Genotype-specific complementation of hepatitis delta virus RNA replication by hepatitis delta antigen

Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.     

Modeling perceived relationships between melody, harmony, and key

A small open reading frame (ORF) was found in the hepatitis delta virus (HDV) antigenomic RNA encoding a short peptide that shares structural similarity with a region of the hepatitis B virus terminal protein. Analysis of all published HDV genome sequences indicates a high degree of conservation for the small ORF. This ORF is located at the 3'-terminal region of the gene encoding the hepatitis delta antigen (HDAg). We speculated that a peptide encoded by this ORF can be represented as the C-terminal domain of a new protein called HDAg'. This protein contains almost the entire sequence represented in the small form of HDAg and a peptide as an additional 'extension' sequence at the C-terminus. Two long synthetic peptides representing the two different types of peptides encoded by the small ORF were synthesized. These peptides were used for the development of an immunoassay for the detection of antibody to the HDAg' specific domain in sera of patients with HDV infection. Among 162 serum samples analyzed, 13 were found to be positive for an antibody reactive with these synthetic peptides. These antibodies were identified in patients with HDV infections and were not found in patients infected with hepatitis B virus, hepatitis C virus, or non-A,non-B,non-C virus. Thus, these data support the identification and existence of a new antigen encoded by the antigenomic RNA of the HDV.     

Immune response to synthetic peptides of hepatitis delta antigen

Hepatitis delta antigen (HDAg) is the only viral protein known to be expressed during hepatitis delta virus (HDV) infection. Detection of antibody to HDAg (anti-HD) is the usual method for diagnosis of HDV infection since viremia lasts only a few weeks. In an effort to identify the major epitopes recognized by humans during natural infection, four oligopeptides including residues 2 to 17 (SP1), 155 to 172 (SP2), 168 to 182 (SP3), and 189 to 211 (SP4) of the HDAg molecule were synthesized and probed by enzyme-linked immunosorbent assay with a panel of 80 serum specimens from 45 patients suffering from either HDV-hepatitis B virus coinfections (n = 17) or HDV superinfections (n = 28). Sera from infected patients recognized these relatively short peptides. Peptide SP2 was the most antigenic; 71% of serum specimens reacted. Antibody to SP2 was also the commonest in sera taken early in the course of the disease. Peptide SP2 corresponds to one of the two regions which is highly conserved between different isolates. Among the 63 serum specimens which scored anti-HD positive by a commercial assay, all but 3 reacted to at least one of the peptides (95% agreement). Peptide assays appeared to be significantly more sensitive than the commercial assay with native HDAg early in the course of HDV infection since 14 of 17 (82%) serum specimens which scored anti-HD negative in the commercial assay reacted to one or more peptides. All serum specimens giving one or more positive results with the various peptides were confirmed as being HDV positive by an inhibition assay with free peptide in solution. The immune response to HDAg peptides vared greatly between individuals. No specific reactivity profile could be assigned to those with either HDV-hepatitis B virus coinfections or HDV superinfections. Overall, HDAg peptides appeared to be convenient reagents in addition to native antigen for the development of new and improved diagnostic tests for HDV infection.     

Human CD4+ T-cell response to hepatitis delta virus: identification of multiple epitopes and characterization of T-helper cytokine profiles

The T-cell-mediated immune response plays a crucial role in defense against hepatotropic viruses as well as in the pathogenesis of viral chronic hepatitides. However, very little is known about the role of specific T cells during hepatitis delta virus (HDV) infection in humans. In this study, the T-cell response to HDV in chronic hepatitis B virus (HBV) carriers with HDV superinfection was investigated at different levels. Analysis of peripheral blood mononuclear cell (PBMC) proliferation in response to a recombinant form of large hepatitis delta antigen (HDAg) revealed that 8 of 30 patients studied (27%) specifically responded to HDAg. By employing synthetic peptides spanning the entire HDAg sequence, we found that T-cell recognition was directed against different antigenic determinants, with patient-to-patient variation in the pattern of response to peptides. Interestingly, all responders had signs of inactive HDV-induced disease, while none of the patients with active disease and none of the control subjects showed any significant proliferation. More accurate information about the specific T-cell response was obtained at the clonal level. A panel of HDAg-specific CD4+ T-cell clones from three HDV-infected individuals and fine-specificity analysis revealed that the clones tested individually recognized four epitopes corresponding to amino acids (aa) 26 to 41, 50 to 65, 66 to 81, or 106 to 121 of HDAg sequence. The study of human leukocyte antigen (HLA) restriction revealed that peptides 50 to 65 and 106 to 121 were presented to specific T cells in association with multiple class II molecules. In addition, peptide 26 to 41 was efficiently generated after processing of HDAg through the endogenous processing pathway. Cytokine secretion analysis showed that all the CD4+ T-cell clones assayed were able to produce high levels of gamma interferon (IFN-gamma), belonging either to T helper-1 (Th1) or Th0 subsets and that some of them were cytotoxic in a specific assay. This study provides the first evidence that detection of a specific T-cell response to HDAg in the peripheral blood of individuals with hepatitis delta is related to the decrease of HDV-induced disease activity. The HDAg epitopes identified here and particularly those recognized by CD4+ T cells in association with multiple major histocompatibility complex class II molecules may be potentially exploited for the preparation of a vaccine for prophylaxis and therapy of HDV infection.     

