Fig 2: Model for nuclear translocation of histones H3 and H4 as monomers and processing in the nucleus Imp5 imports histone H3 and H4 as monomers whilst Imp4 has a preference for dimers, albeit with some redundancy.Upon nuclear translocation, RanGTP induces cargo dissociation with H3 being transferred to sNASP and H4 to the HAT1 complex. These chaperones may have a holdase function that buffers imbalances in individual histone supply. H3 and H4 fold and enter the deposition pathway via a number of distinct NASP-bound complexes.

Fig 3: HAT1 is recruited to cccDNA minichromosome by lncRNA HULC-scaffold HBc. (A) The combination of HAT1 and HBc was measured 8 dpi by immunoprecipitation assays in the HBV-infected dHepaRG cells. (B) The deposition of HAT1 and acetylation of histone including AcH3 and AcH4 on cccDNA minichromosome or the promoter of CCNA2 were verified 8 dpi by ChIP-qPCR in HBV-infected dHepaRG cells continuously transfected with pcDNA 3.1-HBC (2 µg/well) at -4, 0, and 4 dpi. (C) The interaction of HULC with HAT1 was examined by RIP-qPCR assays in the indicated cells. (D) The deposition of HAT1 and acetylation of histone including AcH3 and AcH4 on cccDNA minichromosome or the promoter of CCNA2 were analyzed 8 dpi by ChIP-qPCR in HBV-infected dHepaRG cells continuously transfected with siHULC (100 nM) at -4, 0, and 4 dpi. (E) The combination of HULC with HBc or HBx was examined by RIP-qPCR assays 8 dpi in HBV-infected dHepaRG cells. (F) The interaction of HULC with HBc or HAT1 was assessed 8 dpi by RNA pull-down assays followed by Western blot analysis in HBV-infected dHepaRG cells. (G) The combination of HAT1 and HBc was analyzed 8 dpi by immunoprecipitation assays in HBV-infected dHepaRG cells transfected with siHULC. (H) The levels of lncRNA HULC were examined in the liver of human liver-chimeric mice (n=3) and HBV-infected human liver-chimeric mice (n=3) by RT-qPCR. Mean ± SD of at least three experiments are shown, in which each experiment was designed by three replicates. Statistical significant differences are indicated: **P<0.01; ***P<0.001; ns, no significance.

Fig 4: Validation of import cargo candidates. The analysis of potential import cargo candidates was performed as described in Fig. 4 and included the human proteins Hat1 (O14929), Nampt (P43490), Smug1 (Q53HV7), Hmbs (P08397), and Hdac8 (Q9BY41). Note that the anti-Xpo7 nanobodies shift the here-validated import cargoes to a more cytoplasmic localization. Error bars represent SD. ***, p < 10-6. Bar, 20 µm.

Fig 5: HAT1 promotes histone acetylation on HBV cccDNA minichromosome. (A) The deposition of HAT1 on cccDNA was measured by ChIP-qPCR in the HBV-infected dHepaRG and HepG2-NTCP cells. (B) The deposition of HAT1 and HBc on cccDNA was analyzed by ChIP-qPCR at indicated time in the HBV-infected dHepaRG cells. (C) The colocalization of HAT1 and HBc was assessed 8 dpi by confocal microscopy in the dHepaRG cells. (D-G) The acetylation of histone, including AcH3, AcH4, H3K27ac, H4K5ac and H4K12ac, associated to cccDNA minichromosome or the promoter of CCNA2 was verified 8 dpi by ChIP-qPCR in the dHepaRG and HepG2-NTCP cells. (H) The acetylation of histone H3K27, H4K5 and H4K12 were examined by Western blot analysis in the liver of human liver-chimeric mice (n=3) and HBV-infected human liver-chimeric mice (n=3). (I) A model for the function of HAT1 in promoting histone acetylation of cccDNA minichromosome was shown. Mean ± SD of at least three experiments are shown, in which each experiment was designed by three replicates. Statistical significant differences are indicated: **P<0.01; ***P<0.001; ns, no significance.