Identification of Antinorovirus Genes in Human Cells Using Genome-Wide CRISPR Activation Screening

Orchard, Robert C.; Sullender, Meagan E.; Dunlap, Bria F.; Balce, Dale R.; Doench, John G.; Virgin, Herbert W.

doi:10.1128/jvi.01324-18

Cited by 47 publications

(50 citation statements)

References 39 publications

(51 reference statements)

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“…Proteins in selected, mutually exclusive gene ontology categories are plotted in different colours as depicted. ( B ) A number of factors previously identified as MNV host factors using proteomics or CRISPR approaches (Orchard et al, 2019; Chung et al, 2014; Vashist et al, 2012b) copurified in pulldowns of NS1/2 and NS4 (highlighted red). ( C ) Novel putative MNV host factors highly enriched (Log 2 SILAC ratio >4 for either protein) by NS1/2 and NS4 are plotted in black.…”

Section: Resultsmentioning

confidence: 99%

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation

Hosmillo

Jia

McAllaster

et al. 2019

eLife

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Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating ribosome recruitment. Our data uncovers a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifies the first host factor with pan-norovirus pro-viral activity.

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Section: Resultsmentioning

confidence: 99%

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation

Hosmillo

Jia

McAllaster

et al. 2019

eLife

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“…GBPs are targeted to the MNV replication complex in an interferon-dependent manner that requires components of the autophagy pathway and exert their antiviral activity via an unknown mechanism (71). GBP2 was also identified as a norovirus restriction factor in a CRISPR-based activation screen where it was found to have potent antiviral activity against two strains of MNV (72). Further studies will be required to determine if GBPs have similar antiviral effects during HuNoV infection.…”

Section: Discussionmentioning

confidence: 99%

Norovirus Replication in Human Intestinal Epithelial Cells Is Restricted by the Interferon-Induced JAK/STAT Signaling Pathway and RNA Polymerase II-Mediated Transcriptional Responses

Hosmillo

Chaudhry

Nayak

et al. 2020

mBio

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Human noroviruses (HuNoV) are a leading cause of viral gastroenteritis worldwide and a significant cause of morbidity and mortality in all age groups. The recent finding that HuNoV can be propagated in B cells and mucosa-derived intestinal epithelial organoids (IEOs) has transformed our ability to dissect the life cycle of noroviruses. Using transcriptome sequencing (RNA-Seq) of HuNoV-infected intestinal epithelial cells (IECs), we have found that replication of HuNoV in IECs results in interferon (IFN)-induced transcriptional responses and that HuNoV replication in IECs is sensitive to IFN. This contrasts with previous studies that suggested that the innate immune response may play no role in the restriction of HuNoV replication in immortalized cells. We demonstrated that inhibition of Janus kinase 1 (JAK1)/JAK2 enhanced HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular RNA polymerase II-mediated transcription was not detrimental to HuNoV replication but instead enhanced replication to a greater degree than blocking of JAK signaling directly. Furthermore, we demonstrated for the first time that IECs generated from genetically modified intestinal organoids, engineered to be deficient in the interferon response, were more permissive to HuNoV infection. Taking the results together, our work revealed that IFN-induced transcriptional responses restrict HuNoV replication in IECs and demonstrated that inhibition of these responses mediated by modifications of the culture conditions can greatly enhance the robustness of the norovirus culture system. IMPORTANCE Noroviruses are a major cause of gastroenteritis worldwide, and yet the challenges associated with their growth in culture have greatly hampered the development of therapeutic approaches and have limited our understanding of the cellular pathways that control infection. Here, we show that human intestinal epithelial cells, which represent the first point of entry of human noroviruses into the host, limit virus replication by induction of innate responses. Furthermore, we show that modulating the ability of intestinal epithelial cells to induce transcriptional responses to HuNoV infection can significantly enhance human norovirus replication in culture. Collectively, our findings provide new insights into the biological pathways that control norovirus infection but also identify mechanisms that enhance the robustness of norovirus culture.

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“…This might be caused by differences in chromatin state, the availability of different essential activation factors, or other differences between the cell lines. CRISPR activation was recently used to identify genes inhibiting murine norovirus infection in HeLa cells using a different library (47). Several ISGs were identified among the genes rescuing cells from norovirus infection.…”

Section: Discussionmentioning

confidence: 99%

A CRISPR Activation Screen Identifies Genes That Protect against Zika Virus Infection

Dukhovny

Lamkiewicz

Qian

et al. 2019

J Virol

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Zika virus (ZIKV) is an arthropod-borne emerging pathogen causing febrile illness. ZIKV is associated Guillain-Barré syndrome and other neurological complications. Infection during pregnancy is associated with pregnancy complications and developmental and neurological abnormalities collectively defined as congenital Zika syndrome. There is still no vaccine or specific treatment for ZIKV infection. To identify host factors that can rescue cells from ZIKV infection, we used a genomescale CRISPR activation screen. Our highly ranking hits included a short list of interferon-stimulated genes (ISGs) previously reported to have antiviral activity. Validation of the screen results highlighted interferon lambda 2 (IFN-2) and interferon alpha-inducible protein 6 (IFI6) as genes providing high levels of protection from ZIKV. Activation of these genes had an effect on an early stage in viral infection. In addition, infected cells expressing single guide RNAs (sgRNAs) for both of these genes displayed lower levels of cell death than did the controls. Furthermore, the identified genes were significantly induced in ZIKV-infected placenta explants. Thus, these results highlight a set of ISGs directly relevant for rescuing cells from ZIKV infection or its associated cell death and substantiate CRISPR activation screens as a tool to identify host factors impeding pathogen infection. IMPORTANCE Zika virus (ZIKV) is an emerging vector-borne pathogen causing a febrile disease. ZIKV infection might also trigger Guillain-Barré syndrome, neuropathy, and myelitis. Vertical transmission of ZIKV can cause fetus demise, stillbirth, or severe congenital abnormalities and neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We used a genome-wide CRISPR activation screen, where genes are activated from their native promoters to identify host cell factors that protect cells from ZIKV infection or associated cell death. The results provide a better understanding of key host factors that protect cells from ZIKV infection and might assist in identifying novel antiviral targets.

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Identification of Antinorovirus Genes in Human Cells Using Genome-Wide CRISPR Activation Screening

Cited by 47 publications

References 39 publications

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation

Norovirus Replication in Human Intestinal Epithelial Cells Is Restricted by the Interferon-Induced JAK/STAT Signaling Pathway and RNA Polymerase II-Mediated Transcriptional Responses

A CRISPR Activation Screen Identifies Genes That Protect against Zika Virus Infection

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