Putative reverse transcriptase intermediates of human hepatitis B virus in primary liver carcinomas.

Nucleocapsid-pol fusion proteins have been detected by serological screening hepatocellular carcinoma tissues that contain hepatitis B virus (HBV) DNA. The existence of these fusion proteins suggests that HBV may synthesize its reverse transcriptase in a fashion analogous to the way that retroviruses synthesize and process a precursor. The accumulation of HBV reverse transcriptase intermediates in tumorous tissues and not in other tissues may be related to the absence of viral core particles and possibly contributes to tumor development.

Detection of antibodies to proteins encoded by the X-region of hepatitis B virus (HBV) in sera of patients with chronic HBV infection: correlation with other HBV markers.

The genome of hepatitis B virus (HBV) contains a fourth open reading frame (ORF), designated X-region. It was the aim of our study to test sera of patients with chronic HBV infection for the presence of antibodies reactive with a 17 kd gene product of the X-ORF. For this purpose, a 35S-X-ORF-encoded protein, synthesized in vitro, was applied as antigen for the detection of antibodies to HBx proteins in sera of 86 individuals. Antibodies reacting with a gene product of the X-ORF were present in 10 out of 24 HBsAg-positive patients with hepatocellular carcinoma (PLC) or liver cirrhosis and in one out of 8 HBsAg-carriers. In addition, the antibodies could also be detected in 6 out of 35 sera from patients with PLC or cirrhosis negative for HBsAg but positive for anti-HBc and anti-HBs. Antibodies to a gene product of the X-ORF can be detected in sera of patients with chronic HBV-related liver disease, independently of HBsAg and the HBeAg/anti-HBe system.

Pre-S1 proteins in sera of patients positive for HBsAg and antibodies to hepatitis delta virus.

Infection with the hepatitis Delta virus results in a reduction in hepatitis B virus replication. To study the question as to whether expression of large surface (pre-S1) protein is changed in patients with previous and chronic hepatitis Delta virus infection, sera of 25 HBsAg- and anti-HD-positive patients were analyzed by the Western blot technique using an antibody directed against a pre-S1 fusion protein. Pre-S1 proteins were present only in 3 of the 25 sera. This finding suggests that the expression of pre-S1 proteins and HBsAg is regulated independently, and that pre-S1 proteins are not necessarily required for the envelope of hepatitis Delta virus.

CD11c as a transcriptional biomarker to predict response to anti-TNF monotherapy with adalimumab in patients with rheumatoid arthritis.

We performed transcription profiling using monocytes to identify predictive markers of response to anti-tumor necrosis factor (anti-TNF) therapy in patients with rheumatoid arthritis (RA). Several potential predictors of response were identified, including CD11c. Validation in samples from independent cohorts (total of n = 27 patients) using reverse transcription-PCR confirmed increased expression of CD11c in responders to adalimumab (100% sensitivity; 91.7% specificity, power 99.6%; alpha = 0.01). Pretherapy CD11c levels significantly correlated with the response criteria as defined by the American College of Rheumatology (ACR) (r = 0.656, P < 0.0001). However, CD11c was neither predictive of response to methotrexate (MTX) alone (n = 34) nor to MTX in combination with adalimumab (n = 16). Clinical responders revealed a reset to a normal expression pattern of resident/inflammatory monocyte markers, which was absent in nonresponders. Therefore, an analysis of key cell types identifies potentially predictive biomarkers that may help to restrict the use of adalimumab to therapy responders. Larger studies, including studies of monotherapy with other drugs, are now needed to confirm and validate the specificity of CD11c for anti-TNF biologics.

Expression of the hepatitis B virus core gene in vitro and in vivo.

The core gene of hepatitis B virus contains two in-phase AUG codons which may both be used in the viral life cycle. By in vitro translation of transcripts produced in vitro, we investigated the corresponding core gene products and their counterparts in vivo. Depending on the location of the 5' end of the transcripts, two major core gene-derived proteins were obtained. In transcripts with both in-phase AUGs, only the first one was efficiently used and resulted in synthesis of a 25-kilodalton protein (precore). This protein contains a leader sequence and could be cotranslationally processed to a protein of 22.3 kilodaltons. Translation of transcripts lacking the first AUG of the core gene produced a core protein of 21.5 kilodaltons which comigrated with the core antigen expressed in infected livers. These data suggest that the major nucleocapsid protein expressed in vivo is initiated at the second ATG of the C gene and that a precore protein is probably synthesized as a precursor protein which is cotranslationally processed. Proteins consistent in size with processed and unprocessed precore proteins detected in woodchuck hepatitis virus-infected livers support this conclusion.

Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus.

The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. The single conformational determinant responsible for HBc antigenicity in the assembled core (HBc) and a linear HBe-related determinant (HBe1) were both mapped to an overlapping hydrophilic sequence around amino acid 80; a second HBe determinant (HBe2) was assigned to a location in the vicinity of amino acid 138 but found to require for its antigenicity the intramolecular participation of the extended sequence between amino acids 10 and 140. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis B virus nucleocapsid.

The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation.

