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.     

Effect of hepatitis B virus X protein on the expression of retinoblastoma gene product

Consistent reports from several recent studies suggest that users of third generation oral contraceptives (OCs) containing gestodene and desogestrel may be at increased risk of venous thromboembolic disease (VTE). Paradoxically, other reports indicate that these users may be at decreased risk of acute myocardial infarction (MI) compared with users of second generation OCs. To determine whether the potentially increased risk of VTE would outweigh the potentially reduced risk of MI in users of third generation OCs, we conducted an analysis to quantify the trade-offs providers and users may be faced to make between these formulations. The baseline rates of VTE and MI among non-users were calculated using US data on incidence and mortality of these conditions and estimates of the proportion of women exposed to these formulations in the US. These were multiplied by relative risks published in recent studies on third generation progestins to produce age- and formulation-specific risks. Results indicate that there would be small differences in disease burden between users of second and third generation OCs under the model assumptions at younger ages. However, among women 35-44 years of age, modeling results indicate that the potentially decreased incidence of MI among users of third generation OCs more than offsets the potentially increased risk of VTE at this age.     

Dual topology of the large envelope protein of duck hepatitis B virus: determinants preventing pre-S translocation and glycosylation

The biosynthesis and topology of the large envelope protein (L protein) of hepadnaviruses was investigated using the duck hepatitis B virus (DHBV) model, which also allows the study of hepadnavirus morphogenesis in experimentally infected hepatocytes. Results from proteolysis of virus particles and from the analysis of topology and posttranslational modification of L chains synthesized in vivo or in a cell-free system both support the presence of a mixed population of L-protein molecules with their N-terminal pre-S domain located either inside or outside the virus particle. During L biosynthesis and DHBV morphogenesis, pre-S, together with the neighboring transmembrane domain (TM-I), initially remained cytoplasmically disposed and was translocated only posttranslationally. Delayed pre-S translocation into a post-endoplasmic reticulum compartment is also indicated by the absence of glycosylation at a modification-competent pre-S glycosylation site. Major features of L-protein biosynthesis and of the resulting dual topology appear to be conserved between avian and mammalian hepadnaviruses, supporting the model that pre-S domains function in part either as an internal matrix for capsid envelopment or externally as a ligand for cellular receptor binding. However, differences in the mechanisms controlling pre-S translocation were revealed by the results of mutational analyses identifying and characterizing cis-acting determinants in pre-S that delay its cotranslational translocation. Our data from DHBV demonstrate the negative influence of a cluster of positively charged amino acid residues next to TM-I, a motif that is conserved among the avian but absent from mammalian hepadnaviruses. Additional control elements, which are apparently shared between both virus groups and which may serve in chaperone binding, were mapped by deletion analysis in the central part of pre-S.     

Host cell-virus cross talk: phosphorylation of a hepatitis B virus envelope protein mediates intracellular signaling

Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S-->A) or mimicking (S-->D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell.     

Hepatosplenic gamma delta T-cell lymphoma associated with hepatitis B virus infection

Hepatitis B virus (HBV) infection has been implicated in the development of hepatocellular and hematopoietic malignancies. We describe a patient with chronic hepatitis B who developed hepatosplenic gamma delta T-cell lymphoma. A 45-year-old woman presented with marked hepatosplenomegaly and hepatic failure during the course of chronic hepatitis B. Peripheral blood examination revealed 57% abnormal lymphoid cells which expressed the gamma delta T-cell receptor. The cytogenetic analysis of tumor cells showed an abnormal karyotype; 47, XX, -13, +2mar in all 20 metaphases examined. A clonal rearrangement of the T-cell receptor genes was demonstrated by Southern blot analysis, showing monoclonal expansion of tumor cells. A liver biopsy specimen showed fibrosis of the portal areas and sinusoidal infiltration of tumor cells. HBV infection was documented by the presence of IgG anti-HBc and anti-HBs antibodies in serum. Although HBV-DNA was not detected in tumor cells by polymerase chain reaction analysis, there is a possibility that proliferation of gamma delta T cells in response to HBV infection played a role in the pathogenesis of hepatosplenic gamma delta T-cell lymphoma.     

