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

Abstract

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 hostdependency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.CRISPR is revolutionizing the use of genetic screens because the ability to completely knockout genes substantially increases the robustness of the phenotypes 5,6 . We compared the CRISPR approach in the hepatocyte cell line Huh7.5.1 with an alternative method to generate knockout alleles on a genome-wide scale: insertional mutagenesis in human haploid cells (HAP1) 8,9 (Fig. 1a). Both methods generate libraries of cells with knockout mutations in all non-essential genes. To comprehensively identify cellular genes with crucial roles in the Flaviviridae life cycles, we first infected pools of mutagenized cells with DENV serotype 2 (DENV-2). The two types of genetic screening methods showed a strong concordance in the genes enriched in the DENV-2-resistant population. Many could be functionally classified into three distinct categories, each important for proper expression of ER-targeted glycoproteins (Fig. 1b, c, Supplementary Tables 1, 2). The translocon associated protein (TRAP) complex (containing subunits SSR1, SSR2 ...

Reliance of Wolbachia on High Rates of Host Proteolysis Revealed by a Genome-Wide RNAi Screen of Drosophila Cells

“…Particularly striking are the recent findings that positive-strand RNA viruses specifically target and subvert the ERAD pathway to facilitate replication and assembly (Noack et al 2014). For example, the ERAD pathway was identified in a recent genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) screen to identify host factors essential to the dengue virus (Marceau et al 2016). Our finding that Wolbachia also relies on the ERAD pathway for replication and produces dramatic alterations in ER morphology suggests that Wolbachia and positive-strand RNA viruses may be competing for the same intracellular niche.…”

Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

“…Our arrayed screens are compatible with these screening platforms. Currently, most CRIPSR screens are conducted in a pooled format, using the lentiviral system to identify viral host factors (Ma et al 2015;Marceau et al 2016;Savidis et al 2016;Zhang et al 2016;Kim et al 2017;Park et al 2017). Although these screens have been used successfully, only a few significant factors were selected, even though a genome-wide sgRNA library covering over 10,000 genes was used.…”

RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

“…Many of the identified host factors are components of the translation machinery, including RPs. Other research groups have also performed genome-scale screens to identify host factors for flaviviruses by using siRNA or CRISPR and identified components of the translation machinery, but follow-up experiments of these components were limited (24)(25)(26)(27)(28). Among the top candidate proteins identified as host factors for flavivirus infection of human cells in our screens were RPLP1 and RPLP2 (RPLP1/2), which form a stable heterodimer that is tethered to the ribosome by a third RP, RPLP0, to form a conserved structure known as the ribosomal stalk.…”