Efficient pyrophosphorolysis by a hepatitis B virus polymerase may be a primer-unblocking mechanism

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 β-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.

Evolutionary changes in the expression pattern of a developmentally essential gene in three Drosophila species.

The hypothesis that morphological evolution may largely result from changes in gene regulation rather than gene structure has been difficult to test. Morphological differences among insects are often apparent in the cuticle structures produced. The dopa decarboxylase (Ddc) and alpha-methyldopa hypersensitive (amd) genes arose from an ancient gene duplication. In Drosophila, they have evolved nonoverlapping functions, including the production of distinct types of cuticle, and for Ddc, the production of the neurotransmitters, dopamine and serotonin. The amd gene is particularly active in the production of specialized flexible cuticles in the developing embryo. We have compared the pattern of amd expression in three Drosophila species. Several regions of expression conserved in all three species but, surprisingly, a unique domain of expression is found in Drosophila simulans that does occur in the closely related (2-5 million years) Drosophila melanogaster or in the more remote (40-50 million years) Drosophila virilis. The "sudden" appearance of a completely new and robust domain of expression provides a glimpse of evolutionary variation resulting from changes in regulation of structural gene expression.