Centrifugal inoculation of HBV.

<p>HepG2-NTCP12 cells in 24-well-plate were inoculated by HBV (500 vge/cell) without centrifugation or at different centrifugal force (g) for 30 min. After spinoculation, the cells were transferred to regular culture condition. 7 days later, the infected cells were analyzed by HBcAg immunofluorescence (A), the levels of intracellular HBV RNA transcription and DNA replication were determined by Northern and Southern blot, respectively (B).</p

Effect of centrifugation time on HBV spinoculation.

<p>HepG2-NTCP12 cells were inoculated with HBV (500 vge/cell) by centrifugation at 1,000×g for different time as indicated. 8 days post inoculation, HBV infection was analyzed by HBcAg immunofluorescence (A), viral RNA and core DNA hybridization (B), and HBV cccDNA produced by 60-min-spinoculation was detected by Southern blot and validated by heat denature and EcoR I linearization (C).</p

HBV infection of HepG2-NTCP12 cells with different viral inoculum size.

<p>Cells were mock infected or infected with HBV at indicated inoculum size (vge/cell) in the presence of 2% DMSO. Seven days later, the cells were subjected to HBcAg immunofluorescence microscopy (A). The intracellular HBV RNA and core DNA were analyzed by Northern and Southern blot, respectively (B).</p

DMSO enhances HBV infection in HepG2-NTCP12 cells.

<p>Approximately 5×10⁵ HepG2-NTCP12 cells were mock infected or infected with HBV (100 g.e/cell) in the absence or presence of 2% DMSO. DMSO was added to the PMM medium 24 h prior to the infection and remained present in the entire culture period until the cells were harvested. (A) Cells were immunostained with antibodies against HBV capsid protein (HBcAg) and visualized under fluorescence microscopy. Nuclei were counterstained with DAPI. The image shown represents five different microscopic fields, the percentage of HBcAg-positive cells was indicated (Mean ± SD) (similarly hereinafter). (B) HBV RNA were extracted from the infected cells and analyzed by Northern blot hybridization by using an [α-32P] UTP-labeled plus-strand-specific full-length HBV riboprobe. HBV 3.5 kb precore/pregenomic RNA and the 2.4/ 2.1 kb subgenomic RNA are labeled. Cellular 28S and 18S ribosomal RNA served as loading control. C9-tagged NTCP expression was detected by Western blot with β-actin serving as loading control.</p

Quantification of the genome equivalent of HBV virion DNA in concentrated HBV particles.

<p>(A) HBV virions and naked capsids in the indicated volume of virus stock were separated by native agarose gel electrophoresis, and viral DNA was detected by hybridization. The virion DNA to capsid DNA ratio was calculated by ImageQuant IQTL software using the hybridization signal intensity. (B) HBV DNA were extracted from 10 μl of virus stock and subjected to Southern blot analysis, 100 pg of 3.2 kb HBV linear DNA (approximately 3.1×10⁷ HBV DNA copies quantified by qPCR) served as loading marker. HBV DNA replicative intermediates, including relaxed circular (RC) DNA and single stranded (SS) DNA were labeled. The copy number of total HBV DNA was quantified by using the HBV DNA loading marker as standard. (C) Formula for calculation of HBV virion DNA genome equivalent (v.g.e).</p

Spinoculation-mediated HBV infection is NTCP-dependent.

<p>HepG2 and HepG2-NTCP12 cells were spinoculated with HBV (500 vge/cell, 1,000×g, 60 min) in the absence or presence of indicated compounds. 8 days post inoculation, the infected cells were analyzed by HBcAg immunofluorescence.</p

Inhibition of Hepatitis B Virus Replication by the Host Zinc Finger Antiviral Protein

