Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Sep 9;95(19):e0044421.
doi: 10.1128/JVI.00444-21. Epub 2021 Sep 9.

RNA Helicase DDX17 Inhibits Hepatitis B Virus Replication by Blocking Viral Pregenomic RNA Encapsidation

Richeng Mao  1   2 , Minhui Dong  2   3   4 , Zhongliang Shen  2 , Hu Zhang  1   3   4 , Yuanjie Liu  1   3 , Dawei Cai  1 , Bidisha Mitra  1   3   4 , Jiming Zhang  2 , Haitao Guo  1   3   4
Affiliations

RNA Helicase DDX17 Inhibits Hepatitis B Virus Replication by Blocking Viral Pregenomic RNA Encapsidation

Richeng Mao et al. J Virol. .

Abstract

DDX17 is a member of the DEAD-box helicase family proteins involved in cellular RNA folding, splicing, and translation. It has been reported that DDX17 serves as a cofactor of host zinc finger antiviral protein (ZAP)-mediated retroviral RNA degradation and exerts direct antiviral function against Raft Valley fever virus through binding to specific stem-loop structures of viral RNA. Intriguingly, we have previously shown that ZAP inhibits hepatitis B virus (HBV) replication through promoting viral RNA decay, and the ZAP-responsive element (ZRE) of HBV pregenomic RNA (pgRNA) contains a stem-loop structure, specifically epsilon, which serves as the packaging signal for pgRNA encapsidation. In this study, we demonstrated that the endogenous DDX17 is constitutively expressed in human hepatocyte-derived cells but dispensable for ZAP-mediated HBV RNA degradation. However, DDX17 was found to inhibit HBV replication primarily by reducing the level of cytoplasmic encapsidated pgRNA in a helicase-dependent manner. Immunofluorescence assay revealed that DDX17 could gain access to cytoplasm from nucleus in the presence of HBV RNA. In addition, RNA immunoprecipitation and electrophoretic mobility shift assays demonstrated that the enzymatically active DDX17 competes with HBV polymerase to bind to pgRNA at the 5' epsilon motif. In summary, our study suggests that DDX17 serves as an intrinsic host restriction factor against HBV through interfering with pgRNA encapsidation. IMPORTANCE Hepatitis B virus (HBV) chronic infection, a long-studied but yet incurable disease, remains a major public health concern worldwide. Given that HBV replication cycle highly depends on host factors, deepening our understanding of the host-virus interaction is thus of great significance in the journey of finding a cure. In eukaryotic cells, RNA helicases of the DEAD box family are highly conserved enzymes involved in diverse processes of cellular RNA metabolism. Emerging data have shown that DDX17, a typical member of the DEAD box family, functions as an antiviral factor through interacting with viral RNA. In this study, we, for the first time, demonstrate that DDX17 inhibits HBV through blocking the formation of viral replication complex, which not only broadens the antiviral spectrum of DDX17 but also provides new insight into the molecular mechanism of DDX17-mediated virus-host interaction.

