Journal Article

Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens

Majzoub, Karim, Carette, Jan E., Brewer, Susan M., Ooi, Yaw Shin, Marceau, Caleb D., Mata, Miguel A., Sarnow, Peter, Puschnik, Andreas S., Fuchs, Gabriele, Swaminathan, Kavya, Elias, Joshua E.

2016

Overview

A CRISPR screening approach shows that endoplasmic reticulum (ER)-associated protein complexes, including the oligosaccharyltransferase (OST) protein complex, are important for infection by dengue virus and other related mosquito-borne flaviviruses, whereas hepatitis C virus is dependent on distinct entry factors, RNA binding proteins and FAD biosynthesis. Host factors required for flavivirus infection Jan Carette and colleagues use a CRISPR screening approach to identify cellular genes with important roles in the lifecycle of two important human flaviviruses: dengue virus and hepatitis C virus. The authors show that endoplasmic-reticulum-associated protein complexes, including the oligosaccharyltransferase (OST) protein complex, are important for infection by dengue virus and other related mosquito-borne flaviviruses, whereas hepatitis C virus is dependent on distinct entry factors, RNA binding proteins and FAD biosynthesis. Also in this issue of Nature , Michael Diamond and colleagues report that the endoplasmic-reticulum-associated signal peptidase complex is required for infection by numerous flaviviruses, including West Nile, dengue and Zika viruses, but not for infection by other types of virus or for host protein synthesis. The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide 1 . No approved antivirals are available to date, and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates 2 . Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available 3 . Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies 4 . A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy 5 , 6 to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N -linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects 7 . By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-dependency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 13/106 / 14/19 / 45/41 / 45/70 / 631/326/596/1413 / 631/326/596/1905 / 631/326/596/2557 / 631/61/191 / 82/1 / 82/58 / 96/44 / Birth defects / CRISPR-Cas Systems - genetics / Dengue Virus - genetics / Dengue Virus - growth & development / Dengue Virus - physiology / DNA sequencing / Drug Discovery / Endoplasmic Reticulum - metabolism / Endoplasmic Reticulum-Associated Degradation / Enzymes / Flavin-Adenine Dinucleotide - biosynthesis / Flavin-Adenine Dinucleotide - metabolism / Flavivirus Infections - genetics / Flavivirus Infections - virology / Genes / Genetic aspects / Genetic screening / Genome, Human - genetics / Genomes / Glycosylation / Hepacivirus - physiology / Hexosyltransferases - deficiency / Hexosyltransferases - genetics / Hexosyltransferases - metabolism / Host-Derived Cellular Factors - genetics / Host-Pathogen Interactions - genetics / Host-virus relationships / Humanities and Social Sciences / Humans / letter / Membrane Proteins - deficiency / Membrane Proteins - genetics / Membrane Proteins - metabolism / Methods / MicroRNAs / Molecular Targeted Therapy / multidisciplinary / Mutation / Nucleotide sequencing / Pathogens / Protein Binding / Protein Sorting Signals / Proteins / Receptors, Virus - genetics / RNA-Binding Proteins - genetics / Science / Vector-borne diseases / Viral Nonstructural Proteins - metabolism / Virus Replication / Viruses / Zika Virus - metabolism