposed.     

Characterization of pseudotype VSV possessing HCV envelope proteins

The genome of hepatitis C virus (HCV) encodes two envelope glycoproteins (E1 and E2), which are thought to be responsible for receptor binding and membrane fusion resulting in virus penetration. To investigate cell surface determinants important for HCV infection, we used a recombinant vesicular stomatitis virus (VSV) in which the glycoprotein gene was replaced with a reporter gene encoding green fluorescent protein (GFP) and produced HCV-VSV pseudotypes possessing chimeric HCV E1 or E2 glycoproteins, either individually or together. The infectivity of the pseudotypes was determined by quantifying the number of cells expressing the GFP reporter gene. Pseudotypes that contained both of the chimeric E1 and E2 proteins exhibited 10--20 times higher infectivity on HepG2 cells than the viruses possessing either of the glycoproteins individually. These results indicated that both E1 and E2 envelope proteins are required for maximal infection by HCV. The infectivity of the pseudotype virus was not neutralized by anti-VSV polyclonal antibodies. Bovine lactoferrin specifically inhibited the infection of the pseudotype virus. Treatment of HepG2 cells with Pronase, heparinase, and heparitinase but not with phospholipase C and sodium periodate reduced the infectivity. Therefore, cell surface proteins and some glycosaminoglycans play an important role in binding or entry of HCV into susceptible cells. The pseudotype VSV possessing the chimeric HCV glycoproteins might offer an efficient tool for future research on cellular receptors for HCV and for the development of prophylactics and therapeutics for hepatitis C.     

Nonneutralizing human antibody fragments against hepatitis C virus E2 glycoprotein modulate neutralization of binding activity of human recombinant Fabs.

Evidence from clinical and experimental studies indicates that hepatitis C virus E2 (HCV/E2) glycoprotein is the major target of a putatively protective immune response. However, even in the presence of a vigorous production of anti-HCV/E2 antibodies, reinfection can occur. Dissection of the human immune response against HCV/E2 indicated that blocking of binding of HCV/E2 to target cells [neutralization of binding (NOB) activity] varies widely among antibody clones. Moreover, in vivo, simultaneous binding of antibodies to distinct epitopes can induce conformational changes and synergies that may be relevant to understanding the anti-HCV immune response. In this study, human recombinant Fabs were generated by affinity-selecting a phage display repertoire library with antibody-coated HCV/E2. These Fabs, which share the same complementarity-determining region DNA sequences, had higher affinity than other anti-HCV/E2 Fabs but showed no NOB activity even at the highest concentrations. Binding of Fabs to HCV/E2 caused conformational changes modifying Fab-binding patterns and reducing, with a negative synergistic effect, Fab-mediated NOB activity. These data suggest that some antibody clones have the potential to modify HCV/E2 conformation and that, in this state, binding of this glycoprotein to its cellular target is less prone to inhibition by some antibody clones. This can explain why high anti-HCV/E2 antibody titers do not directly correlate with protection from infection. Information on the interactions among different antibody clones can contribute to understanding virus-host interplay and developing more effective vaccines.     

Enveloped particles in the serum of chronic hepatitis C patients

HCV particles were isolated from the plasma of chronically infected patients. The virus was analysed by sucrose density gradient centrifugation. The fractions were tested for viral RNA, core antigen and envelope proteins by using a monoclonal antibody directed against the natural E1E2 complex (D32.10). Two populations of particles containing RNA plus core antigen were separated: the first with a density of 1.06-1.08 g/ml did not contain the envelope proteins; the second with a density between 1.17 and 1.21 g/ml expressed both E1 and E2 glycoproteins. Electron microscopy of the enveloped population after immunoprecipitation with D32.10 showed spherical particles with a rather featureless surface and with a diameter around 40 nm. Immuno-gold staining gave evidence that the E1E2 complex was indeed positioned at the surface of these particles.     

Ultrastructural localization of the C-terminus of the 43-kd dystrophin-associated glycoprotein and its relation to dystrophin in normal murine skeletal myofiber.