Hepatitis delta virus antigen forms dimers and multimeric complexes in vivo

Although the hepatitis delta virus genome contains multiple open reading frames, only one of these reading frames is known to be expressed during replication of the virus. This open reading frame encodes two distinct molecular species of hepatitis delta antigen (HDAg), p24 delta and p27 delta, depending on the location of the stop codon which terminates translation. We found antibody specific for p27 delta to be capable of precipitating p24 delta in extracts of infected liver, indicating that p27 delta and p24 delta form heterologous complexes in vivo. After cross-linking with 0.05% glutaraldehyde, specific HDAg dimers were detected in antigen prepared from both the liver and serum of an HDV-infected woodchuck carrier of woodchuck hepatitis virus. Guanidine HCl-denatured HDAg extracted from liver and dialyzed against phosphate-buffered saline sedimented in rate-zonal sucrose density gradients as 15S multimeric complexes. These 15S multimers were stable in the presence of 1.2% Nonidet P-40. After RNase digestion, the 15S complex was reduced to a 12S complex without associated RNA, while boiling for 3 min in 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol further reduced the 15S complex to 3S HDAg monomers. In the absence of glutaraldehyde cross-linking, HDAg extracted from liver migrated as monomer species in reducing and nonreducing gels, suggesting that the conserved cysteine residue present in p27 delta does not play a role in the formation of either dimers or multimers. On the other hand, an amino-terminal chymotrypsin-digested HDAg fragment, with a predicted length of 81 or less amino acids, retained the ability to form dimers, consistent with the hypothesis that a coiled-coil motif present between residues 27 and 58 may play a role in HDAg protein interactions in vivo.     

Molecular biology of hepatitis D virus: research and potential for application

Superinfection by hepatitis D virus (HDV) leads to acute hepatitis and causes progression to liver cirrhosis in a significant proportion of hepatitis B surface antigen (HBsAg) carriers. Current regimens (interferon) to treat hepatitis D patients has only transient but no lasting effects. New approaches are, therefore, warranted. Recently, several laboratory studies have discovered interesting properties of HDV that may become targets for antiviral chemicals. Viral replication requires the small hepatitis delta antigen (s-HDAg). The s-HDAg is a nuclear phosphoprotein. There is evidence indicating that phosphorylation is important for HDV replication. A second step of replication requires HDV-RNA self-cleavage and self-ligation. Interestingly, one group of antibiotics, the aminoglycosides, exerts strong suppression effects on HDV ribozyme activities. In the following stage of viral assembly, two post-translational modifications, namely isoprenylation of large HDAg and glycosylation of HBsAg are involved. Agents capable of blocking the two modifications should reduce viral production. These four possible targets are reviewed. For prevention, effective vaccines are not yet available. Two novel approaches are discussed. The first demonstrates the immunogenicity of a nucleic acid vaccine in mice. The second approach assembled an empty HDV particle in yeast. Advances on such laboratory investigations may provide new methods for the control of hepatitis D in the future.     

