Chronic ethanol consumption stimulates hepatitis B virus gene expression and replication in transgenic mice.

Epidemiologic observations show a higher frequency of hepatitis B virus (HBV) serologic markers in chronic alcoholics compared with the general population. This may be the result of an increased susceptibility of alcoholics to infection and/or to an ethanol-mediated stimulation of HBV gene expression and replication. To test the latter hypothesis, HBV transgenic SCID mice, which support consistent levels of virus replication, were fed with a standard Lieber-DiCarli or isocaloric diet for 5 weeks. In ethanol-fed mice, the levels of hepatitis B surface antigen (HBsAg) and viral DNA in serum increased by up to 7-fold compared with mice fed the control diet. Ethanol-treated mice also had elevated HBV-RNA levels, and increased expression of surface, core, and X antigens in the liver, especially in the pericentral regions. None of these changes were observed in transgenic mice fed isocaloric diets. Thus, chronic alcohol consumption alters the patterns of HBV gene expression and replication in the serum and liver of HBV transgenic SCID mice, and may provide a partial explanation for the increased frequency of HBV markers among alcoholics.

A cellular gene up-regulated by hepatitis B virus-encoded X antigen promotes hepatocellular growth and survival.

Polymerase chain reaction (PCR) select complementary DNA (cDNA) subtraction of hepatitis B x antigen (HBxAg)-positive compared with -negative HepG2 cells resulted in the up-regulated expression of a cellular gene that encodes a transcript of 745 bases and a polypeptide 99 amino acids long. GenBank analysis revealed extensive homology with the amino terminal domain of cellular multidrug resistant proteins (MRP), although overexpression of this gene did not confer an MRP phenotype. In situ hybridization and immunostaining showed colocalized expression with HBxAg in the liver of hepatitis B carriers. Overexpression of this protein stimulated the growth of HepG2 cells in serum-free medium, and partially protected cells from anti-Fas-mediated killing, but did not promote growth in soft agar or tumor formation in nude mice. Introduction of the dominant negative inhibitor of nuclear factor kappaB (IkappaBalpha) into HBxAg-positive HepG2 cells decreased the levels of messenger RNA (mRNA) and protein, suggesting that its up-regulation is nuclear factor kappaB (NF-kappaB) dependent. Hence, HBxAg activation of NF-kappaB may result in the up-regulation of a cellular protein that promotes growth factor-independent survival and protects against Fas-mediated killing. This factor may contribute to the persistence of infected hepatocytes during chronic infection, which is important for the later development of hepatocellular carcinoma (HCC).

Identification of a protein isolated from senescent human cells that binds to hepatitis B virus X antigen.

Hepatitis B virus-encoded X antigen contributes to the development of hepatocellular carcinoma. Given that X antigen functions by binding to other proteins, additional X-binding proteins were sought from an adult human liver cDNA library in a yeast two-hybrid system. The results yielded a clone encoding a 55-kd protein that is associated with replicative senescence (p55sen). Binding of p55sen to X antigen was confirmed in vitro by immunoprecipitation and affinity chromatography. The expression of endogenous p55sen inversely correlated with cell growth. Transient transfection of X antigen or p55sen into HepG2 cells stimulated DNA synthesis by twofold to threefold, whereas cotransfection did not, suggesting that these molecules functionally interact. The detection of p55sen in embryonic mouse liver, its absence in adult mouse and human livers, and its reappearance in livers from carriers with chronic liver disease, suggest that it may play important roles in the regulation of liver cell growth. The similarity between p55sen and a notch ligand, which is involved in cell fate determinations during embryogenesis, implies that the binding of p55sen by X antigen may also contribute to an alteration in cell fate, which is characteristic of carcinogenesis.

Partial characterization of the woodchuck tumor suppressor, p53, and its interaction with woodchuck hepatitis virus X antigen in hepatocarcinogenesis.

