Effect of alpha interferon on the hepatitis C virus replicon.

Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-alpha reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-alpha independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-alpha. Finally, under our conditions, selection for IFN-alpha-resistant variants did not occur.

Unique properties of the large antigen of hepatitis delta virus.

The large form of the hepatitis delta virus (HDV) protein (L) can be isoprenylated near its C terminus, and this modification is considered essential for particle assembly. Using gel electrophoresis, we separated L into two species of similar mobilities. The slower species could be labeled by the incorporation of [(14)C]mevalonolactone and is interpreted to be isoprenylated L (L(i)). In serum particles, infected liver, transfected cells, and assembled particles, 25 to 85% of L was isoprenylated. Isoprenylation was also demonstrated by (14)C incorporation in vitro with a rabbit reticulocyte coupled transcription-translation system. However, the species obtained migrated even slower than that detected by labeling in vivo. Next, in studies of HDV particle assembly in the presence of the surface proteins of human hepatitis B virus, we observed the following. (i) Relative to L, L(i) was preferentially assembled into virus-like particles. (ii) L(i) could coassemble the unmodified L and the small delta protein, S. (iii) In contrast, a form of L with a deletion in the dimerization domain was both isoprenylated and assembled, but it could not support the coassembly of S. Finally, to test the expectation that the isoprenylation of L would increase its hydrophobicity, we applied a phase separation strategy based on micelle formation with the nonionic detergent Triton X-114. We showed the following. (i) The unique C-terminal 19 amino acids present on L relative to S caused a significant increase in the hydrophobicity. (ii) This increase was independent of isoprenylation. (iii) In contrast, other, artificial modifications at either the N or C terminus of S did not increase the hydrophobicity. (iv) The increased hydrophobicity was not sufficient for particle assembly; nevertheless, we speculate that it might facilitate virion assembly.

Electrophoretic analysis of the ribonucleoproteins of hepatitis delta virus.

Replication of hepatitis delta virus (HDV) is dependent on delta antigen (deltaAg), an HDV-encoded protein, which binds to HDV RNA and is capable of multimerization. To characterize HDV-specific ribonucleoprotein complexes (RNP) we used electrophoresis into non-denaturing agarose gels followed by northern analysis, to detect HDV RNA, and immunoblot, to detect deltaAg. We studied RNP from three sources: (i) vRNP, disrupted virions obtained from infected woodchuck serum; (ii) sRNP, disrupted particles secreted from transfected cultured cells; and (iii) cRNP, isolated from cells in which HDV genome replication was occurring. sRNP were approximately 28% smaller than vRNP. Treatment of vRNP with aurin tricarboxylic acid disrupted both deltaAg-deltaAg and deltaAg-RNA interactions while vanadyl ribonucleosides released the RNA without causing detectable disruption of the multimeric deltaAg complex. cRNP were smaller and more heterogeneous than vRNP and sRNP, and probably contained host components. The application of these electrophoretic procedures, and especially the use of prior treatments with vanadyl ribonucleoside complexes have provided valuable information on the RNP of HDV, and we expect they should find applicability in RNP studies of other RNA viruses.

Initiation of hepatitis delta virus genome replication.

The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (deltaAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant deltaAg-S and HDV RNA synthesized in vitro to assemble high-molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected HDV genome replication, as assayed by Northern analysis or immunofluorescence microscopy. Our interpretation is that the input deltaAg-S is necessary for the RNA to undergo limited amounts of RNA-directed RNA synthesis, RNA processing, and mRNA formation, leading to de novo translation of deltaAg-S. It is this second source of deltaAg-S which then goes on to support genome replication. This assay made it possible to manipulate in vitro the composition of the RNP and then test in vivo the ability of the complex to initiate RNA-directed RNA synthesis and go on to achieve genome replication. For example, both genomic and antigenomic linear RNAs were acceptable. Substitution for deltaAg-S with truncated or modified forms of the deltaAg, and even with HIV nucleocapsid protein and polylysine, was unacceptable; the exception was a form of deltaAg-S with six histidines added at the C terminus. We expect that further in vitro modifications of these RNP complexes should help define the in vivo requirements for what we define as the initiation of HDV genome replication.

Differentiation of core gene products of the hepatitis B virus in infected liver tissue using monoclonal antibodies.