Immunohistochemical study of hepatitis B and D virus antigens (HBsAg, HBcAg, HDAg) in chronic liver diseases

Authors have investigated the hepatitis B and D virus antigens in the liver tissue of 30 HBsAg and/or anti-HD seropositive patients (23 males, 7 females, age: 20-65, mean: 44 years) by immunohistochemical method. The immunohistochemical identification of HBsAg, HBcAg and HDAg in 42 liver sample (36 obtained by percutaneous biopsy, 6 from dissection) of 30 patients was performed with Dako and Sorin Biomedica kits. The detailed virus serologic examinations were carried out with Biomedica and Abbott kits by radioimmunoassay and ELISA methods. Examining 36 liver tissue samples of 27 HBsAg seropositive patients, HBsAg could be demonstrated in 31 cases. Each patient suffering of active HBV and/or HDV replication was HBsAg positive by immunohistochemistry, while the tissue samples of patients in integrational phase of HBV infection were positive in only 9 cases of 14. There was no HBsAg tissue positivity in HBsAg seronegative cases. 7 of 16 tissue samples of 12 patients classified to active HBV replication state were HBcAg positive by immunohistochemistry. HBcAg could be detected in the liver tissue of each HBe seropositive patient, while in only 3 of 8 cases with only IgM anti-HBc seropositivity (indicating low level of HBV replication). Tissue HBcAg positivity, indicating active virus replication, was verified in 2 of 11 patients classified to HBV integration state by serology. Authors detected HDAg tissue positivity only in cases with serologically active HDV replication (IgM anti-HD seropositive) and HDAg could also be identified from liver tissue in each IgM anti-HD seropositive case. No HBsAg, HBcAg and HDAg tissue positivity was observed in HBsAg negative cases. Authors emphasise mainly the importance of immunohistochemical detection of HBcAg and HDAg completing the serologic diagnosis of chronic HBV and HDV infections, helping the verification or exclusion of active virus replication being essential for selecting adequate therapy.     

Recurrence of hepatitis D (delta) in liver transplants: histopathological aspects

BACKGROUND: The viral/pathological correlates of recurrent hepatitis delta virus (HDV) disease in orthotoptic liver transplants are reported. METHODS: We examined the histological features of recurrent HDV disease in nine patients with transplants for terminal HDV cirrhosis were examined; intrahepatic HDV and hepatitis B virus (HBV) antigens were detected by immunoperoxidase techniques. Sera were tested for the battery of HDV and HBV markers. RESULTS: In four patients, HDV reinfection was accompanied by the recurrence of an HBV infection with features of active viral replication. In the other five, HDV reinfection was accompanied by an atypical recurrence of HBV infection without evidence of active HBV replication (no expression of intrahepatic hepatitis B core antigen). In four of the latter patients, the atypical HBV pattern changed during the follow-up into a pattern of active viral replication accompanied by chronic necroinflammation detected during histology. CONCLUSION: The pattern of recurrent HBV infection can influence the pathological aspects of the relapses of HDV disease in liver grafts.     

Inhibition of Sindbis virus replication by cyclopentenone prostaglandins: a cell-mediated event associated with heat-shock protein synthesis

Cyclopentenone prostaglandins (PGs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report on the effect of prostaglandin A1 (PGA1) on the multiplication of a positive strand RNA virus, Sindbis virus, in Vero cells under one-step multiplication conditions. PGA1 was found to inhibit Sindbis virus production dose-dependently, and virus yield was reduced by more than 90% at the concentration of 8 micrograms/ml, which was non-toxic to the cells and did not inhibit DNA, RNA or protein synthesis in Vero cells. The cyclopentenone prostaglandin delta 12-PGJ2 was also shown to be a potent inhibitor of Sindbis virus replication. Virus-induced reduction of [3H]uridine uptake by cells was partially prevented by PGA1 treatment, which also caused a 1 h delay in the peak of virus RNA synthesis. SDS-PAGE analysis of [35S]methionine-labeled proteins showed that PGA1 moderately inhibited the synthesis of the viral structural proteins E1, E2 and C, and induced the synthesis of a 72 kDa M(r) protein, identified as a heat-shock protein related to the HSP70 group, in both virus-infected and uninfected cells. Actinomycin D treatment completely prevented PGA1-antiviral activity, indicating that a cellular product is responsible for this action. PGA1-induced HSP70 is a good candidate for this role.     

Identification and characterization of the RNA chaperone activity of hepatitis delta antigen peptides

In this study, we identified an activity of the hepatitis delta antigen that both modulates the cis-cleaving activities of hepatitis delta virus (HDV) genomic RNA fragments and facilitates the trans-cleavage reactions between hammerhead ribozymes and the cognate substrates of various lengths in vitro. Hepatitis delta antigen peptides exert their effect by accelerating the unfolding and refolding of RNA molecules and by promoting strand annealing and strand dissociation. In addition, the stimulatory effect of hepatitis delta antigen peptide on hammerhead catalysis is observed whether the peptide is removed or not by phenol/chloroform extraction prior to the initiation of trans-cleavage reaction. Therefore, hepatitis delta antigen peptide acts as an RNA chaperone. The RNA chaperone domain of hepatitis delta antigen overlaps with the coiled-coil domain that is rich in lysine residues. The RNA binding domains of hepatitis delta antigen previously identified are not required for the RNA chaperone activity identified herein. The RNA chaperone activity of hepatitis delta antigen may be important for the regulation of HDV replication in vivo.     