Full length cDNAs for p53 were made by reverse transcription-polymerase chain reaction of total RNA from two normal woodchuck livers. Two randomly chosen clones from each liver were sequenced and shown to be identical. This sequence revealed 80% or more identity with p53 sequences from human, monkey, and mouse. The cDNA was translated into a 55 kD protein in vitro that was immunoprecipitated by antibodies to p53. Cotranslation of woodchuck p53 with woodchuck hepatitis virus X antigen, followed by immunoprecipitation suggested X/p53 complex formation. Similar complexes were also immunoprecipitated from extracts of infected liver, but not from uninfected liver. The finding of X/p53 complexes in vivo and in vitro in the woodchuck hepadnavirus system, combined with analogous data with hepatitis B, suggests a common mechanism by which these viruses contribute to hepatocellular transformation.

Polymerase-related polypeptides associated with woodchuck hepatitis core antigen (WHcAg) particles.

The woodchuck hepatitis virus (WHV) polymerase (pol)-encoded polypeptide(s), obtained from purified virus nucleocapsid particles, have been characterized by Western blotting. Peptide antibodies to amino-terminal (residues 32-45, WHV pol-6) and carboxy-terminal (residues 861-879, WHV pol-1) sequences were used, in addition to monoclonal antibodies made from purified woodchuck hepatitis core antigen (WHcAg) particles. One of the monoclonal antibodies, WC pol-11, specifically bound WHV pol-1. Both peptide and monoclonal anti-WHV pol-1 also bound a recombinant DNA-produced WHV polymerase polypeptide. These antibodies specifically detected WHcAg-associated polymerase polypeptides at 65,000 (p65) and 31,000 (p31) Da by Western blotting. These results support the conclusion that WHV pol-11 has anti-pol reactivity and that it binds the carboxyl-terminal sequences of the WHV polymerase. The finding that these reagents also specifically bind to corresponding sequences from the carboxy terminus of the hepatitis B virus polymerase suggests that these viral polymerases are cross reactive. Finally, anti-WHV pol-6 did not bind either WHcAg p65 or p31, suggesting that both of these polypeptides have different amino-terminal but the same carboxy-terminal sequences.

X antigen/antibody markers in hepadnavirus infections. Antibodies to the X gene product(s).

Antibodies to the X antigen of hepatitis B virus and woodchuck hepatitis virus were assayed in serial sera from infected individuals and compared with other markers of infection. Antibody to the X antigen was found in 11 of 17 (65%) patients and 17 of 40 (42%) woodchucks that were surface-antigen positive. In comparison, this antibody was found in 5 of 14 (36%) patients and in none of 4 woodchucks that were surface-antigen negative. In 5 of 6 patients showing seroconversion from hepatitis B e antigen to antibody, antibody to X appeared at or near the time of seroconversion. In patients persistently positive for e antigen, X antibody often appeared when viral DNA became undetectable in the serum. In 14 of 17 (82%) woodchucks positive for antibody to X antigen, it also appeared near or after the time that viral DNA in serum disappeared. X antibodies were detected with great frequency only in populations with high frequencies of other hepatitis B virus markers. The results are consistent with the conclusion that antibody to X antigen is a marker of hepadnavirus infections that seems to be associated with a decrease in viral replication. Antibodies to the X antigen, then, may be a host response to the replication complex of the virus.

Monoclonal antibodies raised to purified woodchuck hepatitis virus core antigen particles demonstrate X antigen reactivity.

Woodchuck hepatitis core antigen (WHcAg) particles purified from the liver of chronically infected animals were used for monoclonal antibody production. Most of the putative clones demonstrated anti-WHc specificity. However, the supernatants from several putative clones bound X antigen sequences from woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV). One monoclonal antibody, designated WC9-85 (an IgM), specifically bound hepatitis B X antigen (HBxAg) residues spanning positions 115-131 (peptide 100). WC9-85 also specifically detected liver-derived WHcAg and duck hepatitis B core antigen (DHBcAg) particles in the same CsCl density gradient fractions as did specific anticore and cross-reactive polyclonal anti-x. WC9-85 did not bind to HBcAg particles made by recombinant DNA techniques, in which only the C-gene sequences are expressed, but did bind to liver-derived HBcAg in identical assays. A second monoclonal anti-x, WC8-62, had similar characteristics. Identification of the immunoreactive species in liver-derived core particles by Western blotting showed that WC9-85 bound the major DHBcAg polypeptide having an apparent molecular weight of 35,000 Da. WC9-85 also bound WHcAg-associated bands at approximately 37,000 and 27,000 Da, but little or no binding at the apparent molecular weight of the major WHcAg polypeptide (about 21,000 Da) was observed. These results are consistent with the conclusions that X determinants are associated with core particles purified from naturally infected livers, that such determinants are associated with the major DHBcAg polypeptide and at least two minor WHcAg-associated polypeptides, and that X reactivity is distinct from core and/or e reactivity in hepadnavirus core particles.