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.     

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.     

Computer-aided studies on the spatial structure of the small hepatitis B surface protein

Progressive Multifocal Leukoencephalopathy (PML) is a subacute demyelinating opportunistic infection of the central nervous system which frequently occurs in AIDS. CT and MR, together with clinical and virological findings, can suggest a correct diagnosis in most cases, thus avoiding stereotactic biopsy which is too invasive considering the lack of therapy and the poor prognosis of this disease. In this study we reviewed the CT and MR findings of 16 proved AIDS-related PML cases. PML lesions appeared as hypodense on CT, hypointense on T1w, hyperintense on PDw and T2w MR images. CT was less sensitive than T2w MR images and underestimated the number of lesions and/or disease extent. On the basis of our findings during the progression of the lesions we observed two different patterns of PML presentation and evolution i.e., "single" and "multifocal". Single lesions generally involve subcortical white matter (arcuate fibers) of parietal lobe and spread to the contralateral hemisphere across the corpus callosum; multiple "patchy" lesions can be localized variably in the cerebral hemispheres and also in the brainstem and cerebellar hemispheres.     

Alpha interferon therapy in children with chronic active hepatitis B and delta virus infection

Two children with chronic hepatitis B virus and delta virus infection were treated with alpha interferon. Both tolerated the therapy without complications, and one demonstrated clearance of the infection and development of immunity.     

Mapping of homologous interaction sites in the hepatitis B virus core protein

Hepatitis B virus consists of an outer envelope and an inner capsid, or core, that wraps around the small genome plus the viral replication enzyme. The icosahedrally symmetric nucleocapsid is assembled from multiple dimeric subunits of a single 183-residue capsid protein, which must therefore contain interfaces for monomer dimerization and for dimer multimerization. The atomic structure of the protein is not known, but electron microscopy-based image reconstructions suggested a hammerhead shape for the dimer and, very recently, led to a tentative model for the main chain trace. Here we used a combination of interaction screening techniques and functional analyses of core protein variants to define, at the primary sequence level, the regions that mediate capsid assembly. Both the two-hybrid system and the pepscan technique identified a strongly interacting region I between amino acids (aa) 78 and 117 that probably forms part of the dimer interface. Surprisingly, mutations in this region, in the context of a C-terminally truncated but assembly-competent core protein variant, had no detectable effect on assembly. By contrast, mutations in a second region, bordered by aa 113 and 143, markedly influenced capsid stability, strongly suggesting that this region II is the main contributor to dimer multimerization. Based on the electron microscopic data, it must therefore be located at the basal tips of the dimer, experimentally supporting the proposed main chain trace.     

The defective nature of hepatitis delta virus

Forty-nine out of 66 patients with Turner's syndrome and different karyotypes underwent full cardiological evaluations (physical examination, electrocardiography, chest X-ray and echocardiography). Congenital cardiovascular anomalies were found in 11 patients (22.4%). Among the cardiac anomalies in patients with Turner's syndrome, aortic malformations (aortic coarctation [27%], aortic stenosis [18%] and bicuspid aortic valve [18%]) were the most frequent. We observed that the most severe malformations were found in those with karyotype 45,X or 45,X/46,XX. No anomalies were detected in patients with the X isochromosome or those mosaic with the Y component.     

Point mutations in the S and pre-S2 genes observed in two hepatitis B virus carriers positive for antibody to hepatitis B surface antigen

Two hepatitis B virus (HBV) carriers who had antibodies to HBV surface antigen (anti-HBs) were studied. Case 1 was a 47 year old woman positive for hepatitis B e antigen (HBeAg), and case 2 was a 61 year old man positive for antibody to HBeAg (anti-HBe) and DNA-polymerase (DNA-p). Neither case had received the HBV vaccine. The nucleotide sequences of the HBV-DNA extracted from the patients' sera were determined within the pre-S2 and S genes. Seven out of nine S gene clones from case 1 and six out of nine S gene clones from case 2 had an amino acid replacement from Thr or Ile to Ser at codon 126 in the alpha-determinant of the S gene. Amino acid substitution of codon 145 of the S gene previously reported was not observed. Although two previous reports on HBV escape mutant carriers with both anti-HBs and HBeAg described some deletions in the pre-S2 gene, our cases did not show these deletions. Our analysis indicated that carriers with the HBV escape mutant did not always have pre-S2 gene deletions. We found two HBV escape mutant carriers who had amino acid substitutions at codon 126 in the S gene due to point mutation without any deletions in the pre-S2 gene.     

