A genome-wide CRISPR screen identifies ZCCHC14 as a host factor required for hepatitis B surface antigen production

SUMMARY A hallmark of chronic hepatitis B virus (CHB) infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A novel small molecule RG7834 has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase protein 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential novel host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identified novel factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.

[1]  J. Young,et al.  PAPD5/7 Are Host Factors That Are Required for Hepatitis B Virus RNA Stabilization , 2019, Hepatology.

[2]  Xingchun Han,et al.  Discovery of RG7834: The First-in-Class Selective and Orally Available Small Molecule Hepatitis B Virus Expression Inhibitor with Novel Mechanism of Action. , 2018, Journal of medicinal chemistry.

[3]  V. Kim,et al.  Mixed tailing by TENT4A and TENT4B shields mRNA from rapid deadenylation , 2018, Science.

[4]  George-Lucian Moldovan,et al.  PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress , 2018, bioRxiv.

[5]  F. Nigsch,et al.  Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy , 2017, Proceedings of the National Academy of Sciences.

[6]  Wenzhe Lu,et al.  A novel orally available small molecule that inhibits hepatitis B virus expression. , 2017, Journal of hepatology.

[7]  F. Zoulim,et al.  Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B. , 2017, Journal of hepatology.

[8]  D. Durantel,et al.  Interplay between the Hepatitis B Virus and Innate Immunity: From an Understanding to the Development of Therapeutic Concepts , 2017, Viruses.

[9]  Gregory McAllister,et al.  Identification of a novel NAMPT inhibitor by CRISPR/Cas9 chemogenomic profiling in mammalian cells , 2017, Scientific Reports.

[10]  Craig M. Crews,et al.  Induced protein degradation: an emerging drug discovery paradigm , 2016, Nature Reviews Drug Discovery.

[11]  T. Pihlajaniemi,et al.  Small-Molecule Chemical Probe Rescues Cells from Mono-ADP-Ribosyltransferase ARTD10/PARP10-Induced Apoptosis and Sensitizes Cancer Cells to DNA Damage. , 2016, Cell chemical biology.

[12]  John A. Tallarico,et al.  Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62 , 2016, eLife.

[13]  C. Kuny,et al.  Virus–Host Interactions and the ARTD/PARP Family of Enzymes , 2016, PLoS pathogens.

[14]  Meghan M. Holdorf,et al.  Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation , 2015, Proceedings of the National Academy of Sciences.

[15]  O. Hofmann,et al.  VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research , 2016, Nucleic acids research.

[16]  M. Nassal.  HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B , 2015, Gut.

[17]  F. Zoulim,et al.  Antiviral therapies and prospects for a cure of chronic hepatitis B. , 2015, Cold Spring Harbor perspectives in medicine.

[18]  H. Chan,et al.  Hepatitis B virus infection , 2014, The Lancet.

[19]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[20]  Piero Carninci,et al.  PAPD5-mediated 3′ adenylation and subsequent degradation of miR-21 is disrupted in proliferative disease , 2014, Proceedings of the National Academy of Sciences.

[21]  C. Semenkovich,et al.  Peroxisomes: a nexus for lipid metabolism and cellular signaling. , 2014, Cell metabolism.

[22]  A. Bertoletti,et al.  Innate and adaptive immune responses in chronic hepatitis B virus infections: towards restoration of immune control of viral infection , 2011, Gut.

[23]  Megha K. Purohit,et al.  Air1 Zinc Knuckles 4 and 5 and a Conserved IWRXY Motif Are Critical for the Function and Integrity of the Trf4/5-Air1/2-Mtr4 Polyadenylation (TRAMP) RNA Quality Control Complex* , 2011, The Journal of Biological Chemistry.

[24]  K. Borroto-Esoda,et al.  HBV DNA replication mediated by cloned patient HBV reverse transcriptase genes from HBV genotypes A-H and its use in antiviral phenotyping assays. , 2011, Journal of virological methods.

[25]  S. Hamill,et al.  Structure and function of the polymerase core of TRAMP, a RNA surveillance complex , 2010, Proceedings of the National Academy of Sciences.

[26]  R. Brodsky.  How I treat paroxysmal nocturnal hemoglobinuria. , 2009, Blood.

[27]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[28]  D. Tollervey,et al.  The Many Pathways of RNA Degradation , 2009, Cell.

[29]  J. Hagman,et al.  The 'zinc knuckle' motif of Early B cell Factor is required for transcriptional activation of B cell-specific genes. , 2008, Molecular immunology.

[30]  R. König,et al.  A probability-based approach for the analysis of large-scale RNAi screens , 2007, Nature Methods.

[31]  Keith Bowman,et al.  Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs , 2005, Nature Biotechnology.

[32]  E. Petfalski,et al.  RNA Degradation by the Exosome Is Promoted by a Nuclear Polyadenylation Complex , 2005, Cell.

[33]  W. Keller,et al.  A New Yeast Poly(A) Polymerase Complex Involved in RNA Quality Control , 2005, PLoS biology.

[34]  S. Goff,et al.  Inhibition of Retroviral RNA Production by ZAP, a CCCH-Type Zinc Finger Protein , 2002, Science.

[35]  Alexander Bürkle,et al.  Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly(ADP‐ribosyl)ation , 2002, Hepatology.

[36]  H. Tang,et al.  Transcriptional regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[37]  J. Peters,et al.  In Vivo Regulation of Hepatitis B Virus Replication by Peroxisome Proliferators , 1999, Journal of Virology.

[38]  T. Yen,et al.  Hepatitis B virus RNA element that facilitates accumulation of surface gene transcripts in the cytoplasm , 1994, Journal of virology.

[39]  T. Liang,et al.  A novel hepatitis B virus (HBV) genetic element with Rev response element-like properties that is essential for expression of HBV gene products , 1993, Molecular and cellular biology.

[40]  S. Fuller,et al.  Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment , 1992, The Journal of cell biology.

[41]  M. Umeda,et al.  Lipid composition of hepatitis B virus surface antigen particles and the particle-producing human hepatoma cell lines. , 1990, Journal of lipid research.

[42]  D. Ganem,et al.  Sequences 5' to the polyadenylation signal mediate differential poly(A) site use in hepatitis B viruses. , 1990, Genes & development.

[43]  V. Lingappa,et al.  Secreted hepatitis B surface antigen polypeptides are derived from a transmembrane precursor , 1988, The Journal of cell biology.

[44]  A. Levinson,et al.  Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids , 1983, Molecular and cellular biology.

[45]  A. Lee,et al.  HBsAg mRNA degradation induced by a dihydroquinolizinone compound depends on the HBV posttranscriptional regulatory element , 2018, Antiviral research.

[46]  Jeremy L Jenkins,et al.  Identifying compound efficacy targets in phenotypic drug discovery. , 2016, Drug discovery today.