n-Butyrate, a cell cycle blocker, inhibits early amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of duck hepatocytes.

During chronic hepadnavirus infection, virus persistence depends on the regulation of the pool of covalently closed circular DNA (cccDNA), which is the template for transcription of viral RNA species. The development of in vitro infection of duck hepatocyte primary cultures by duck hepatitis B virus (DHBV) provides a unique opportunity to study the regulation of cccDNA synthesis. After DHBV in vitro infection, cccDNA is detected 1 day later and is amplified to a high copy number after 1 week in culture. We studied whether this amplification occurs during cell cycle progression of duckling hepatocytes. By using [3H]thymidine incorporation, we found that hepatocytes obtained from 3-week-old ducklings spontaneously entered the S phase of the cell cycle when cultured in serum-free medium without added growth factors. Bromodeoxyuridine labeling confirmed that cellular DNA synthesis took place in more than 50% of parenchymal cells. Cytofluorometry analysis revealed the presence of asynchronous populations and polyploidization processes. The addition of a cell cycle blocker, n-butyrate, completely inhibited [3H]thymidine incorporation and blocked duckling hepatocytes in the G1 phase of the cell cycle. Simultaneously, butyrate inhibited cccDNA amplification and allowed the establishment of DHBV infection, as demonstrated by the detection of a basal level of cccDNA in treated hepatocytes. Both effects were reversible since active cell DNA synthesis was restored and cccDNA accumulated after drug withdrawal.

Synthesis and antiviral activity of new carbonylphosphonate 2',3'-dideoxy-3'-thiacytidine conjugates.

The synthesis of new potential PFA-BCH-189 conjugate analogues is described and their molecular structure clearly identified through NMR and mass spectra techniques. The anti-HIV-1 activity was determined according to the inhibition of syncytium formation in MT-4 cells, while the anti-HBV activity was determined in infected duck hepatocytes. Both antiviral activities of the PFA-BCH-189 conjugates were much lower than those of the parent BCH-189 (2',3'-dideoxy-3'-thiacytidine) (1). Whereas a prodrug effect, following cleavage and release of the free BCH-189 and PFA, cannot be ruled out, poor cellular permeation of the drug seems to be the most likely reason for the reduced activities against HIV and DHBV. The presence of the PFA moiety appears to be detrimental for both the anti-HIV and anti-DHBV activity of PFA-BCH-189 cases.

Direct cloning and expression of PCR amplified DNA and RNA sequences: application to the hepadnaviruses nucleocapsid proteins.

Gene amplification may benefit from the construction of primers that augments the speed at which cloning and protein expression proceeds. Such primers include EcoRI or HindIII linkers as well as an in phase initiation or termination codon. PCR was carried out directly from viral particles of human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) without DNA purification and from RNA extracted from WHV infected liver. Amplified products were directly cloned in the pKK223-3 expression vector under the control of the tac promoter. The characterization of the recombinant clones expressing the nucleocapsid protein (C protein) was done by direct incubation of the filter with 125I-labelled anti-HBc and confirmed by radioimmunoassay and Western-blot analysis. This procedure allows easy selection of recombinant clones expressing a given protein and could be applied to many other genes.