The intrahepatic expression of the hepatitis B virus in chronic delta hepatitis

In order to evaluate the interference of hepatitis delta virus (HDV) in hepatitis B viral particle (HBsAg, HBcAg) expression in the liver of chronic HDV patients, 39 and 81 liver biopsies of HBsAg carriers seropositive for anti-HDV and anti-HDV negative controls, respectively, were studied. HBcAg was positive in 16.7% of the HBeAg-positive patients with HDAg in the liver and in 91,4% of controls. In contrast, in HBeAg- and anti-HDV negative patients the intrahepatic expression of HBcAg was detected in 32.6%. In anti-HDV negative patients the HBcAg liver expression correlated significantly with the HBeAg in serum (p < 0.00001). The distribution of HBcAg was exclusively cytoplasmatic in 30% of HDV-infected patients but mixed nuclear and cytoplasmic in 38.3% of the controls. The nuclear expression of HBcAg was decreased in chronic HDV infection. HBsAg was positive in 70.3% of patients who were anti-HDV positive and in 82.3% of controls. The membranous expression of HBsAg was detected less frequently in HDV-infected patients (p < 0.05) than in controls, while associated with HBeAg in serum of HBV carriers without HDV superinfection (p < 0.00001). The prevalence and the HBsAg cytoplasmic expression was not different for the chronic HDV infection or controls. Our results show: 1) decreased intrahepatic expression of HBcAg and membranous HBsAg in HBV carriers superinfected with HDV, suggesting decreased HBV replication in the liver of these patients. 2) the changing of HBcAg and HBsAg expression in the liver of HDV-infected patients, suggest not so much a decrease but rather a modulation in HBV replication.     

Intracellular expression of cellular eIF-5A mutants inhibits HIV-1 replication in human T cells: a feasibility study

Previously, we described two mutants of the cellular Rev co-factor, eukaryotic initiation factor 5A (eIF-5A M13 and M14), which suppress human immunodeficiency virus type 1 (HIV-1) SF2 replication in clonal T cell lines. This study introduced the notion that it is possible to develop gene therapies against infectious agents on the basis of mutant host factors required for viral replication. In this report, we provide further evidence to support this new paradigm and describe murine leukemia virus (MLV)-based retroviral vectors expressing three different eIF-5A mutants from the viral long terminal repeat (LTR). HIV-1 replication (SF2, HXB-3) was reduced up to 2 orders of magnitude in transduced, polyclonal T cell populations. All eIF-5A mutants also showed antiviral activity (approximately seven-fold reduction) in a chronic HIV-1 infection model. Expression of eIF-5A mutant M13 delta in peripheral blood lymphocytes (PBLs) showed no difference in proliferation and metabolic activity as determined in a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT)-assay, suggesting that expression of this type of mutant protein is not associated with cellular toxicity. In summary, these data suggest that gene therapy for HIV-1 infection can be developed on the basis of mutants of the Rev co-factor eIF-5A.     

Effect of mutations in the small envelope protein of hepatitis B virus on assembly and secretion of hepatitis delta virus

The gene coding for the small (S) envelope protein of hepatitis B virus was mutated to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. This study was focused on a domain of the S protein that is exposed in the cytoplasm during synthesis and thereby represented a good candidate for interaction with the viral nucleocapsid during virion assembly. The mutations consisted of deletion/insertions spanning the entire cytosolic domain of S between amino acid residues 24 and 80. Although the expression of mutants clustered between residues 59 and 80 could not be obtained, we demonstrated that a large part of the cytosolic loop, from residues 29-47 and 49-59, does not contain motifs essential for production of hepatitis B virus subviral particles or HDV virions. However, deletion of residues 24-28 led to the synthesis of S protein mutant, which was competent for secretion of subviral particles but deficient for production of HDV. We concluded that the sequence between Arg-24 and Ile-28 located at the carboxyl boundary of the transmembrane signal I for S contains residue or residues important for HDV particle assembly.