We have used the endogenous reverse transcriptase reaction of viral core particles from duck liver to elucidate the mechanism of inhibition of duck hepatitis B virus (DHBV) replication by the nucleoside analog (؊)-␤-L-2,3-dideoxy-3-thiacytidine (3TC). As is the case in human immunodeficiency virus replication, 3TC-5-triphosphate (3TC-TP) acts as a chain terminator for the DNA polymerase activities. The results of several different experiments support this conclusion, which explains the potent activity of 3TC against the hepadnaviruses. In isolated DHBV core particles, 3TC-TP inhibited the reverse transcriptase in a manner that resembled competitive inhibition with respect to dCTP. However, the kinetics of inhibition was not linear on a double-reciprocal plot for the highest concentrations of 3TC-TP and the lowest concentration of dCTP. This anomaly would be expected if binding to the nucleotide site was followed by DNA chain termination. Calculations that used only the linear part of the curve yielded a K i of 0.78 ؎ 0.10 M 3TC-TP. The inhibition of core particles incubated in vitro with 3TC-TP was not reversed by removal of the free inhibitor. 3TC-TP inactivated the reverse transcriptase activity in a concentration-dependent manner. The K m of the chain termination reaction was calculated at 0.71 ؎ 0.05 M. Similar competitive kinetics and irreversible inhibition were also obtained on the endogenous DNA polymerase from viral particles from serum, suggesting that 3TC-TP also acts as a chain terminator of the DNA-directed DNA polymerase of DHBV replication. Core particles purified from DHBV-infected hepatocytes treated with 3TC also showed irreversible inhibition of the endogenous reverse transcriptase activity, indicating that chain termination is the mechanism of inhibition in the presence of the normal deoxynucleotide pools within cells. Endogenous sequencing of duck liver viral core particles with 3TC-TP as a chain terminator shows a pattern of bands similar to that obtained with ddCTP, and [ 32 P]3TC-TP labeled the growing DNA chain at the C position.) is a deoxycitidine analog in which the 3Ј carbon is replaced by a sulfur atom with loss of the 3Ј OH group necessary for the elongation of the DNA chain. 3TC was shown to be an effective inhibitor of human immunodeficiency virus (HIV) replication (7,13,28,33), and it was recently approved for compassionate use as an anti-HIV agent.Previous work showed that 3TC is also a very effective agent against human hepatitis B virus (5, 10, 32) and duck hepatitis B virus (DHBV [32]). In vivo and in infected hepatocytes, 3TC rapidly reduces the amount of viral DNA within days of the start of treatment, and it does not show significant toxicity (reference 5 and our unpublished results). 3TC is currently undergoing phase II clinical trials as an antiviral agent for human hepatitis B virus.For both HIV and hepadnaviruses, the mechanism of action of 3TC requires phosphorylation to 3TC-5Ј-triphosphate (3TC-TP), which in turn specifically inhibits the viral polymerases (3, 6, 15...

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