6, 7 Several studies suggest that a primary function of HBx in the HBV life cycle is to promote viral gene expression.8-10 Perhaps most compelling is the recent finding that primary human hepatocytes infected with HBx-deficient HBV particles show normal levels of cccDNA but essentially no viral gene expression.11 The underlying mechanism whereby HBx promotes viral messenger RNA (mRNA) synthesis remains elusive. In cell culture, HBx behaves as a pleiotropic transactivator capable of stimulating a variety

of cellular and Anti-infection Compound Library molecular weight viral promoters.12, 13 Although typically modest, the transactivation activity of HBx is likely biologically relevant. It is conserved among the HBx proteins encoded by HBV, woodchuck hepatitis virus, and ground squirrel hepatitis virus.8 Furthermore, the ability of HBx to stimulate reporter gene expression and HBV replication correlate.10, 14 The current explanation for the pleiotropic transactivation effects of HBx is that the protein Sunitinib can interact with numerous cellular proteins and has functions in both the cytoplasm and the nucleus of cells. Thus, HBx has been

proposed to activate diverse signal transduction pathways in the cytoplasm,12 whereas in the nucleus it is believed to function by way of direct interaction with transcription factors15, 16 (and references therein), components of the basal transcription machinery (reviewed12, 17), as well as DNA- and histone-modifying enzymes.18-20 That HBx may have so many activities is puzzling, especially because the HBx gene largely

overlaps the polymerase gene on the viral genome, a situation that has likely limited its potential to evolve multiple functions. In the present study we provide an alternative explanation for the pleiotropic transactivation properties of HBx. Previous work has established that HBx and WHx bind to host cell protein UV-damaged DNA binding protein 1 (DDB1) and Bacterial neuraminidase likely function as viral substrate-recruiting subunits of the DDB1-containing E3 ubiquitin ligase complex.14, 21 We show here that through its interaction with the E3 ligase, HBx up-regulates luciferase reporter and HBV gene expression by a mechanism that operates selectively on extrachromosomal DNA templates irrespective of the nature of the promoter sequences and cognate activators. cccDNA, covalently closed circular DNA; DDB1, UV-damaged DNA binding protein 1; GFP, green fluorescent protein; HBV, hepatitis B virus; HBx, hepatitis B virus X. GFP-HBx, GFP-HBx(R96E),22 GFP-SV5V,23 and the HBx(R96E)-DDB1 fusions14, 23 have been described and are all expressed at detectable levels.14, 22, 23 GFP-WHx was generated by amplifying the woodchuck WHx coding region by polymerase chain reaction (PCR) from a WHV genomic construct (OHVCGA prototype). The proteins were produced from the episomal vectors KEBOB-PL24 in Fig. 1A and EBS-PL24 in Figs.