Crystal structure of a hepatitis delta virus ribozyme

The self-cleaving ribozyme of the hepatitis delta virus (HDV) is the only catalytic RNA known to be required for the viability of a human pathogen. We obtained crystals of a 72-nucleotide, self-cleaved form of the genomic HDV ribozyme that diffract X-rays to 2.3 A resolution by engineering the RNA to bind a small, basic protein without affecting ribozyme activity. The co-crystal structure shows that the compact catalytic core comprises five helical segments connected as an intricate nested double pseudoknot. The 5'-hydroxyl leaving group resulting from the self-scission reaction is buried deep within an active-site cleft produced by juxtaposition of the helices and five strand-crossovers, and is surrounded by biochemically important backbone and base functional groups in a manner reminiscent of protein enzymes.     

Suppressors of cleavage-site mutations in the p62 envelope protein of Semliki Forest virus reveal dynamics in spike structure and function

The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature form, E2. Cleavage is not necessary for particle formation or release but is necessary for infectivity. Previous results had shown that phenotypic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations in the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, secondary mutations in another distant domain of the E2 protein appeared instead, all of which still caused cleavage-deficient mutations. Such mutants grew very poorly and were inefficient in virus entry and release. The mutated sites define domains of the spike protein which probably interact to regulate its structure and function. Because of their highly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, thus increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.     

The GDPAL region of the pre-S1 envelope protein is important for morphogenesis of woodchuck hepatitis virus

The pre-S envelope protein of duck hepatitis B virus (DHBV) contains a region, Asp-Asp-Pro-Leu-Leu (DDPLL), that is specifically required for virus assembly and secretion (Lenhoff and Summers, J Virol 1994;68:4565-4571). We found that amino acids 201 to 205 of the pre-S envelope protein of woodchuck hepatitis virus (WHV) form a conserved amino acid cluster, Gly-Asp-Pro-Ala-Leu (GDPAL), which resembles the DDPLL sequence of DHBV. To determine whether the GDPAL region was functionally equivalent to the DDPLL region, we deleted this region from the pre-S protein of WHV or mutated individual amino acids within the region. The mutant DNA was transfected into human hepatoma cell line Huh7, and the medium was assayed for virion production by immunoprecipitation and Southern blot analysis. We found that an in-frame deletion of this small region inhibited virion formation, suggesting that the GDPAL region of the pre-S envelope protein was required for virus assembly and/or secretion of WHV. Individual replacement of alanine 204, leucine 205, or serine 206 with other amino acid residues did not affect virus production. However, substitution of either aspartic acid 202 with valine or proline 203 with leucine dramatically inhibited WHV production. Furthermore, the GDPAL mutants were individually tested for their abilities to complement a pre-S1 defective genome. The results showed that the GDPAL region functioned as part of the pre-S1 protein but was not required to function as part of the pre-S2 protein.     

Effect of human leukocyte interferon on hepatitis B virus infection in patients with chronic active hepatitis

Four patients with chronic hepatitis B infection and chronic active hepatitis were treated with human leukocyte interferon. Three of them had consistently elevated levels of circulating Dane-particle markers, including Dane-particle-associated DNA polymerase activity, hepatitis B core antigen and Dane-particle-associated DNA. Parenteral interferon administration at a dosage between 6.0 X 10(3) and 17 X 10(4) U per kilogram per day was associated with a rapid and reproducible fall in all Dane-particle markers in the three patients. The suppressive effect was transient when the interferon was given for 10 days or less but appeared to be more permanent when administration was prolonged for a month or more. In addition, long-term interferon therapy was associated with a marked fall in hepatitis B surface antigen in two of three patients and a disappearance of e antigen in two of two patients. Interferon may be useful in limiting carrier infectivity or eradicating chronic infection.