Hepatitis B virus (HBV) core gene translational products were localised previously in the cytoplasm and/or in the nuclei of infected cells. We investigated in naturally infected human hepatocytes whether this variation in the subcellular expression is due to differences in the presence of assembled core particles and other core gene derived proteins, the expression of HBeAg and the processing of liver tissue. By immunostaining of liver specimens infected with HBeAg-positive and HBeAg-minus variants of HBV, using monoclonal antibodies specific for assembled core particles and for various epitopes on denatured core protein, it was shown that virtually all immunoreactive core gene products are assembled into core particles. The latter are present both in the nuclei and in the cytoplasm of hepatocytes, independent of the infecting virus strain. A marked reduction or absence of immunoreactivity, observed with some monoclonal antibodies, was shown to result from nucleotide sequence variations within or close to the corresponding epitope. These results demonstrate that immunoreactive products, derived from the HBV core gene, in the nuclei and cytoplasm of human hepatocytes represent assembled core particles and that monoclonal antibodies with known recognition sites can reveal region-specific core gene variation of the infecting HBV population.

Phosphorylation of the hepatitis delta virus antigens.

We used two-dimensional electrophoresis (nonequilibrium pH gradient electrophoresis followed by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis) coupled with 32P labeling and immunoblotting detection with 125I-protein A to detect and quantitate phosphorylation of the large and small forms of the delta antigen (deltaAg-L and deltaAg-S, respectively). Analysis of deltaAg species from the serum and liver of an infected woodchuck as well as deltaAg species expressed in and secreted from transfected Huh7 cells revealed the following. (i) No detectable phosphorylation of deltaAg-S occurred. (ii) In virions from the serum of an infected animal and in the particles secreted from cotransfected cells, none of the deltaAg-L was phosphorylated. (iii) Only in the infected liver and in transfected cells was any phosphorylation detected; it corresponded to a monophosphorylated form of deltaAg-L. Given these results, we carried out serine-to-alanine mutagenesis of the deltaAg-L to determine whether the monophosphorylation was predominantly at a specific site on the unique 19-amino-acid (aa) extension. We mutated each of the two serines, aa 207 and 210, on this extension and also the serine at aa 177. These three mutations had no significant effect on phosphorylation. In contrast, mutagenesis to alanine of the cysteine at aa 211, which normally acts as the acceptor for farnesylation, completely inhibited phosphorylation. Our interpretation is that the site(s) of phosphorylation is probably not in the 19-aa extension unique to deltaAg-L and that phosphorylation of deltaAg-L may depend upon prior farnesylation. The possible significance of the intracellular phosphorylated forms of deltaAg-L is discussed.

Redistribution of the delta antigens in cells replicating the genome of hepatitis delta virus.

When the small form of the delta antigen (deltaAg-S) was expressed from a cDNA expression plasmid and subsequently detected by immunofluorescence, it was found localized to the nucleoli. However, if the cDNA was cotransfected with a cDNA expressing a mutated hepatitis delta virus (HDV) genome that could only replicate by using the deltaAg-S provided by the first plasmid, then most of the deltaAg-S was redistributed to the nucleoplasm, largely to specific discrete nucleoplasmic sites or speckles; this pattern was stable for at least 50 days after transfection. These speckles coincided with those detected with an antibody to SC35, an essential non-small nuclear ribonucleoprotein splicing factor. Others have shown that SC35 speckles correspond to active sites of DNA-directed transcription by RNA polymerase II and also of RNA processing. We also found, in contrast to the cotransfections with the mutant HDV and the deltaAg-S provided in trans, that cells transfected with wild-type HDV showed a variable pattern of staining. The SC35-like speckle pattern of accumulation of delta antigen deltaAg was maintained for only 6 days, after which the pattern began to change. By 18 days posttransfection, a variety of different deltaAg staining patterns were observed. This pattern of change occurs at a time when the large form of the delta antigen deltaAg-L appears and HDV RNA synthesis begins to shut down. Our studies therefore support the interpretation that HDV RNA and deltaAg-S accumulate at SC35 speckle sites in the nucleoplasm. We speculate that these may be the sites at which HDV RNA is transcribed by RNA polymerase II and/or sites of HDV RNA processing. Furthermore, when deltaAg-L, as well as other mutant deltaAg accumulate, the speckle association is disrupted, thereby stopping HDV RNA replication.