Analysis of the serum of duck hepatitis B virus (DHBV)-infected ducks has revealed the presence of C-terminally truncated viral core proteins (e antigens). These proteins are glycosylated and therefore were not released from infected cells by lysis but rather by active secretion, indicating that the DHBV core protein can be synthesized alternatively as a cytoplasmic or a secretory protein. Transient expression of cloned wild-type DHBV DNA and of a specifically designed viral mutant in a human hepatoma cell line (Hep-G2) showed that the DHBV core gene promoter is active in differentiated human liver cells and that synthesis and secretion of the processed core proteins are dependent on the expression of the pre-C region, a small open reading frame which precedes the core gene. In addition, these experiments showed that the mechanism of core protein processing and secretion is conserved between DHBV and the human hepatitis B virus and therefore might be important for the hepatitis B virus life cycle in general. In spite of this, intrahepatic injection of the pre-C mutant into uninfected ducks resulted in viremia without concomitant e-antigen synthesis, indicating that virus formation is independent of pre-C expression.

Expression and structural and functional properties of human ferritin L-chain from Escherichia coli.

The human ferritin L-chain cDNA was cloned into a vector for overproduction in Escherichia coli, under the regulation of a lambda promoter. The plasmid obtained contains the full L-chain coding region modified at the first two codons. It is able to direct the synthesis of the L-chain which can constitute up to 15% of the total soluble protein of bacterial extract. The L-chains assemble to form a ferritin homopolymer with electrophoretic mobility, molecular weight, thermal stability, spectroscopic, and immunological properties analogous to natural ferritin from human liver (95% L-chain). This recombinant L-ferritin is able to incorporate and retain iron in solution at physiological pH values. At variance with the H-ferritin, the L form does not uptake iron at acidic pH values and does not show detectable ferroxidase activity. It is concluded that ferritin L-chain lacks the ferroxidase site present in the H-chain and that the two chains may have specialized functions in intracellular iron metabolism.

Detection of hepatitis B virus core gene products in sera and liver of HBV-infected individuals.

The C gene of hepatitis B virus (HBV) codes for at least two different proteins (p 21c and p 17e). To investigate the expression of C-gene-encoded proteins in vivo, serum and liver samples from HBsAg-positive patients as well as serial serum samples from an HBV-transfected chimpanzee were studied. Antibodies directed against bacterially synthesized C-fusion proteins were used in Western blots to test for the presence of p 21c and p 17e. In serial serum samples from the chimpanzee, p 21c and p 17e were detected concomitantly during the acute phase of the infection. When sera of patients with chronic HBV infection were studied, all sera containing p 17e were found to be positive also for p 21c. Sera positive for HBV DNA but negative for HBeAg were only positive for p 21c, indicating that HBeAg/p 17e is not an absolutely reliable marker for infectivity. In liver tissue specimens from 20 patients with HBV-related liver diseases, p 21c was detected in five cases, indicating viral replication. The p 17e antigen, however, was present only in low amounts in three of these five, suggesting that synthesis of p 21c and p 17e is not strictly coupled. C/Pol-gene-encoded fusion proteins were found in the liver tissue of only one patient with cirrhosis, supporting our previous finding that detectable levels of these proteins are expressed rarely.

Synthesis of the X-protein of hepatitis B virus in vitro and detection of anti-X antibodies in human sera.

A protein of 154 amino acids, predicted to be encoded by the X-open reading frame of the hepatitis B virus (HBV) genome, was synthesized in an in vitro translation system from SP6 transcripts containing the X-coding sequence. As characterized by SDS-PAGE and immunoprecipitation this X-protein possesses the expected molecular weight of 17 kDa and reacts specifically with rabbit antisera directed against a fusion protein from Escherichia coli that contained 145 of the 154 amino acids from the X-sequence. The X-protein, radiolabeled with [35S]methionine, provided a sensitive and specific antigen to screen for anti-X antibodies in sera from HBV patients. Positive signals were obtained preferentially in subjects suffering from HBV-induced liver cirrhosis or primary hepatocellular carcinoma (PHC), i.e., individuals that had been exposed for an extended time period to HBV gene products. Carefully controlled experiments failed to reveal the presence of X-related proteins specific to liver specimens from HBV patients.

Detection of pre-S1 proteins in serum and liver of HBsAg-positive patients: a new marker for hepatitis B virus infection.

The presence of pre-S1 proteins in serum and liver of individuals with acute and chronic hepatitis B virus infection was investigated in Western blots using antibodies against a fusion protein, containing amino acids 20-120 of the pre-S region. Pre-S1 proteins were present in 20 of 38 HBsAg-positive sera. All sera positive for pre-S1 proteins were also positive for hepatitis B virus DNA indicating the presence of hepatitis B virions, and 16 of these sera were also positive for HBeAg. In five sera positive for hepatitis B virus DNA, pre-S1 proteins were not found. In an additional study, pre-S1 proteins could be detected in 4 of 6 patients with acute hepatitis B virus infection during the first 2 weeks after admission to the hospital. The presence of pre-S1 proteins showed a good correlation with the detection of hepatitis B virus DNA. After seroconversion from HBeAg to anti-HBe, both hepatitis B virus DNA and pre-S1 proteins were no longer detectable. Pre-S1 proteins were present in three liver tissue specimens from two patients with acute hepatitis B virus infection and from one patient with cirrhosis of the liver. The proteins were not found in the liver of two HBsAg-positive patients with hepatocellular carcinoma (primary liver carcinoma), negative for HBeAg. Pre-S1 proteins can be detected in serum, positive for hepatitis B virus DNA and in liver tissue of hepatitis B virus-infected individuals. The presence of these proteins appears to correspond with the presence of hepatitis B virus DNA, both markers indicating hepatitis B virus replication.