X antigen polypeptides in the sera of hepatitis B virus-infected patients.

Studies were carried out to test the hypothesis that the X antigen product(s) of hepatitis B virus (HBV) appeared in serum during infection. Consequently, when serial sera from HBV-infected renal dialysis patients were tested for X antigen (HBxAg) by ELISA, many were positive. Sera from several positive patients were further characterized by immunoprecipitation followed by SDS/PAGE and Western blotting to discern the number and size of immunoreactive polypeptides. The dominant polypeptide observed in positive sera was approximately 17,000 Da (p17), which is compatible with the full-length size of the X gene product potentially encoded by HBV. Some sera contained another polypeptide species, approximately 13,000 Da (p13) in size. HBxAg was present most often in sera positive for HBeAg and/or HBV DNA or apparently complexed to anti-HBx in sera lacking these markers. Sera from HBV negative individuals were negative for these polypeptides. It appears, then, that HBxAg can be found in the serum of some HBV-infected patients as one or more polypeptide species associated with other markers of virus replication. In the presence of anti-HBx, HBxAg can be found after the peak of virus replication and may be the only detectable antigen in the blood of some chronically infected patients.

X antigen/antibody markers in hepadnavirus infections. Presence and significance of hepadnavirus X gene product(s) in serum.

The finding that X antigen is associated with hepatitis B core antigen particles and that serum hepatitis B e antigen derives from the cleavage of one or more core associated polypeptides raises the question as to whether core associated X antigen could be similarly generated and released into serum. To test this hypothesis, antisera raised to X antigen peptides were used to construct an enzyme-linked solid-phase immunoassay to detect X antigen in sera from patients infected with hepatitis B virus and from woodchucks infected with woodchuck hepatitis virus. X antigen was present in more than half of the individuals tested. There was a significant association between X antigen and markers of viral replication. Most individuals destined to become surface-antigen carriers had X antigen appearing before surface antigen, as did a smaller proportion of individuals transiently positive for surface antigen. A high frequency of X antigen was observed only in sera from human populations with a high frequency of other hepatitis B virus markers. These results suggest that X is a newly identified serum marker of viral replication that often appears before surface antigen in productive infections.

Activation of the complement system in the adult respiratory distress syndrome.

The adult respiratory distress syndrome (ARDS) is an acute pulmonary disorder characterized by the accumulation of neutrophils within the lower respiratory tract. Because activation of the complement system can generate C5a, a potent neutrophil chemoattractant, complement activation was assessed in both serum and bronchoalveolar lavage fluid obtained from 10 patients with ARDS and compared with that from normal control subjects. Crossed immunoelectrophoresis was used to determine activation of the complement components C3 and properdin factor B (PFB), and radioimmunoassay was used to determine the presence of C5a. Complement activation was not detected either in the plasma or in the lung epithelial lining fluid of the control subjects. In contrast, evidence of C3 activation was found in the plasma of 50% of the patients with ARDS when initially studied; likewise, C3 activation, PFB activation, and C5a could all be detected in the epithelial lining fluid of all patients with ARDS with a single exception. Follow-up bronchoalveolar lavages revealed decreased amounts of C3 activation and C5a 2 to 7 days after the onset of ARDS, and the complement activation had resolved when the patients with ARDS had completely recovered. To determine if the C5a in bronchoalveolar lavage fluid could be responsible for the influx of neutrophils observed in ARDS, epithelial lining fluids obtained from both normal control subjects and from patients with ARDS were fractionated by molecular sieve chromatography. Two distinct fractions of chemotactic activity were found in the ARDS bronchoalveolar lavage fluid.(ABSTRACT TRUNCATED AT 250 WORDS)