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Key Points Viruses are obligate intracellular pathogens that depend on host cellular components for replication. Genetic screens are an unbiased and comprehensive method to uncover host cellular components that are critical for the infection with viruses. Loss-of-function screens result in the genome-wide disruption of gene expression, whereas gain-of-function screens rely on large-scale overexpression of host genes. Genetic knockout screens can be conducted using haploid insertional mutagenesis or the CRISPR–Cas system. Genetic screens using the CRISPR–Cas system have provided crucial insights in the host determinants of infections with important human pathogens such as dengue virus, West Nile virus, Zika virus and hepatitis C virus. CRISPR–Cas-based techniques additionally provide ways to generate both in vitro and in vivo models to study viral pathogenesis, to manipulate viral genomes, to eradicate viral disease vectors using gene drive systems and to advance the development of antiviral therapeutics. In this Review, Puschnik and colleagues discuss the technical aspects of using CRISPR–Cas technology in genome-scale knockout screens to study virus–host interactions, and they compare these screens with alternative genetic screening technologies. Viruses depend on their hosts to complete their replication cycles; they exploit cellular receptors for entry and hijack cellular functions to replicate their genome, assemble progeny virions and spread. Recently, genome-scale CRISPR–Cas screens have been used to identify host factors that are required for virus replication, including the replication of clinically relevant viruses such as Zika virus, West Nile virus, dengue virus and hepatitis C virus. In this Review, we discuss the technical aspects of genome-scale knockout screens using CRISPR–Cas technology, and we compare these screens with alternative genetic screening technologies. The relative ease of use and reproducibility of CRISPR–Cas make it a powerful tool for probing virus–host interactions and for identifying new antiviral targets.

Many viruses interface with the autophagy pathway, a highly conserved process for recycling cellular components. For three viral infections in which autophagy constituents are proviral (poliovirus, dengue, and Zika), we developed a panel of knockouts (KOs) of autophagy-related genes to test which components of the canonical pathway are utilized. We discovered that each virus uses a distinct set of initiation components; however, all three viruses utilize autophagy-related gene 9 (ATG9), a lipid scavenging protein, and LC3 (light-chain 3), which is involved in membrane curvature. These results show that viruses use noncanonical routes for membrane sculpting and LC3 recruitment. By measuring viral RNA abundance, we also found that poliovirus utilizes these autophagy components for intracellular growth, while dengue and Zika virus only use autophagy components for post-RNA replication processes. Comparing how RNA viruses manipulate the autophagy pathway reveals new noncanonical autophagy routes, explains the exacerbation of disease by starvation, and uncovers common targets for antiviral drugs.

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.

Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase β adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology.

RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.

Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two ER-associated lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. TMEM41B is also a recently validated host factor required by flaviviruses and coronaviruses. However, the exact underlying mechanism of TMEM41B in promoting viral infections remains an open question. Here, we validated that both TMEM41B and VMP1 are essential host dependency factors for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43), but not chikungunya virus (CHIKV). While HCoV-OC43 failed to replicate entirely in both TMEM41B- and VMP1-deficient cells, we detected diminished levels of DENV infections in these cell lines, which were accompanied by upregulation of the innate immune dsRNA sensors, RIG-I and MDA5. Nonetheless, this upregulation did not correspondingly induce the downstream effector TBK1 activation and Interferon-beta expression. Despite low levels of DENV replication, classical DENV replication organelles were undetectable in the infected TMEM41B-deficient cells, suggesting that the upregulation of the dsRNA sensors is likely a consequence of aberrant viral replication rather than a causal factor for reduced DENV infection. Intriguingly, we uncovered that the inhibitory effect of TMEM41B deficiency on DENV replication, but not HCoV-OC43, can be partially reversed using exogenous fatty acid supplements. In contrast, VMP1 deficiency cannot be rescued using the metabolite treatment. In line with the observed phenotypes, we found that both TMEM41B- and VMP1-deficient cells harbor higher levels of compromised mitochondria, especially in VMP1 deficiency which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, we revealed distinctive global dysregulations of the cellular metabolome, particularly lipidome, in TMEM41B- and VMP1-deficient cells. Our findings highlight a central role for TMEM41B and VMP1 in modulating multiple cellular pathways, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses.

The phospholipase PLA2G16 is required for the entry of picornaviruses, and in its absence, virus infection is prevented by a galectin-8-mediated process. The role of PLA2G16 in picornavirus replication Thijn Brummelkamp and colleagues use a genome-wide haploid genetic screen to identify the host factors required for the replication of picornaviruses. They identify the small phospholipase PLA2G16 as being required for the cytoplasmic delivery of the viral genome. In a second screen to find mutants that restored virus susceptibility to PLA2G16-deficient cells, the authors identify galectin-8, a sensor previously implicated in the autophagic clearance of intracellular bacteria. The precise function of PLA2G16 remains unclear, but the authors suggest that it facilitates the displacement of the viral genome from galactin-8-positive vesicles. Picornaviruses are a leading cause of human and veterinary infections that result in various diseases, including polio and the common cold. As archetypical non-enveloped viruses, their biology has been extensively studied 1 . Although a range of different cell-surface receptors are bound by different picornaviruses 2 , 3 , 4 , 5 , 6 , 7 , it is unclear whether common host factors are needed for them to reach the cytoplasm. Using genome-wide haploid genetic screens, here we identify the lipid-modifying enzyme PLA2G16 (refs 8 , 9 , 10 , 11 ) as a picornavirus host factor that is required for a previously unknown event in the viral life cycle. We find that PLA2G16 functions early during infection, enabling virion-mediated genome delivery into the cytoplasm, but not in any virion-assigned step, such as cell binding, endosomal trafficking or pore formation. To resolve this paradox, we screened for suppressors of the Δ PLA2G16 phenotype and identified a mechanism previously implicated in the clearance of intracellular bacteria 12 . The sensor of this mechanism, galectin-8 (encoded by LGALS8 ), detects permeated endosomes and marks them for autophagic degradation, whereas PLA2G16 facilitates viral genome translocation and prevents clearance. This study uncovers two competing processes triggered by virus entry: activation of a pore-activated clearance pathway and recruitment of a phospholipase to enable genome release.

Genetic screens in cultured human cells represent a powerful unbiased strategy to identify cellular pathways that determine drug efficacy, providing critical information for clinical development. We used insertional mutagenesis-based screens in haploid cells to identify genes required for the sensitivity to lasonolide A (LasA), a macrolide derived from a marine sponge that kills certain types of cancer cells at low nanomolar concentrations. Our screens converged on a single gene, LDAH , encoding a member of the metabolite serine hydrolase family that is localized on the surface of lipid droplets. Mechanistic studies revealed that LasA accumulates in lipid droplets, where it is cleaved into a toxic metabolite by LDAH. We suggest that selective partitioning of hydrophobic drugs into the oil phase of lipid droplets can influence their activation and eventual toxicity to cells. Lasonolide A is hydrolyzed into a cytotoxic metabolite by a lipid droplet-associated orphan serine hydrolase, showing that lipid droplets can promote drug toxicity by both accumulating and metabolizing drugs in cells.

A combination of shRNA- and CRISPR-Cas9-based gene editing screens, corroborated by a metabolite suppression experiment identifies the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) as the target of the broad-spectrum antiviral compound GSK983. Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.