We used single and double immunogold labeling electron microscopy to investigate ultrastructural localization of the C terminus of the 43-kd dystrophin-associated glycoprotein (43-DAG) and its relationship to dystrophin in normal murine skeletal myofibers. Single immunolabeling localized the antibody against the C terminus of 43-DAG to the inside surface of the muscle plasma membrane and the sarcoplasmic side of plasma membrane invaginations. Double immunolabeling co-localized antibodies against dystrophin and the C terminus of 43-DAG to the same site noted in the single immunolabeling localization of 43-DAG. In particular, dystrophin and the C-terminal 43-DAG antibody signals were often observed as doublets separated by less than 30 nm. We compared these results with those obtained from double immunogold labeling with anti-dystrophin and anti-beta-spectrin, as well as anti-C-terminal 43-DAG and anti-beta-spectrin antibodies. The antibodies against dystrophin and beta-spectrin, or beta-spectrin and 43-DAG, also co-localized to similar sites in skeletal muscle fibers. Signals of doublet formations were noted but their frequency was significantly lower than the doublet frequency of antidystrophin and anti-43-DAG antibodies. The results support the presence of dystrophin and 43-DAG linkage at the inside surface of the murine skeletal muscle plasma membrane.     

Preferential immune response to virion surface glycoproteins by caprine arthritis-encephalitis virus-infected goats.

Six months after inoculation with caprine arthritis-encephalitis virus, the serum and synovial fluid of virus-infected goats had antibodies to [35S]methionine-labeled viral proteins with apparent molecular weights of 125,000, 90,000, 28,000, and 15,000. The 125,000-, 90,000-, and 15,000-molecular-weight methionine-labeled proteins were identified as virion surface glycoproteins by lactoperoxidase iodination and galactose oxidase-boro[3H]hydride reduction labeling techniques. Radioimmunoassay antibody titers to purified p28, the most abundant viral structural protein, averaged 1:182 in synovial fluid and 1:67 in serum 6 months after inoculation. High dilutions of serum and synovial fluid reacted with gp90 and gp125 electroblotted onto nitrocellulose paper from polyacrylamide gels. Anti-gp90 activity was detected at dilutions with an immunoglobulin G content of 0.02 to 11 micrograms, whereas antibody to p28, when detectable on Western blots, was present in samples with an immunoglobulin G content of 0.1 to 2 mg, representing 100- to 1,000-fold-greater titers of antibody to the surface glycoprotein. Synovial fluids often contained more anti-gp90 antibody than did sera. Immunoprecipitation of lactoperoxidase-iodinated virus confirmed the presence of high antibody titers to the two virion surface glycoproteins. Because antiviral gp90 and gp125 antibody is abundant in the synovial fluid of infected goats, it probably contributes to the high immunoglobulin G1 concentrations seen at this site 6 months after caprine arthritis-encephalitis virus infection.     

Evidence for a polytopic form of the E1 envelope glycoprotein of Hepatitis C virus

The polyprotein precursor of the Hepatitis C virus (HCV) contains multiple membrane-spanning domains that define the membrane topology and subsequent maturation of the viral structural proteins. In order to examine the biogenesis of the E1-E2 heterodimeric complex, we inserted an affinity tag (S-peptide) at specific locations within the envelope glycoproteins. In particular, and based on the prediction that the E1 glycoprotein may be able to assume a polytopic topology containing two membrane-spanning domains, we inserted the affinity tag within a putative cytoplasmic loop of the E1 glycoprotein. The HCV structural polyprotein containing this tag (at amino acids 295/296) was highly expressed and able to form a properly processed and noncovalently associated E1-E2 complex. This complex was bound by murine and conformation-dependent human monoclonal antibodies (MAbs) comparably to the native untagged complex. In addition, MAb recognition was retained upon reconstituting the tagged E1-E2 complex in lipid membrane as topologically constrained proteoliposomes. Our findings are consistent with the model of a topologically flexible E1 glycoprotein that is able to adopt a polytopic form. This form of the E1-E2 complex may be important in the HCV life cycle and in pathogenesis.     

The significance of carbohydrate trimming for the antigenicity of the Semliki Forest virus glycoprotein E2.

