RNA of Australia Antigen

ALTHOUGH the exact nature of Australia (Au) antigen is not resolved, increasing evidence suggests that it is the causal agent of viral hepatitis. This supposition is based chiefly on the frequent occurrence of Au antigen in the sera of patients with viral hepatitis^1–4 and on its virus-like appearance under the electron microscope^5–7. Biochemical studies have shown that Au antigen consists largely of protein, with a minor lipid moiety^{8, 9}. So far, however, no genetic material has been detected in the Au antigen and it has been suggested that the Au antigen might be “a unique infectious particle with little or no nucleic acid”¹⁰. We wish to present evidence, however, that RNA is an essential component of Au antigen.     

Biophysical Properties of Australia Antigen

Biophysical studies with Australia complement-fixing (CF) antigen showed it to be a particle with a buoyant density of 1.20 g/cm(3) in CsCl, a sedimentation coefficient of 110, and an average diameter of 25 nm. The CF antigen was not inactivated by ether, 1% deoxycholate, 1% Tween 80 or overnight heating at 56 C. The antigen was unstable when treated with 1% sodium dodecyl sulfate. A procedure is described for the isolation and partial purification of Australia antigen from serum by using isopycnic banding and rate separation techniques. Treatment of the 1.20 g/cm(3) Australia antigen with 1% Tween 80 yielded a minor peak of CF activity with a buoyant density of 1.39 g/cm(3) in CsCl.     

Specificity and Sensitivity of Radioimmunoassay for Hepatitis B Antigen

Sera from a survey of 6,026 people were tested for hepatitis B surface antigen by using radioimmunoassay and counterelectrophoresis. Forty-eight sera (0.79%) were positive by counterelectrophoresis and 152 sera (2.52%) were positive by radioimmunoassay, using the most liberal of the recommended criteria for positivity (i.e., counts 3 standard deviations above the mean). Absorption tests performed on the 152 radioimmunoassay-positive sera showed that 10 (6.6%) were false-positive reactions to guinea pig protein, 74 (48.6%) were due to false-positive reaction(s) with other protein(s) in the test system, and 68 (44.8%) were true positives. There was a strong correlation between the degree of elevation of radioactive counts and the proportions of sera that were true positives; all 49 sera with counts >50 standard deviation units above the mean were true positives, but only 19 (18.4%) of the 103 sera with counts <50 standard deviation units were true positives. A few sera with high counts required absorption with type-specific (type D) antisera. The following conclusions were reached from this study: (i) absorption tests should be run on all radioimmunoassay-positive, counterelectrophoresis-negative sera; (ii) most (about 90%) false positives are not due to anti-guinea-pig protein reactions; and (iii) radioimmunoassay, in combination with absorption tests, yields a modest increase (about 35%) in detection of true positives over use of counterelectrophoresis alone.     

Australia Antigen and Hepatitis in Accra,Ghana

Viral hepatitis in young adults in Accra, Ghana, is associated with Australia antigen (H.A.A.). Sera from 85 patients in hospital with viral hepatitis were available for determinations of H.A.A. Of the 16 patients whose serum was obtained within the first week of symptoms, 15 were positive. The only factor related to finding H.A.A. was the time between onset of symptoms and the collection of the serum sample. Persistence of H.A.A. was associated with persistence of jaundice in men but not in women. Previous epidemiological studies in Accra found no evidence for parenteral transmission of viral hepatitis and showed a shanty-town predilection pointing to faecal-oral transmission. It thus seems that H.A.A.-associated hepatitis is transmitted in West Africa either faecal-orally or by shanty-town associated arthropods. The finding that H.A.A. hepatitis is the usual hepatitis in young adults in Accra is in accord with the high prevalence of H.A.A. elsewhere in the general population in Africa and may be related to the high rate of cirrhosis and hepatoma in Africa.     

Removal of Anti-complementary Material and Australia Antigen from Immunoglobulin

Inmunoglobulin isolated from human sera, be it by the cryo-alcohol, rivanol, multi membrane electrodecantation or polyethylene glycol process, alvays contains denatured material. This may result from the influence either singly or in combination, of acme of the follwing factors:

1. inefficiency of the purification procedure;

2. surface denaturation;

3. imperfect freeze-drying of the final product; and

4. factors yet unknown vhich cause alteration in the immoglobulins or other protein components not ellminated by the purification procedures.

     

Preparation of Australia Antigen Free Human Albumin with Polyethylene Glycol

Albumin was prepared from human plasma diluted In 0.05M phosphate buffer pH 8.6, by the precipitation of approximately 95% of the associated plasma proteins with 357. polyethylene glycol. More than 70% of the albumin In the original plasma was recoverable as a viscous liquid on lowering the pH to 5.5. The albumin prepared by this technique is associated with 5 to 10% or and B globulin. Plasma, positive for Australia antigen (Au(SH)ag) yields an albumin preparation negative for the antigen.     

Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity

Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.Human hepatitis delta virus (HDV) is an unusual subviral agent that increases the severity of acute and chronic liver disease in those infected with its helper, hepatitis B virus (23). The HDV genome is a 1,680-nucleotide (nt) single-stranded circular RNA that is replicated by a double-rolling-circle mechanism (reviewed in references 15 and 28). Both the genome and antigenome RNAs form a characteristic unbranched rod structure due to 70% sequence complementarity between the noncoding and coding regions of the RNA (10, 11, 31). HDV encodes just one protein, hepatitis delta antigen (HDAg), which forms ribonucleoprotein (RNP) complexes with both the genome and the antigenome in cells replicating HDV (3, 5, 30). These complexes play fundamental roles in viral RNA replication and packaging and their characterization is essential for understanding these processes, which are not well characterized.HDAg has been shown to form dimers and higher order multimers, even in the absence of HDV RNA (25, 30, 32). The multimerization activity has been localized to the amino-terminal third of the 195-amino-acid (aa) protein (12, 24, 30, 32). X-ray crystallographic analysis of a peptide comprised of aa 12 to 60 indicated that antiparallel dimers are stabilized by a coiled coil (aa 16 to 48), as well as a hydrophobic core region (aa 50 to 60) that also stabilizes interactions between dimers such that an octameric structure may form (35). Zuccola et al. found that bacterially expressed HDAg could be cross-linked in an octameric structure, and Cornillez-Ty et al. obtained evidence supporting such a structure in cells replicating HDV (7, 35). Site-directed mutations of HDAg amino acids critical for dimerization and/or multimerization abolish the ability of HDAg to support RNA replication (18, 32), indicating that the formation of HDAg multimers is essential for this process.We recently showed that bacterially expressed, C-terminally truncated HDAg forms stable RNP complexes in vitro with segments of HDV RNA that form unbranched rod structures (8). No particular sequences or structures in the RNA, other than the HDV unbranched rod, were essential for complex formation, but, remarkably, binding required that the RNA have a minimum length of at least about 300 nt. Overall, the results were consistent with the formation of a large RNP containing multiple copies of the 19-kDa protein that bound to the RNA either in a highly cooperative manner or as a preformed multimer. On the other hand, based on indirect measures of the RNA-binding activity of site-directed HDAg mutations in cells, others have found that HDAg multimerization might not be required for RNA-binding activity (18).Here, we directly analyze the role of HDAg multimerization in the formation of the HDV RNP complex. We find that HDAg binds to HDV unbranched rod RNA as a preformed multimer. Site-directed mutations that disrupted protein multimerization did not abolish binding but profoundly altered the nature of the RNA-protein complex. In particular, we found that multimerization is associated with RNA-binding specificity, including the RNA length requirement for binding. For the wild-type protein, RNP formation was found to be strongly temperature dependent, suggesting that conformational changes occur on binding, and providing a plausible explanation of the RNA length requirement for binding. Furthermore, we show that the protein binds as multiple multimeric units on longer RNAs, provided the length of the RNA is sufficient. We conclude that the HDAg multimer plays a critical role in the formation of properly structured HDV RNPs.     

Preparation and Standardization of an Australia Antigen Antibody of Equine Origin

A horse has been immunized with Australia antigen (Au/SH) purified 20-fold by a procedure employing gel filtration of Cohn fraction IV derived from an Au/SH-positive human plasma pool. Hyperimmunization was initiated by the intramuscular injection of 20 ml of a mixture of equal parts of purified Au/SH and complete Freund's adjuvant. The 20-ml volume was divided into four 5-ml doses, two of which were administered on each side of the horse's neck. Booster doses of antigen alone were given as follows: 10 ml intravenously 30 days later and 5 ml intramuscularly on each of days 77 and 205. Au/SH antibody formed readily, beginning on day 17, and was demonstrated by the agar gel double-diffusion technique and the complement fixation test during the subsequent 6 months. Antihuman plasma protein antibodies were effectively removed from the horse serum by one absorption with 1 to 3 volumes of normal human plasma. Abrupt rises in anticomplementary activity observed shortly after the third and fourth antigen injections, when the horse had developed elevated and steady levels of Au/SH antibody, could possibly be due to formation of antigen-antibody complexes. After optimal conditions were determined, an Au/SH antibody reagent pool which met official requirements was prepared. It was found equally suitable for the agar gel double-diffusion, complement fixation, and counterimmunoelectrophoresis test procedures.     

Sensitive Matrix Gel Diffusion Test for the Detection of Australia Antigen

A matrix gel diffusion (MGD) procedure with a sensitivity comparable to the complement fixation test (CF) has been developed for detecting Australia antigen in serum. The test utilizes a thin layer of agar (0.1 mm) with an applied plastic matrix. Reactants are introduced directly onto the surface of the agar through wells in the plastic matrix. End points obtained by CF with a panel of 11 sera varied from 1:8 to 1:512. When these sera were tested by MGD, end points for detection of antigen were within one dilution of that obtained by CF.     

Conformational Studies of Australia Antigen by Optical Rotatory Dispersion and Circular Dichroism

The optical rotatory dispersion and circular dichroism of intact, 8 m urea- or sodium dodecyl sulfate-treated, and carbamidomethylated Australia antigen indicated that the antigen possesses a high alpha-helical content similar to human high-density lipoproteins.     

Enhanced Detection of Australia Antigen in Serum Hepatitis Patients by Discontinuous Counter-Immunoelectrophoresis

The use of discontinuous counter-immunoelectrophoresis enhanced the reaction between Au/SH antigen and its antibody in agarose. The ionic strength of the Veronal buffer used in the agarose was 0.015 mu, whereas 0.075 mu Veronal (both pH 8.6) was used for anode and cathode buffers. Electroendosmosis is increased under such conditions. Au/SH antigen and antibody reacted to give sharp lines within 30 to 45 min as compared with conventional counter-immunoelectrophoresis which required 1 to 3 hr or longer.