First Mariner Mos1 transposase inhibitors.

We described chemical inhibitors of Mos1 transposition. Some were already known to affect a related prokaryotic transposase (Tn5) or HIV-1 integrase, whereas the other were new compounds in this field. The new compounds were all organized around a bis-(heteroaryl)maleimides scaffold. Their mechanism of action depended on the chemical substitutions on the scaffold. The cross-activity, between HIV-1 integrase and Mos1 transposase, of the new group of inhibitors showed that Mos1 transposase could constitute an excellent surrogate HIV-1 inhibitor screen.

Animal models for the study of HBV infection and the evaluation of new anti-HBV strategies.

BACKGROUND: Our aim was to evaluate the anti-HBV activity of a novel L-nucleoside analog, 2',3'-dideoxy-2',3'-didehydro-beta-L-5-fluorocytidine (beta-L-Fd4C), in study models of HBV infection. METHOD: Its mechanism of action was evaluated on the in vitro expressed duck HBV (DHBV) reverse transcriptase and in primary hepatocyte cultures of duck and human origin. The capacity of antiviral therapy to clear viral infection was analyzed in vivo in the duck and woodchuck models. RESULTS: beta-L-Fd4C-TP exhibited a more potent inhibitory effect on the RT activity of the DHBV polymerase than other cytidine analogs (lamivudine-TP, ddC-TP, beta-L-FddC-TP). In primary duck hepatocyte cultures, beta-L-Fd4C exhibited a long-lasting inhibitory effect on viral DNA synthesis but could not clear viral cccDNA. In vivo treatment with beta-L-Fd4C in infected ducklings and woodchucks, induced a greater suppression of viremia and intrahepatic viral DNA synthesis than with lamivudine. However, covalently closed circular DNA persistence explained the relapse of viral replication after treatment withdrawal. Viral spread was strongly reduced in the case of early therapeutical intervention, but the number of infected cells did not decline when therapy was started during chronic infection. Liver histology analysis showed a decrease in the inflammatory activity of chronic hepatitis while no ultrastructural modification of liver cells was observed in electron microscopy studies. Furthermore, in human primary hepatocyte cultures, beta-L-Fd4C induced a significant inhibition of HBV DNA synthesis. CONCLUSION: beta-L-Fd4C is a potent inhibitor of hepadnavirus RT and inhibits viral DNA synthesis in hepatocytes both in vitro and in vivo. These experimental studies allowed as to show that beta-L-Fd4C is a promising anti-HBV agent. Combination therapy should be evaluated to eradicate viral infection.

Duck hepatitis B virus polymerase gene mutants associated with resistance to lamivudine have a decreased replication capacity in vitro and in vivo.

BACKGROUND/AIMS: Hepatitis B virus mutants of the polymerase gene are frequently selected during lamivudine therapy for chronic hepatitis B. To study the biology of these mutants, we analyzed their replication capacity in the duck hepatitis B virus (DHBV) infection. METHODS: The B and C domain polymerase mutants corresponding to the clinical isolates were engineered by site directed mutagenesis in the DHBV genome in different expression vectors. RESULTS: The study of the enzymatic activity of the mutated viral polymerase polypeptides analyzed in a cell free system demonstrated a lower priming activity and a decreased capacity of elongation of viral minus strand DNA that was consistent with the lower replication capacity of these mutants in transfected leghorn male hepatoma cells compared to wild type genome. These mutants had a lower replication capacity in primary hepatocytes and in in vivo transfected ducklings. Although resistant to lamivudine, these mutants remained sensitive to PMEA. CONCLUSION: YMDD mutants of the DHBV reverse transcriptase have a decreased replication capacity both in vitro and in vivo, and are not cross-resistant to PMEA. These results may be important to design new antiviral strategies to combat the replication of the lamivudine resistant viral strains.

Bovine thrombospondin-2: complete complementary deoxyribonucleic acid sequence and immunolocalization in the external zones of the adrenal cortex.

Given the variety of biological functions in the adrenal cortex that are controlled by ACTH, we hypothesized that some extracellular proteins act as biological relays for this systemic hormone. One candidate protein [corticotropin-induced secreted protein (CISP)] was purified from the conditioned medium of bovine adrenocortical cells on the basis of a 5- to 14-fold increase in its synthesis after the addition of ACTH. We report here the cloning of overlapping complementary DNAs that span the sequence encoding the full-length protein (1170 amino acids). The deduced CISP protein sequence is 89% identical to that of human thrombospondin-2 (TSP2), but only 61% identical to that of bovine TSP1, confirming that CISP is the bovine ortholog of TSP2. The bovine TSP2 sequence aligned perfectly with human, mouse, and chicken TSP2 sequences, except for a gap of 2 amino acids located in a linker region. All 58 cysteine residues that are conserved in other species were present in the bovine sequence as well as most of the functional domains. Most endocrine tissues (adrenal cortex, testis, ovary, and placenta) appeared to express TSP2, as determined by Western blot analysis. The highest levels of TSP2 protein were found in the adrenal cortex, followed by the heart, spleen, brain, and kidney. A differential extent of N-glycosylation or tissular proteolytic maturation may be responsible for the mol wt differences observed between bovine TSP2 detected in the medium from primary cultures and that in fresh tissue extracts. The immunohistochemical analysis of the distribution of TSP2 in the bovine adrenal gland revealed that the protein is much more abundant in the external zones (zona glomerulosa and zona fasciculata) than in the internal reticularis zone, a pattern similar to that reported for ACTH receptors. This distribution clearly suggests that TSP2 is a candidate relay protein for a subset of ACTH actions in the adrenal cortex.

Inhibitory effect of 2'-fluoro-5-methyl-beta-L-arabinofuranosyl-uracil on duck hepatitis B virus replication.

The antiviral activity of 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU), a novel L-nucleoside analog of thymidine known to be an inhibitor of hepatitis B virus (HBV) replication in hepatoma cells (2.2.1.5 cell line), was evaluated in the duck HBV (DHBV) model. Short-term oral administration (5 days) of L-FMAU (40 mg/kg of body weight/day) to experimentally infected ducklings induced a significant decrease in the level of viremia. This antiviral effect was sustained in animals when therapy was prolonged for 8 days. The histological study showed no evidence of liver toxicity in the L-FMAU-treated group. By contrast, microvesicular steatosis was found in the livers of dideoxycytidine-treated animals. L-FMAU administration in primary duck hepatocyte cultures infected with DHBV induced a dose-dependent inhibition of both virion release in culture supernatants and intracellular viral DNA synthesis, without clearance of viral covalently closed circular DNA. By using a cell-free system for the expression of an enzymatically active DHBV reverse transcriptase, it was shown that L-FMAU triphosphate exhibits an inhibitory effect on the incorporation of dAMP in the viral DNA primer. Thus, our data demonstrate that L-FMAU inhibits DHBV replication in vitro and in vivo. Long-term administration of L-FMAU for the eradication of viral infection in animal models of HBV infection should be evaluated.