Six groups, designated a-f, of noncompeting murine monoclonal antibodies to the envelope glycoprotein E2 of Semliki Forest virus (SFV) have been used to analyze antigenic changes caused by differences in the carbohydrate chain composition of the envelope glycoprotein E2 in the virion. Deletion of terminal sialic acids as observed in virus progeny from mosquito cells did not affect antigenic properties. Inhibition of the trimming pathway in infected chicken cells by the mannosidase I inhibitor dMM led to infectious virus particles containing mannose-rich oligosaccharides of the composition Man9(GlcNAc)2 in the envelope glycoproteins. This alteration had no effect on antigenicity. If inhibition was, however, performed with MdN which acts on alpha-glucosidase giving rise to virions with glycoproteins containing three additional glucose residues in the carbohydrate chains [Glc3Man7,8,9(GlcNAc)2], significant antigenic changes were observed. The six epitopes were differently affected by the underlying structural change and the pattern of exposition of epitopes was not identical with that observed after cleavage of intramolecular disulfide bonds. Concomitantly, the cleavage rate of gp62, the intracellular precursor molecule of the glycoproteins E2 and E3 of the virus particle, was reduced causing a reduction of virus yield. It is concluded that the existence of untrimmed carbohydrate chains is sufficient to allow SFV maturation. The trimming reactions improve this process in a matter suggesting that the carbohydrate chains influence intracellular traffic (addressing) of the respective glycoprotein.     

Protective monoclonal antibodies define maturational and pH-dependent antigenic changes in Sindbis virus E1 glycoprotein

Monoclonal (MC) antibodies specific for either the EI or E2 glycoproteins of Sindbis virus (SIN) were used to probe for differences in the surface topography of SIN epitopes between infected cells and mature virions. Employing an enzyme-linked immunosorbent assay (ELISA) in which binding of individual peroxidase-labeled MC antibodies to immobilized (solid-phase) detergent-disrupted SIN was inhibited specifically by one or more unlabeled antibodies, viral epitopes could be grouped into six spatially distinct antigenic sites--five on E1, designated a through e, and one site on E2. All six sites were represented on the surfaces of SIN-infected cells as shown by the complement (C')-dependent lysis mediated by antibodies of the corresponding epitope specificities. In contrast, virus-neutralizing (NT) activity was restricted to antibodies specific for epitopes on E2 and on site c of E1, irrespective of the presence of added C' and an antiserum against mouse immunoglobulins. That E1 sites a, b, d, and e became inaccessible to antibody binding was shown by a competitive-inhibition ELISA. Whereas all MC antibodies were inhibited from binding to solid-phase SIN when premixed with detergent-treated virions, only those having NT activity could be competitively inhibited by intact virions. Sites E1-d and E1-e could be exposed not only by detergent disruption but also by lowering the virion pH from 7.2 to 6.0. These collective results indicate that a majority of immunologically relevant E1 epitopes present on SIN-infected cell surfaces become cryptic during SIN maturation and, except at low pH, remain undetectable on virion surfaces.     

Distribution and localization of caveolin-1 in sinusoidal cells in rat liver

Caveolin, the principal structural protein in caveolae, is involved in signal transduction. The aim of the present study was to clarify the distribution and ultrastructural localization of caveolin-1 in hepatic sinusoidal endothelial cells (SECs) and hepatic stellate cell (HSCs) by confocal microscopy and the electron immunogold method. Liver tissue sections were prepared from male Wistar rats. SECs and HSCs were isolated from rat livers by collagenase infusion. For immunohistochemistry, liver sections were reacted with anticaveolin-1 antibody. The localization and distribution of caveolin-1 were identified by confocal immunofluorescence. The ultrastructural localization of caveolin-1 on SECs and HSCs was identified by electron microscopy using the immunogold method. Immunohistochemical studies using liver tissues localized caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein, and hepatic artery. By confocal microscopy, caveolin-1 was mainly demonstrated at the Golgi complex in SECs and HSCs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae (SEF) and vesicles in SECs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of caveolae and vesicles in HSCs. We concluded that caveolin-1 is localized from SEFs to the Golgi complex in SECs and from caveolae to the Golgi complex in HSCs.     