Epitopes exposed on hepatitis delta virus ribonucleoproteins.

A total of 17 antibodies, raised in several nonhuman species and specific for different regions on the delta antigen (delta Ag), were used to map, via immunoprecipitation, those domains exposed on the surface of the viral ribonucleoprotein (RNP). These studies showed that the domains for the nuclear localization signal and the C-terminal extension, unique to the large form of delta Ag, are exposed. Also exposed is the C-terminal region of the small form of delta Ag. In contrast, reactivity was not found with the coiled-coil domain needed for protein dimerization. When the hepatitis delta virus (HDV) RNA was released by treatment of viral RNP with vanadyl ribonucleoside complexes, no change in the pattern of delta Ag epitope presentation was detected, consistent with the interpretation that a multimeric protein structure persists in the absence of RNA. These RNP studies have implications not only for understanding of the process of HDV assembly but also for evaluation of the immune responses of an infected host to HDV replication.

A novel form of hepatitis delta antigen.

Hepatitis delta virus (HDV) is known to express a protein termed the small delta antigen, a structural protein which is also essential for genome replication. During replication, posttranscriptional RNA editing specifically modifies some of the HDV RNA, leading to the production of an elongated form of the delta antigen, the large form, which is essential for virus assembly. The present study showed that yet another form of HDV protein is expressed during genome replication. This novel form is not produced in all infected cells, but it arises during replication in transfected cells and in infected woodchucks, and as was previously reported, patients infected with HDV do make antibodies directed against it. These findings are an indicator of the complexity of gene expression during HDV infection and replication.

Hepatitis delta virus mutant: effect on RNA editing.

During the replication cycle of hepatitis delta virus (HDV), RNA editing occurs at position 1012 on the 1679-nucleotide RNA genome. This changes an A to G in the amber termination codon, UAG, of the small form of the delta antigen (delta Ag). The resultant UGG codon, tryptophan, allows the translation of a larger form of the delta Ag with a 19-amino-acid C-terminal extension. Using HDV cDNA-transfected cells, we examined the editing potential of HDV RNA mutated from G to A at 1011 on the antigenome, adjacent to normal editing site at 1012. Four procedures were used to study not only the editing of the A at 1012, but also that of the new A at 1011: (i) nucleotide sequencing, (ii) a PCR-based RNA-editing assay, (iii) immunoblot assays, and (iv) immunofluorescence. Five findings are reported. (i) Even after the mutation at 1011, editing still occurred at 1012. (ii) Site 1011 itself now acted as a novel RNA-editing site. (iii) Sites 1011 and 1012 were edited independently. (iv) At later times, both sites became edited, thereby allowing the synthesis of the large form of the delta Ag (delta Ag-L). (v) Via immunofluorescence, such double editing became apparent as a stochastic event, in that groups of cells arose in which the changes had taken place. Evaluation of these findings and of those from previous studies of the stability of the HDV genomic sequence (H.J. Netter et al., J. Virol. 69:1687-1692, 1995) supports both the recent reevaluation of HDV RNA editing as occurring on antigenomic RNA (Casey and Gerin, personal communication) and the interpretation that editing occurs via the RNA-modifying enzyme known as DRADA.

Introduction of hepatitis delta virus into animal cell lines via cationic liposomes.

Cationic liposomes are known to facilitate efficient transfection of animal cells with DNA and even some viruses. As reported here, we have been able to use such a commercially available formulation (Lipofectamine) and introduce human hepatitis delta virus (HDV) into lines of cultured cells and demonstrate replication of the HDV genome both by immunofluorescence and by Northern (RNA) analysis. As much as 10% of the human hepatoma cell line Huh7 was transfected with HDV. Also transfected were the baby hamster kidney cell line BHK-21 and the Morris rat hepatoma line 7777. Two initial applications of HDV transfection have been made. (i) The ribonucleoprotein structure of HDV was isolated from disrupted virions and demonstrated as being sufficient to transfect Huh7 cells. In contrast, naked HDV RNA was not sufficient. (ii) From a study of cells transfected with HDV particles, it was found that, even after as long as 7 weeks and the associated replication of the transfected cells, the HDV RNA genome was still replicating. Apparently, HDV, in the absence of helper virus and in the absence of virus assembly, can maintain persistent replication and expression of the HDV genome. Transfection was also achieved with woodchuck hepatitis virus introduced into Huh7 cells. In summary, this transfection procedure should be of use for the study of these and maybe other recalcitrant animal viruses.