Keywords: DDX17; encapsidation; hepatitis B virus; pgRNA; viral replication.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Assessment of DDX17 expression in cell cultures. (A) HepG2, Huh7, HEK293T, and uninduced HepDES19 cells were harvested 36 h after reaching confluence. Endogenous DDX17 levels were detected by Western blotting. β-Actin served as a loading control. (B) HepG2 cells were transfected with control plasmid or FLAG-DDX17 for 4 days. The endogenous DDX17 and FLAG-tagged DDX17 were detected by Western blotting using antibodies against DDX17 and FLAG epitope, respectively. (C) HepG2 and PHH cells were left untreated or were treated with IFN-α (1,000 IU/ml) for 48 h. The expression levels of DDX17 and ISG56 were determined by Western blotting.
FIG 2
FIG 2
DDX17 is dispensable for ZAP-mediated HBV RNA degradation. Control vector or HA-ZAP-S was cotransfected with pHBV1.3 into HepG2-NTCP-shControl and HepG2-NTCP-shDDX17 cells. Five day after transfection, cells were harvested for HBV total RNA analysis by Northern blotting. The 3.5-kb precore mRNA and pgRNA and 2.4/2.1-kb surface mRNA are indicated. rRNA (28S and 18S) served as an RNA loading control. The protein expression levels of DDX17 and HA-ZAP-S were detected by Western blotting. β-Actin served as a protein loading control.
FIG 3
FIG 3
Overexpression of DDX17 inhibits HBV replication primarily by blocking pgRNA encapsidation in a dose-dependent manner. HepG2 cells were cotransfected with 0.6 μg of pHBV1.3 and the indicated amounts of FLAG-DDX17. Control vector was supplemented to normalize the total amount of transfected plasmids to 1.8 μg/transfection. Cells were harvested at day 5 posttransfection, and HBV total RNA and cytoplasmic encapsidated pgRNA were analyzed by Northern blotting. The lower-molecular-weight encapsidated pgRNA species represent the degradation intermediates catalyzed by the RNase activity of HBV pol during viral minus-strand DNA synthesis. HBV precore (p22) and core proteins and FLAG-tagged DDX17 protein were detected by Western blotting. HBV cytoplasmic core DNA was detected by Southern blotting. RC, relaxed circular DNA; SS, single-stranded DNA.
FIG 4
FIG 4
Knockdown of DDX17 promotes HBV pgRNA encapsidation in HBV stable cell line. HBV pgRNA transcription and DNA replication in HepDES19-shControl and HepDES19-shDDX17 cell lines were induced for 5 days after tetracycline (tet) withdrawal. HBV total RNA and cytoplasmic encapsidated pgRNA were analyzed by Northern blotting, HBV core protein and FLAG-tagged DDX17 protein were detected by Western blotting. HBV cytoplasmic core DNA was detected by Southern blotting.
FIG 5
FIG 5
Knockdown of DDX17 promotes HBV infection in vitro. HepG2-NTCP-shControl and HepG2-NTCP-shDDX17 cells were infected by HBV at an MOI (multiplicity of infection) of 100 for 9 days. (A) HBV total RNA and cytoplasmic encapsidated pgRNA were analyzed by Northern blotting, and HBV cytoplasmic core DNA was detected by Southern blotting. FLAG-tagged DDX17 proteins were detected by Western blotting. (B) HBV cccDNA was quantified by qPCR and plotted as relative levels of control (fold change of 1) (mean ± standard deviation, n = 3).
FIG 6
FIG 6
RNA-binding activity is required for DDX17-mediated inhibition of pgRNA encapsidation. (A) Schematic diagram of DDX17. The ATP-binding, RNA-binding, and DEAD domains of DDX17 are indicated by shaded boxes. Point mutations are indicated by the residue number of the wild-type residue and the alteration. (B) HepG2 cells were cotransfected with pHBV1.3 and equal amounts of control vector (lanes 1), FLAG-DDX17 (lanes 2), Myc-DDX17K142R (lanes 3), or FLAG-DDX17S277L (lanes 4). Cells were harvested 5 days posttransfection, followed by Northern and Southern blotting of viral total RNA, encapsidated pgRNA, and core DNA, respectively. HBV precore/core protein and epitope-tagged wild-type and mutant DDX17 protein were analyzed by Western blotting.
FIG 7
FIG 7
Subcellular localization of DDX17 in the absence and presence of HBV. HepG2 cells were cotransfected with FLAG-DDX17 plus control vector (top), pCMVHBV (middle), or pCMVHBVΔORF (bottom) for 3 days, and the intracellular localization of FLAG-DDX17 was detected by immunofluorescence. Cell nuclei were stained by DAPI.
FIG 8
FIG 8