Characterization of a major virion envelope glycoprotein complex of murine cytomegalovirus and its immunological cross-reactivity with human cytomegalovirus

Three glycoproteins on the murine cytomegalovirus (MCMV) virion with apparent molecular weights of 150K (gp 150), 105K (gp 105), and 52K (gp52) were immunoprecipitated by two monoclonal antibodies (MAbs) 8G5.12A and 2E.12A. However, only 8G5.12A was able to neutralize MCMV infectivity in the presence of complement. The accessibility of these three glycoproteins to radiolabeling by surface-iodination reactions suggested that they were exposed on the surface of the virion. Western blot analysis of the three glycoproteins showed that gp150 shared antigenic determinants with gp105 and gp52. Briefly, the MAb 8G5.12A reacted with gp150 and gp105, whereas the MAb 2E8.12A reacted with gp150 and gp52. A third MAb 3H2.12A was also found to be reactive with gp150 and gp105 in Western blots, but was unable to immunoprecipitate these glycoproteins. Data from pluse-chase experiments suggested that all three virion glycoproteins were synthesized from a common 128K precursor, providing a partial explanation of their antigenic relatedness. Furthermore, we have demonstrated the presence of high-molecular-weight complexes formed by disulfide bonding between gp150, gp105, and gp52. Lastly, the MAb 8G5.12A was able to immunoprecipitate 84K and 99-110K glycoproteins from human CMV-infected WI-38 cells, demonstrating that conserved determinants exist between murine and human CMV envelope glycoproteins.     

A prime-boost strategy using virus-like particles pseudotyped for HCV proteins triggers broadly neutralizing antibodies in macaques

Chronic hepatitis C virus (HCV) infection, with its cohort of life-threatening complications, affects more than 200 million persons worldwide and has a prevalence of more than 10% in certain countries. Preventive and therapeutic vaccines against HCV are thus much needed. Neutralizing antibodies (NAbs) are the foundation for successful disease prevention for most established vaccines. However, for viruses that cause chronic infection such as HIV or HCV, induction of broad NAbs from recombinant vaccines has remained elusive. We developed a vaccine platform specifically aimed at inducing NAbs based on pseudotyped virus-like particles (VLPs) made with retroviral Gag. We report that VLPs pseudotyped with E2 and/or E1 HCV envelope glycoproteins induced high-titer anti-E2 and/or anti-E1 antibodies, as well as NAbs, in both mouse and macaque. The NAbs, which were raised against HCV 1a, cross-neutralized the five other genotypes tested (1b, 2a, 2b, 4, and 5). Thus, the described VLP platform, which can be pseudotyped with a vast array of virus envelope glycoproteins, represents a new approach to viral vaccine development.     

Immunoreactivity of HCV/HBV epitopes displayed in an epitope-presenting system

It has been demonstrated that the immunodominant region of the HCV core protein and the hepatitis B surface antigen (HBsAg) have high degree of reactivity. In order to construct a chimeric protein that carries HCV and HBV epitopes and possesses immunogenicity to both HCV and HBV, four epitopes derived from residues aa2-21 (epitope C1), aa22-40 (epitope C2) of the core protein, residues aa315-328 (epitope E) of E1 protein of HCV, and residues aa124-147 (epitope S) of HBsAg were chosen to be displayed in a conformation-specific manner on the outer surface of the Flock House virus capsid protein and expressed in E. coli cells. The reactivity of these epitopes with antisera from hepatitis C and hepatitis B patients and induction of immune response in guinea pigs were determined. The results showed that when displayed in this system, the chimeric protein carrying only epitope S could react with anti-HBsAg positive human sera, elicit an anti-HBsAg response in guinea pigs. The chimeric protein carrying epitopes C1, C2 and E could react with antibodies to different HCV genotypes, elicit an anti-HCV response in guinea pigs. The chimeric protein carrying epitopes C1, C2, E, and S could react with antibodies against HCV and HBV, elicit anti-HCV and anti-HBsAg responses in guinea pigs. The results suggested that these epitopes displayed in this form could be considered for development of epitope-based vaccines against HCV/HBV infections.     

