Heinrich G. Köchel

Association of hepatitis C virus in human sera with β-lipoprotein

Hepatitis C virus (HCV)-RNA in sera of patients with viral hepatitis C is supposed to be included, at least partially, into HCV particles. We found that the density of HCV-RNA-carrying material was variable, as determined by sucrose gradient density centrifugation (1.03–1.20 g/cm3). In some of the sera examined HCV-RNA was restricted to low densities between 1.03 and 1.08 g/cm3. In other sera additional densities of HCV-RNA were found distributed over the whole gradient with peaks at 1.12 and 1.17 and at 1.19–1.20 g/cm3. HCV-RNA banding at low densities could be completely co-precipitated with anti-β lipoprotein, whereas HCV-RNA fractions of higher densities were only partially precipitated or not at all. In 8 of 20 sera directly examined, HCV-RNA could be completely and in 9 sera only partially co-precipitated by anti-β lipoprotein. In 3 sera no significant precipitation could be observed.

Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein

Hepatitis B virus (HBV) DNA contains a precore (pre-c) sequence of 29 codons with unknown function upstream of its gene for the major core protein. Its significance was studied by expression of core proteins with and without pre-c in Escherichia coli. Core protein without pre-c, P22c, assembled spontaneously to core particles and formed core antigen. It had the same size and antigenicity as core particles from infected liver. Core protein with pre-c, P25e, instead formed membrane-associated e antigen (HBeAg). The data suggest that pre-c functions as a signal peptide for the attachment of core protein P25e to cellular membranes. This hypothesis can explain the not yet understood relation between viremia and HbeAg and the protective role of anti-HBe antibody.

Assay of hepatitis B viras genome titers in sera of infected subjects

A method for quantitative standardization of the DNA hybridization assay for hepatitis B virus (HBV) DNA protein complex in serum is described. This method was used to determine the titer of HBV DNA in various groups of subjects with HB surface antigen (HBsAg) in order to ascertain its accuracy as an index of infectivity. The methods detection limit was 105 genome equivalents or 0.3 pg DNA per ml. Titers of 5×107 to 5×108 genome equivalents per ml were found to be typical for persistent massive viremia, which occurred more frequently in symptomatic (30 of 48) than in asymptomatic (24 of 72) carriers positive for HBe antigen (HBeAg). Moderate viremia (10s5−5×107) was usually found in patients eliminating the virus from the blood. Patients with resolving acute hepatitis B were frequently positive at the onset (18 of 26) with moderate titers, but became negative within several weeks. In 11 patients who developed chronic hepatitis B, titers increased until typical massive viremia was evident. Whereas healthy HBsAg carriers with anti-HBe always had negative genome titers (144 of 144), symptomatic carriers with anti-HBe often had moderate genome titers (9 of 30). It is recommended that genome titers be monitored in HBeAg-positive and in symptomatic anti-HBe positive virus carriers in order to distinguish between virus carriers with high (>5×107), moderate (105−5×107) and low (<105) infectivity.

Antibodies to human papillomavirus type-16 in human sera as revealed by the use of prokaryotically expressed viral gene products

Open reading frames of human papillomaviruses were expressed in Escherichia coli as beta-galactosidase fusion proteins. These bacterially derived papillomaviral gene products were used to examine sera from 67 women (63 healthy subjects, 4 patients with genital carcinoma) for antibodies to papillomavirus type-16 antigens (E1, E2, E4, E5, E6, E7, L1, L2) and the L2 proteins of HPV-6b and HPV-18 by Western-blot analysis. The serologic data were compared with cytological findings classified according to Papanicolaou and with nucleic acid hybridization data from cervical smears of the same individuals. Twenty-three of the normal individuals showed antibodies exclusively directed against L2 gene products; whereas in the sera from the four genital cancer patients, antibodies to the early gene products E4 and/or E7 could be detected. In one case these antibodies were found to be combined with antibodies to L2 of HPV-16 and -18 and in another case with those to E1 and E2 of HPV-16. In none of the sera examined could antibodies to L1, E5 or E6 be identified. Three of the antibody positive normal women were found to be also positive for HPV-16/18 DNA, while all of the 40 seronegative women were HPV-16/18 DNA negative. These data indicate that serology may be a valuable means to study the epidemiology of genital human papillomavirus infection.

Characterization of the endogenous protein kinase activity of the hepatitis B virus.

During the assembly of the nucleocapsid of the hepatitis B virus a protein kinase, probably of cellular origin, is encapsidated. This enzyme phosphorylates serine residue(s) localized within the lumen of the particle. By using purified, liver-derived core particles, we characterized the protein kinase activity in the presence of different ions and inhibitors. Controls were performed with cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) and recombinant core particles. We showed that the endogenous protein kinase of the core particles was not inhibited by H89, a specific inhibitor of PKA. Staurosporine, a selective inhibitor of PKC inhibited the endogenous kinase activity only within the first minutes of the reaction. In contrast, quercetine, a selective inhibitor of the protein kinase M (PKM) did not inhibit during the first minutes but inhibited efficiently during later phases of incubation. PKM represents an enzymatically active proteolytic fragment of PKC. These results suggest that PKC is encapsidated into human core particles and is converted to PKM during the in vitro reaction. This conclusion implies the association of a protease activity localized with the HBV nucleocapsid inside liver-derived core particles.

Identification of a binding site in the hepatitis B virus RNA pregenome for the viral Pol gene product

The hepatitis B virus, although containing a DNA genome, replicates by reverse transcription of an RNA pregenome. The viral Pol gene encodes the reverse transcriptase which catalyzes viral DNA synthesis. To study the interaction of this protein with HBV RNA, the entire Pol gene product was expressed except its eight amino-terminal codons in Escherichia coli as fusion protein with beta-galactosidase. In the absence of competing nucleic acids full-length expression products were able to nonspecifically bind in vitro synthesized HBV RNAs of different polarity and length. However, if competed with an excess of unspecific RNA, only those HBV RNAs were bound which contained besides the direct repeats 1 and 2 nucleotide sequences downstream of direct repeat 1. The corresponding binding site was found to be located within the adjacent 134 nucleotides downstream of DR1. We conclude from our data that this region which is in part homologous to the U5 region of retroviral genomes may be important for the binding of the HBV Pol gene product to the viral pregenome.