Marita Büscher

To study the replication strategy of the human hepatitis B virus, the 5' end of the RNA pregenome and the initiation sites of DNA plus and minus strands have been mapped. The RNA pregenome was found to be terminally redundant by 120 nucleotides; it is initiated within the pre-C region and may also function as mRNA for synthesis of the major core protein and the hepatitis B virus reverse transcriptase. The hepatitis B virus DNA minus strand is initiated within the direct repeat sequence DR1, it contains a terminal redundancy of up to eight nucleotides, and its synthesis does not require any template switch. The DNA plus strand is primed by a short oligoribonucleotide probably derived from the 5' end of the RNA pregenome, and its synthesis is initiated close to the direct repeat sequence DR2. For its elongation to pass the discontinuity in the DNA minus strand an intramolecular template switch occurs using the terminal redundancy of this template. Thus, the route of reverse transcription and DNA replication of hepatitis B viruses is fundamentally different from that of retroviruses.

In NIH3T3 cells stably transfected with the human c-fos gene, serum, platelet derived growth factor (PDGF), phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, TPA), ultraviolet irradiation (UV) and 3'-5'-cyclic adenosine monophosphate (cAMP) cause a transient and rapid activation of both the endogenous and the transfected c-fos genes. While serum, TPA, UV and PDGF dependent activation of the gene is severely impaired, when the serum responsive element from position -319 to -300 (SRE, Treisman, 1985) is destroyed, a full response to cAMP is retained. Insertion of a synthetic oligonucleotide corresponding to the SRE element upstream of position -96 restores the responses to TPA and serum, and large parts of the responses to UV and PDGF. The signal transduction chains elicited by UV and TPA are blocked by an inhibitor of protein kinase. Only TPA, however, causes the translocation of protein kinase C to the membrane. UV and TPA treated cells become refractory to a second stimulation by the same agent at 3 or 24 hours after the first treatment. Alternating the agents, however, leads to full responses. In addition, saturating doses of UV and TPA are at least additive. Ca-ionophores severely reduce only UV induced c-fos expression. These data indicate, that different signal transduction pathways elicited by growth promoting agents and by UV induced stress converge onto the same enhancer element.

The low basal expression of Fos and the rapid and effective turn-off of serum induced Fos transcription is due to autoregulation. Fos and Jun/AP-1 protein cooperate in the repression mechanism. Overexpressions of Fos and Jun decrease basal and induced transcription from Fos-CAT constructs and from the endogenous gene in NIH3T3 cells. The introduction into cells of either antisense Fos or antisense Jun sequences leads to elevated basal Fos promoter activity. Gel retardation experiments with synthetic oligonucleotides define two target sequences in the Fos promoter which bind Fos-Jun/AP-1 (centering at about -296 and -60). In vivo competition with these oligonucleotides relieves repression.