Identification of hepatitis B virus core protein regions exposed or internalized at the surface of HBcAg particles by scanning with monoclonal antibodies.

Hepatitis B virus (HBV) core antigen (HBcAg) particles purified from Escherichia coli were probed in a competition enzyme immunoassay (EIA) with a panel of 16 murine monoclonal antibodies (MAbs) directed to different forms of core protein. The linear binding sites of the MAbs were mapped by combination of solid-phase and competition EIA using synthetic peptides covering the complete sequence of HBV core protein. Relative accessibilities of the linear binding sites at the HBcAg surface were investigated by comparing reactivities in solution of the MAbs to (i) two genetic variants of particulate HBcAg, (ii) denatured core protein, and (iii) synthetic peptides mimicking the appropriate linear binding sites. Further, accessibilities of HBV preS1 and preS2 epitopes (introduced into core protein at positions 77 or 144) at the surface of chimeric HBcAg particles were investigated. The previously described surface localization of core protein region 78-83 at the core particle surface was confirmed. In addition, another region, encompassing residues 127-133, was found to occupy a surface position at particulate HBcAg, whereas regions 9-20 and 133-145 were exposed after denaturation of the core protein and at synthetic peptides but not at particulate HBcAg.

Pathogenesis associated with replication of hepatitis delta virus.

Hepatitis delta virus (HDV) is a subviral satellite of human hepatitis B virus (HBV). HDV was discovered in patients chronically infected with HBV who had a more severe form of disease. Subsequent studies have attempted to understand the cytopathic effects due to HDV, and this article reviews the progress along with newer studies that suggest that HDV genome replication per se causes no more than a moderate inhibition of cellular growth rate. This inhibition nevertheless provides a selective pressure for reduced levels of HDV genome replication. Such a reduction is apparently achieved by a host cell activity that edits the HDV RNA genome.

The affinities of monoclonal antibodies against core antigen of hepatitis B virus.

Four monoclonal antibodies generated against the recombinant core antigen of hepatitis B virus are investigated for antigen binding. All exhibit a similar affinity to polystyrene-sorbed antigen but only one of them interacts with native form of HBcAg (an assembled particle) in solution. The presence of 0.1% sodium dodecylsulphate is required for the binding of other three antibodies. The phenomenon can be interpreted as inaccessibility of the corresponding epitopes unless the multimeric antigen structure is disrupted. The core antigen coated on polystyrene is considered as a similar exposed structure.

RNA editing in the replication cycle of human hepatitis delta virus.

For some time it has been known that the RNA genome of human hepatitis delta virus (HDV) undergoes a specific RNA editing event. This review describes the editing phenomenon and its potential biological significance, and evaluates the data regarding the mechanism involved, including the possible relationship to other RNA editing phenomena.

Immunochemical structure of the carboxy-terminal part of hepatitis B e antigen: identification of internal and surface-exposed sequences.

The C-terminal region of hepatitis B virus (HBV) e antigen (HBeAg), amino acids (aa) 121 to 147, was characterized for reactivity with 15 monoclonal antibodies (MAbs) and sera from 16 chronic carriers on the HB surface antigen with anti-HBe. Recombinant proteins exposing fragments of the HBc/e polypeptide (aa 29 to 176, 60 to 176, 101 to 176, 121 to 176, 134 to 176, 138 to 176, 139 to 176, 140 to 176, 146 to 176 and 156 to 176) fused to the N terminus of the coat protein of RNA phage fr were constructed, as were two sets of synthetic peptides covering residues 121 to 136 and 130 to 147, where each residue was sequentially substituted by alanine. The MAbs were found to recognize overlapping epitopes in the fusion proteins within residues 121 to 176; however, none of the MAbs reacted with proteins covering residues 146 to 176 and 156 to 176. Using the synthetic peptides it was found that the MAbs recognized epitopes at residues 128-TPPAYR-133, 133-RPPNAP-138, 135-PNAPIL-140, 138-PILSTLPE-145 and 143-LPET-146. Only MAbs recognizing the epitope 128-TPPAYR-133 were found to react with both native HBeAg and denatured HBcAG, whereas MAbs recognizing epitopes located closer to the C terminus of HBeAg were reactive only with denatured HBcAg. The recognition sites for the human IgG1 overlapped with the epitopes of the MAbs recognizing native HBeAg. Our interpretation of these findings is that the region 124 to 133 is on the surface of native HBeAg and denatured HBcAg, and that the adjacent region 135 to 147 is not accessible on the surface of native HBeAg, but becomes exposed on denatured HBcAg.

