Effective antiviral agents are thought to inhibit hepatitis B virus (HBV) DNA synthesis irreversibly by chain termination because reverse transcriptases (RT) lack an exonucleolytic activity that can remove incorporated nucleotides. However, since the parameters governing this inhibition are poorly defined, fully delineating the catalytic mechanism of the HBV-RT promises to facilitate the development of antiviral drugs for treating chronic HBV infection. To this end, pyrophosphorolysis and pyrophosphate exchange, two nonhydrolytic RT activities that result in the removal of newly incorporated nucleotides, were characterized by using endogenous avian HBV replication complexes assembled in vivo. Although these activities are presumed to be physiologically irrelevant for every polymerase examined, the efficiency with which they are catalyzed by the avian HBV-RT strongly suggests that it is the first known polymerase to catalyze these reactions under replicative conditions. The ability to remove newly incorporated nucleotides during replication has important biological and clinical implications: these activities may serve a primer-unblocking function in vivo. Analysis of pyrophosphorolysis on chain-terminated DNA revealed that the potent anti-HBV drug -L-(؊)-2 ,3 -dideoxy-3 -thiacytidine (3TC) was difficult to remove by pyrophosphorolysis, in contrast to ineffective chain terminators such as ddC. This disparity may account for the strong antiviral efficacy of 3TC versus that of ddC. The HBV-RT pyrophosphorolytic activity may therefore be a novel determinant of antiviral drug efficacy, and could serve as a target for future antiviral drug therapy. The strong inhibitory effect of cytoplasmic pyrophosphate concentrations on viral DNA synthesis may also partly account for the apparent slow rate of HBV genome replication.antiviral inhibition ͉ core particles ͉ proofreading ͉ lamivudine͞3TC H epatitis B virus (HBV) is a member of the Hepadnaviridae family characterized by small, circular, partially doublestranded DNA genomes that are replicated by reverse transcription (1). Chronic HBV infection causes hepatitis and hepatocellular carcinoma, and remains a significant health problem worldwide (2). Designing antiviral drugs to combat HBV infection is impeded by a poor understanding of the parameters governing viral inhibition. Since currently effective antiviral drugs target the viral reverse transcriptase (RT; reviewed in ref.3), a detailed characterization of the HBV-RT catalytic mechanism is essential to understanding antiviral drug efficacy.The HBV-RT is a multifunctional enzyme with RNA-and DNA-dependent DNA polymerase, RNase H, and proteinpriming activities (reviewed in ref. 4). Once translated, the RT binds the epsilon stem-loop structure of the pregenomic RNA template and, together with bound cellular factors, this replication holoenzyme complex is encapsidated within a coat composed of the viral core protein (5-7). All viral DNA synthesis catalyzed by the RT subsequently proceeds enclosed within these replicating c...
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