<div><p>The zinc finger antiviral protein (ZAP) is a mammalian host restriction factor that inhibits the replication of a variety of RNA viruses, including retroviruses, alphaviruses and filoviruses, through interaction with the ZAP-responsive elements (ZRE) in viral RNA, and recruiting the exosome to degrade RNA substrate. Hepatitis B virus (HBV) is a pararetrovirus that replicates its genomic DNA via reverse transcription of a viral pregenomic (pg) RNA precursor. Here, we demonstrate that the two isoforms of human ZAP (hZAP-L and -S) inhibit HBV replication in human hepatocyte-derived cells through posttranscriptional down-regulation of viral pgRNA. Mechanistically, the zinc finger motif-containing N-terminus of hZAP is responsible for the reduction of HBV RNA, and the integrity of the four zinc finger motifs is essential for ZAP to bind to HBV RNA and fulfill its antiviral function. The ZRE sequences conferring the susceptibility of viral RNA to ZAP-mediated RNA decay were mapped to the terminal redundant region (nt 1820–1918) of HBV pgRNA. In agreement with its role as a host restriction factor and as an innate immune mediator for HBV infection, ZAP was upregulated in cultured primary human hepatocytes and hepatocyte-derived cells upon IFN-α treatment or IPS-1 activation, and in the livers of hepatitis B patients during immune active phase. Knock down of ZAP expression increased the level of HBV RNA and partially attenuated the antiviral effect elicited by IPS-1 in cell cultures. In summary, we demonstrated that ZAP is an intrinsic host antiviral factor with activity against HBV through down-regulation of viral RNA, and that ZAP plays a role in the innate control of HBV replication. Our findings thus shed light on virus-host interaction, viral pathogenesis, and antiviral approaches.</p></div

The enzymatically inactive form of ISG20 (ISG20^D94G) is defective to degrade HBV RNA but retains antiviral activity against pgRNA encapsidation.

<p>HepG2 cells were transfected with pHBV1.3 and equal amount of control vector (lanes 1 & 2) or F-ISG20 (lanes 3 & 4) or F-ISG20^D94G (lanes 5 & 6). Cells were harvested 5 days post-transfection and levels of HBV RNA (1st panel from the top) and encapsidated pgRNA (4th panel from the top) were determined by Northern blot hybridization. The assembled HBV capsid was revealed by native capsid gel EIA assay (3rd panel from the top) and the viral DNA in capsid was detected <i>in situ</i> by hybridization (5th panel from the top). HBV core DNA replicative intermediates were extracted and analyzed by Southern blot (6th panel from the top). Expression of FLAG-tagged ISG20 proteins was revealed by Western blot and β-actin served as loading control (bottom two panels). Results from duplicate experiments are presented.</p

ISG20^D94G physically associates with HBV RNA in cells.

<p>HepG2 cells were co-transfected with plasmid pCMVHBVΔCΔP and either control vector (lane 1) or FLAG-Pol (lanes 2&3), or pCMVHBV with either control vector (lane 4) or F-ISG20^D94G (lanes 5&6). Cells were harvested 4 days post-transfection. Input HBV RNA was determined by Northern blot (top panels). Input FLAG-Pol and F-ISG20^D94G proteins were determined by Western blot using FLAG Ab (top panels). Cell lysates were immunoprecipitated with beads coated with FLAG Ab, the immunoprecipitated Pol and ISG20^D94G were revealed by Western blot using FLAG Ab (lanes 3&6, bottom panel), and the bound RNA was extracted by Trizol and analyzed by Northern blot (lanes 3&6, bottom panel). HA Ab pull-down served as negative controls (lanes 2 & 5, bottom panel). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006296#sec014" target="_blank">Material and Methods</a> for more experimental details.</p

ISG20 overexpression reduces HBV replication in hepatocyte-derived cells.

<p>(A) HepG2 cells were co-transfected with either pHBV1.3 and F-ISG20 or empty vector, or pCMVHBV and F-ISG20 or empty vector, as indicated. Cells were harvested at day 5 post-transfection, and the levels of viral RNA and DNA were determined by Northern (top) and Southern (middle) blot hybridization, respectively. For RNA analysis, each lane was loaded with 10 μg of total RNA and probed with a genome-length, plus-strand-specific HBV riboprobe. Ribosomal RNAs (28S and 18S) are presented as loading controls. The positions of HBV pgRNA (3.5kb) and subgenomic surface RNAs (2.4kb and 2.1kb) are indicated. For DNA analysis, HBV core DNA was probed with genome-length, minus-strand-specific HBV riboprobe. The positions of relaxed circular (RC) and single-stranded (SS) DNAs are indicated. The relative pgRNA, sRNA or total DNA replicative intermediate level in each sample is expressed as the percentage of RNA or DNA of the cells transfected with empty vector. ISG20 overexpression was confirmed by Western blot using monoclonal antibodies against FLAG-tag. β-actin expression was presented as protein loading control (bottom panels). (B) The same experiment was done in Huh7 cells with pHBV1.3 as HBV expression vector.</p