The structure of the variable regions of mouse monoclonal antibodies to hepatitis B virus core antigen.

From a panel of monoclonal antibodies (MoAbs) directed against E. coli-derived native and denatured hepatitis B virus (HBV) core antigen we have selected a set of specific MoAbs which recognize different linear antigenic determinants: MoAb C1-5--cl epitope; MoAb 14K8--less immunogenic N-terminal region; and MoAbs 13C9, 10F10 and 14E11, 14G3--the immunodominant region between amino acids 134 and 140. We have applied the polymerase chain reaction technique to clone Ig VH and VL region genes, and appropriate full-length cDNA clones were obtained and characterized by nucleotide sequence analysis. Among the six heavy chain variable region sequences examined, three VH families were represented. Two of them belong to the 7183 (MoAb C1-5) and 3609 (14B8) families respectively and four, having only two amino acid changes in the CDR2 region, to the J558 family. These four probably are derived from a single expanded B-cell clone. The light chain sequences indicate that their VL are encoded by V kappa 21, V kappa 19 and V kappa 3 germline genes. Unlike VH genes, light chain genes are closely related to known representatives of mouse kappa light chain families and are employed also by MoAbs raised against other antigens.

Epitopes recognized by antibodies to denatured core protein of hepatitis B virus.

Particulate and denatured core protein as well as e-antigen (HBe) of hepatitis B virus (HBV) differ in part immunologically but this has not been studied in sufficient detail. Therefore, in this study the B-cell immune response to native and denatured HBV core protein which both can exhibit HBe-specific epitopes was examined using a panel of mouse MABs and rabbit polyclonal antibodies to native and denatured core protein and polyclonal anti-HBe/anti-HBc antibodies from sera of infected patients. Epitope mapping was performed using a set of partially overlapping synthetic HBc peptides, carboxy-terminally truncated HBc proteins and various HBc fusion proteins. A major immunogenic region between amino acids 134-140 and two less immunogenic regions, one spanning amino acids 2-10 and one with three partially overlapping epitopes between amino acid positions 138 and 154, were defined by mouse MABs. Polyclonal rabbit antibodies to denatured HBc, woodchuck and ground squirrel hepatitis core proteins (WHc and GSHc) recognized similar epitopes but in addition occasionally region 61-85, and the latter was also recognized on particulate HBc. Two antigenic regions (amino acid positions 2-10 and 138-145) were found to be exposed on HBe from human serum, and were recognized by mouse anti-HBe but not by anti-HBc antibodies from sera of infected patients. This study demonstrates a more complex pattern of HBc and HBe epitopes than detected previously and provides tools to study conformational changes which may take place during HBc/HBe processing, transport and core particle assembly.

Tetrapeptide QDPR is a minimal immunodominant epitope within the preS2 domain of hepatitis B virus.

A hepatitis B virus preS2 deletion library with the preS2 sequence fused to the coat protein of the RNA phage fr (fr CP) as a carrier has been constructed and used for the approximate localization of epitope recognized by a panel of murine monoclonal anti-preS2 antibodies. DNA copies of putative preS2 epitopes were synthesized and cloned within the fr CP gene. Tetrapeptide Gln-Asp-Pro-Arg (QDPR) corresponding to the preS (132-135) sequence was found to be the minimal sufficient recognition site for one of the monoclonal antibodies, S26. The closely related tetrapeptide EDPR did not mimic the epitope activity of QDPR.

Expression of hepatitis B virus large envelope protein in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for hepatitis B large envelope protein was cloned under the lac promoter in bacterial vector pUC-8 and under the ADH1 promoter in yeast expression shuttle vector pVT103-U, and expression of HBsAg in bacteria and yeast was determined. The strongest expression of large envelope protein was obtained after transformation of the protease-deficient yeast strain BJ1991. The recombinant large envelope protein did not form complex 22-nm particles and was not secreted into medium.