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Hepatitis B virus (HBV), a DNA virus that replicates through reverse transcription, encodes only 7 primary gene products: the capsid-forming core protein and the precore precursor of hepatitis B e antigen (HBeAg), the reverse transcriptase (P protein), 3 envelope proteins (collectively known as hepatitis B surface antigen [HBsAg]) and the product of the X open reading frame (ORF), HBx. Hundreds of studies, mostly in artificial settings, have concluded that HBx is a promiscuous transcriptional transactivator. In addition, a plethora of other, apparently unrelated interaction partners have been described (for reviews see Murakami 1 and Yen 2 ), yet the role of HBx in the virus life cycle remained mysterious. The study by Bouchard et al. now strongly suggests that a major function of HBx is to mobilize Ca 2Ο© from intracellular stores, triggering activation of the tyrosine kinase Pyk2; eventually this leads to a well detectable enhancement of HBV replication in a transfected human hepatoma cell line, with the major effect on viral DNA synthesis rather than transcription.
While substantial progress has been made in disclosing how HBV replicates (for recent reviews see Nassal 3 and Ganem and Schneider 4 ), few virus proteins have similarly resisted elucidation of their function as the 154 amino acid HBx protein, named so because its sequence did not offer any clues to function. Many viruses encode factors that enable or enhance viral transcription, that drive cells into cell cycle phases optimal for viral replication, or that counteract host defense mechanisms (for review see Goodbourn et al. 5 ). Numerous such functions have been attributed to HBx. In addition to transcriptional transactivation and the induction of major signaling cascades, activation of nitric oxide synthase (NOS) and in vitro interactions with p53, the proteasome, DNA repair proteins, a mitochondrial voltage-dependent anion channel, 6 components of nucleo-cytoplasmic trafficking, plus various others have been reported. Although the relevance in HBV infection of none of these activities is established, they have been taken to indicate a potential involvement of HBx in cell cycle regulation, apoptosis, and hepatocellular carcinoma. 7 However, carcinogenesis may at least as well be explained as a mainly immune-mediated event, with HBx probably having an at most weak pro-oncogenic potential. 8 Attesting physiologic relevance to this bewildering multifunctionality is hampered by the lack of appropriate experimental systems. In HBV-infected patients it is very diffcult to detect the HBx protein, or its mRNA, although some investigators apparently were successful. 9 Similarly, discordant data have been reported in HBV transgenic mice. 10,11 When HBx, or its woodchuck hepatitis virus (WHV) homolog WHx, could be detected, a larger fraction was usually found in the cytoplasm, and a smaller one in the nucleus. The most compelling data regarding the in vivo relevance of HBx have been obtained in the woodchuck. Earlier work 12,13 did not find evidence for infection in the absence of a functional X gene, concluding that WHx, and by inference HBx, is essential for establishing infection. Recent studies suggest that this requirement may not be absolute. 14 Nonetheless, HBx appears to significantly enhance viral replication in vivo-but by what mechanism?
Reproducing, under controlled cell culture conditions, a measurable effect of HBx on replication turned out to be 755