Structure of human ferritin light subunit messenger RNA: comparison with heavy subunit message and functional implications.

Ferritin has a protein shell of 5 X 10(6) Da consisting of 24 subunits of two types, a heavier (H) chain of 21,000 Da and a lighter (L) chain of 19,000 Da. A cDNA clone of the messenger for the L subunit has been isolated from a human monocyte-like leukemia cell line. The clone contains an open reading frame of 522 nucleotides coding for an amino acid sequence matching 97% of the published sequence of human liver ferritin L subunit determined by sequenator, but it corresponds to only 55% of the reported amino acid sequence of a human liver H-subunit clone. Nevertheless, computer analysis of the subunit conformations predicted from the open reading frames of the L and H clones shows that most of the amino acid differences are conservative and would allow both subunits to form the five alpha-helices and beta-turns established by x-ray crystallography for horse spleen ferritin subunits. This suggests that L and H subunits are structurally interchangeable in forming an apoferritin shell. The 5' untranslated region of our human ferritin L clone has considerable homology with that of the rat liver ferritin L clone in the region immediately upstream from the initiator codon, notably showing an identical sequence of 10 nucleotides at the same position in both subunit clones that may participate in regulating the known activation of ferritin mRNA after iron administration. Extensive homology, including several blocks of nucleotides, was identified between the 3' untranslated regions of the human and rat L clones. The common structural features of the H and L subunits lead us to conclude that they have diverged from a single ancestral gene.

A tripartite HIV-1 tat-env-rev fusion protein.

A 26 kd protein reactive with antiserum to the transactivator tat of the Human Immunodeficiency Virus Type 1 (HIV-1) has been detected in virus producing cells. The 26 kd protein is shown to be a tripartite fusion protein including coding sequences of the tat, envelope (env) and regulator of virion expression (rev) genes. Fusion of these coding sequences occurs by use of a previously undescribed exon within env. This 26 kd protein, designated tnv, has tat but no rev activity detectable with the assay used. The existence of other less abundant tat and rev related proteins in HIV-1 producing cells is also noted.

The influence of purified recombinant human heavy-subunit and light-subunit ferritins on colony formation in vitro by granulocyte-macrophage and erythroid progenitor cells.

Purified recombinant human heavy subunit (rHF, acidic) and recombinant human light subunit (rLF, basic) ferritins were assessed for their effects in vitro on colony formation by normal human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells. The purity of the samples was confirmed by electrophoresis in both nondenaturing and denaturing conditions and silver staining. Concentrations of 10(-8) to 10(-10) mol/L rHF caused an approximately 40% significant decrease in colony formation. Some significant activity was detected at 10(-11) mol/L, and activity was lost at 10(-12) mol/L. In contrast, rLF had no significant activity at 10(-8) to 10(-16) mol/L. rHF was significantly active against mouse bone marrow CFU-GM to concentrations as low as 10(-8) to 10(-9) mol/L. The inhibitory activity of rHF was inactivated with three different monoclonal antibodies recognizing the heavy subunit of ferritin, but not with two monoclonal antibodies recognizing the light subunit of ferritin. The inhibitory activity of rHF was similar in the absence or presence of serum, monocytes, and T lymphocytes. We and others have shown an association of a glycosylated natural acidic isoferritin (AIF) with inhibitory activity, but since the rHF was expressed in Escherichia coli and did not bind to concanavalin A, glycosylation of AIF is not an absolute prerequisite for this activity. These results demonstrate that rHF has suppressive activity in vitro and substantiate our original observations using purified natural acidic isoferritins.

Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock.

IL-1 beta-converting enzyme (ICE) cleaves pro-IL-1 beta to generate mature IL-1 beta. ICE is homologous to other proteins that have been implicated in apoptosis, including CED-3 and Nedd-2/lch-1. We generated ICE-deficient mice and observed that they are overtly normal but have a major defect in the production of mature IL-1 beta after stimulation with lipopolysaccharide. IL-1 alpha production is also impaired. ICE-deficient mice are resistant to endotoxic shock. Thymocytes and macrophages from the ICE-deficient animals undergo apoptosis normally. ICE therefore plays a dominant role in the generation of mature IL-1 beta, a previously unsuspected role in production of IL-1 alpha, but has no autonomous function in apoptosis.

Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core-related antigens (HBc/HBeAg) if well-differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH-7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH-7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus-associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS--PAGE after labeling with [35S]methionine. Antisera specific for X-gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV-related gene function and HBV replication.