A novel negative cis-regulatory element on the hepatitis B virus S-(+)-strand.
Hepatitis B virus (HBV) has a double-stranded DNA genome. The minus-strand contains coding regions for all known HBV proteins and most of the cis-regulatory elements. Little is known about transcription from the S-(+)-strand and its regulation. Thus, the presence of regulatory elements located on the S-(+)-strand was investigated by inserting nt 1038-1783 of HBV in both orientations between the human cytomegalovirus (HCMV) promoter and a luciferase gene. Transfection experiments revealed that the plasmid containing this HBV DNA fragment in an orientation allowing expression from the S-(+)-strand (antisense) led to inhibition of luciferase gene expression compared to the plasmid containing this sequence in an orientation that allows gene expression from the L-(-)-strand (sense). Deletion analyses delimit the sequence essential for the inhibitory effect to a 150 bp region that also carries part of the enhancerII/core promoter complex. However, the possible influence of this regulatory element has been excluded in various experiments. The repressing HBV sequence acts in an orientation- and position-dependent manner; no inhibition was observed when this DNA element was inserted upstream of the HCMV promoter or downstream of the luciferase gene. Northern blot analyses revealed reduced luciferase mRNA steady-state levels in cells transfected with constructs containing the essential HBV sequence in antisense orientation compared to plasmids containing this sequence in sense orientation. Since nuclear run-on experiments showed similar transcription initiation rates with these plasmids, the diminished luciferase mRNA steady-state levels must be due to altered stabilities, suggesting that nt 1783-1638 of HBV encode an RNA-destabilizing element.
Wagner
Markus
M
Department of Virus Research, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried , Germany1.
Alt
Michael
M
Department of Virus Research, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried , Germany1.
Hofschneider
Peter Hans
PH
Department of Virus Research, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried , Germany1.
Renner
Matthias
M
Department of Virus Research, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried , Germany1.
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D012333:RNA, Messenger; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012333:RNA, Messenger