Characterization of human herpesvirus-8 K8.1A/B glycoproteins by monoclonal antibodies.

Human herpesvirus-8 K8.1 gene encodes for two immunogenic class I glycoproteins, K8.1A and B, originating from spliced messages [(1998) Virology 243, 208-217]. The 228-amino-acid-long K8.1A open reading frame (ORF) contains four N-glycosylation sites and the 167-amino-acid-long K8.1B ORF contains three N-glycosylation sites, sharing similar amino- and carboxyl-termini with ORF K8.1A but with an in-frame deletion [(1998) Virology 249, 140-149]. To characterize the K8.1A and B glycoproteins in the infected body cavity-based B cell lymphoma (BCBL-1) cells and in the virion envelopes, monoclonal antibodies (MAbs) recognizing only K8.1A protein or both K8.1A and B proteins were generated. These antibodies reacted with the infected cell membranes and virion envelopes. Stable COS-1 transformant cells expressed the K8.1A and B proteins independently on the plasma membranes. MAbs recognized multiple proteins with molecular weights ranging from 23 to 72 kDa from the BCBL-1 cells and COS-1 cells and the 72 to 68 kDa molecular-weight proteins from the virion particles. The K8.1A is the predominant protein affinity purified from the infected BCBL-1 cells. Digestion with glycosidases show that these proteins contain both N- and O-linked sugars, suggesting that the multiple proteins recognized by the MAbs represent the precursor and product forms of K8.1A and B proteins, and the 72 to 68 kDa molecular-weight proteins represent the virion particle-associated mature forms of these glycoproteins.     

Nuclear localization of the Epstein-Barr virus/C3d receptor (CR2) in the human Burkitt B lymphoma cell, Raji.

Epstein-Barr virus/C3d receptor (CR2) is a glycoprotein of mol. wt 140,000 expressed on the surface of Raji cells. We previously isolated phosphorylated CR2 from purified Raji cell nuclei. We have analyzed the nuclear localization of CR2 by electron microscope immunochemistry of thin sections of Raji cells and we have compared the binding properties of CR2 expressed on purified plasma membranes or nuclei. Anti-CR2 mAb immunogold labeling of thin sections of Raji cells identified CR2 at the nuclear surface and also within the nucleus. Nuclear envelope associated CR2 was localized mainly at nuclear pores. Within the nucleus, CR2 was associated with ribonucleoprotein (RNP) interchromatin fibrils. This labeling was preserved in nuclear matrix preparations. CR2 expressed on the surfaces of purified nuclei or on the cell surface interacted with soluble and particle-bound C3bi/C3d. Monoclonal anti-CR2 antibodies, which recognized extracellular domains of CR2, reacted differently with CR2 depending on its subcellular localization. The presence of CR2 in nuclei may be due to translocation of the cell surface CR2 and/or the presence of two distinct intracellular pathways for mature CR2.     

Bluetongue virus: surface exposure of VP7.

The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominantly labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact virions, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface.     

In vivo processing and isolation of furin protease-sensitive alphavirus glycoproteins: a new technique for producing mutations in virus assembly

Sindbis virus particles are composed of three structural proteins (Capsid/E2/E1). In the mature virion the E1 glycoprotein is organized in a highly constrained, energy-rich conformation. It is hypothesized that this energy is utilized to drive events that deliver the viral genome to the cytoplasm of a host cell. The extraction of the E1 glycoprotein from virus membranes with detergent results in disulfide-bridge rearrangement and the collapse of the protein to a number of low-energy, non-native configurations. In a new approach to the production of membrane-free membrane glycoproteins, furin protease recognition motifs were installed at various positions in the E1 glycoprotein ectodomain. Proteins containing the furin-sensitive sites undergo normal folding and assembly in the endoplasmic reticulum and only experience the consequence of the mutation during transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane. Processing of the proteins also impacts the envelopment of the nucleocapsid in the modified plasma membrane. This technique provides a unique method for studying the mechanism of virus assembly and protein structure without altering crucial early events in protein assembly, folding, and maturation.