DDX17 interacts with HBV RNA in cells. Huh7 cells were cotransfected with pCMVHBV and control vector (lane 1), FLAG-DDX17 (lane 2), Myc-DDX17K142R (lanes 3), or FLAG-DDX17S277L (lane 4) for 4 days. (Top) The input HBV RNA and epitope-tagged DDX17 proteins were detected by Northern blotting and Western blotting, respectively. Cell lysates were immunoprecipitated by beads coated with anti-FLAG or anti-Myc antibodies. (Bottom) The immunoprecipitated FLAG-DDX17, Myc-DDX17K142R, and FLAG-DDX17S277L were detected by Western blotting. HBV RNA captured by beads was extracted and subjected to Northern blotting.
FIG 9
FIG 9
HBV pgRNA sequence element responsible for DDX17 binding is located in the terminal redundancy. (A) Diagram of HBV plasmid constructs expressing the full-length wild-type (WT) pgRNA and mutant pgRNA with terminal redundancy (TR) deletions, including the 5′ TR deletion mutant (pg-Δ5TR), 3′ TR deletion mutant (pg-Δ3TR), and double-deletion mutant (pg-Δ3/5TR). The arrow indicates the pgRNA transcription initiation site (nt 1820). pA is the polyadenylation signal (nt 1918). The solid dot indicates the 5′ cap of mRNA, and the sawtooth line represents the poly(A) tail. The 5′ and 3′ TR (nt 1820 to 1918) are indicated. (B) Huh7 cells were transfected with the indicated plasmids for 4 days. (Top) The input HBV RNA and FLAG-tagged DDX17 proteins were detected by Northern blotting and Western blotting, respectively. Cell lysates were immunoprecipitated by beads coated with anti-FLAG antibodies. (Bottom) The immunoprecipitated FLAG-DDX17 was detected by Western blotting. HBV RNA captured by beads were extracted and subjected to Northern blotting.
FIG 10
FIG 10
DDX17 binds to ε RNA in vitro. (A) Indicated amounts of purified His-tagged DDX17 or DDX17S277L proteins were incubated with 100 ng 32P-labeled ε RNA in binding buffer to form nucleoprotein complexes. The samples were analyzed by native PAGE, and the shifted bands were detected by autoradiography (lanes 2 to 7). Probe only served as a negative control (lane 1). (B) Indicated amounts of purified His-tagged DDX17 proteins were incubated with 100 ng 32P-labeled ε RNA in binding buffer to form His-DDX17/ε complexes (lanes 3, 5, and 7). Monoclonal anti-His antibody was used to bind the His-DDX17/ε complex for supershifting (lanes 4, 6, and 8). Excessive amounts (10×, 20×, and 40×) of cold unlabeled ε RNA were used to compete with the 32P-labeled ε RNA for DDX17 binding (lanes 9 to 11). Probe only and probe mixed with anti-His antibody served as negative controls (lanes 1 and 2). The samples were separated by native PAGE and subjected to autoradiography.
FIG 11
FIG 11
DDX17 competes with HBV pol for binding to pgRNA in cell cultures. Huh7 cells in 60-mm dishes were transfected with 1 μg of pCMVHBVΔCΔP and 5 μg control vector (lane 1) or 1 μg of FLAG-Pol plus increased amounts of HA-DDX17 (0 μg, 1 μg, 2 μg, and 4 μg; lanes 2 to 5). Control vector was supplemented to normalize the total amount of transfected plasmids to 6 μg per transfection. Cells were harvested 5 days posttransfection. (Top) The input HBV RNA, FLAG-pol, and HA-DDX17 proteins were detected by Northern and Western blotting, respectively. Cell lysates were immunoprecipitated by beads coated with anti-FLAG or anti-HA antibodies. (Bottom) The immunoprecipitated HBV RNA were extracted and analyzed by Northern blotting.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Revill PA, Chisari FV, Block JM, Dandri M, Gehring AJ, Guo H, Hu J, Kramvis A, Lampertico P, Janssen HLA, Levrero M, Li W, Liang TJ, Lim SG, Lu F, Penicaud MC, Tavis JE, Thimme R, Zoulim F, Members of the ICE-HBV Working Groups, ICE-HBV Stakeholders Group Chairs, ICE-HBV Senior Advisors. 2019. A global scientific strategy to cure hepatitis B. Lancet Gastroenterol Hepatol 4:545–558. 10.1016/S2468-1253(19)30119-0. - DOI - PMC - PubMed
    1. Block TM, Guo H, Guo JT. 2007. Molecular virology of hepatitis B virus for clinicians. Clin Liver Dis 11:685–706. 10.1016/j.cld.2007.08.002. - DOI - PMC - PubMed
    1. Seeger C, Mason WS. 2000. Hepatitis B virus biology. Microbiol Mol Biol Rev 64:51–68. 10.1128/MMBR.64.1.51-68.2000. - DOI - PMC - PubMed
    1. Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H, Fu L, Song M, Chen P, Gao W, Ren B, Sun Y, Cai T, Feng X, Sui J, Li W. 2012. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1:e00049. 10.7554/eLife.00049. - DOI - PMC - PubMed
    1. Marchetti AL, Guo H. 2020. New insights on molecular mechanism of hepatitis B virus covalently closed circular DNA formation. Cells 9:2430. 10.3390/cells9112430. - DOI - PMC - PubMed

Publication types

LinkOut - more resources