31 To address the first two possibilities, we measured the amount of nuclear plasmid recovered from cells expressing HBx or control proteins. To avoid interference with cytoplasmic DNA, we performed the experiment 5 days after reporter gene transfection, a time when HBx shows strong stimulatory effects and the transfected DNA has been eliminated from the cytoplasm.32 Figure 5 shows that under conditions in which it induced strong activation, HBx had no obvious effect on the amount of reporter DNA recovered (Fig. 5A,B). This argues against HBx acting on plasmid nuclear import or copy number. To address whether

HBx would prevent methylation-mediated silencing of the transfected DNA, we examined the ability of HBx to increase BGB324 chemical structure activity of a luciferase learn more reporter construct lacking CpG dinucleotides.33 HBx was similarly efficient at activating the CpG-free reporter gene, thus excluding that HBx functions by preventing plasmid DNA methylation (Fig. 5C). The ability of HBx to enhance expression from extrachromosomal DNA specifically is of special interest, because the HBV genomic template transcribed by RNA Pol II exists as an episomal, nonreplicating cccDNA in the infected cells.34 We therefore wished to assess whether

HBx could discriminate between a chromosomally integrated HBV genome versus the extrachromosomal cccDNA, the natural viral template, in the same cells. For this purpose, Sucrase we designed an integrative HBV genomic construct carrying a defective HBx gene and four consecutive point mutations in the 3′-terminal redundant region. As a result, the mRNAs transcribed from this construct will contain the mutations at their 3′ ends. However, during reverse transcription of the pregenomic RNA by the viral polymerase and its conversion into cccDNA, the mutations will be lost (Fig. 6B; Fig. S3). As a consequence, the mRNAs originating from the cccDNA can be distinguished

from those transcribed from the chromosomal construct by RT-PCR using appropriate primers (Figs. S3, S4). We generated stable HepG2 cell lines containing the integrated HBV genomic construct and producing detectable amounts of cccDNA (Fig. S5). The cells were then transduced at high efficiency with wildtype HBx or the DDB1-binding defective HBx(R96E) mutant, the woodchuck WHx counterpart, or the paramyxovirus SV5-V protein that binds the DDB1 subunit of the E3 ligase the same way but lacks stimulatory activities (Fig. S1).14 As a further control, we transfected the Enhancer I-driven luciferase reporter construct, which is responsive to HBx and WHx in a transient assay (Fig. 1). As shown in Fig. 6A, HBx and WHx exhibited the expected stimulatory effect on transient luciferase expression, whereas HBx(R96E) and SV5-V were inactive.