Kinome siRNA screen identifies novel cell-type specific dengue host target genes

Kwon, Yong-Jun; Heo, Jinyeong; Wong, Hazel E.E.; Cruz, Deu John M.; Velumani, Sumathy; Silva, Camila T. da; Mosimann, Ana Luiza Pamplona; Santos, Claudia Nunes Duarte dos; Freitas‐Junior, Lúcio H.; Fink, Katja

doi:10.1016/j.antiviral.2014.07.006

Cited by 20 publications

(21 citation statements)

References 26 publications

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“…We have developed a new approach, designated viscRNA-Seq, to simultaneously quantify the whole transcriptome and intracellular virus abundance at the single cell level. This approach probes the quantitative gene expression dynamics of virus infections and is therefore complementary to knockout and knockdown genetic screens, which induce a controlled perturbation (Marceau et al 2016;Sessions et al 2009;Kwon et al 2014;Le Sommer et al 2012;Lin et al 2017) . However, unlike those loss-of-function assays, viscRNA-Seq is able to fully discern cell-to-cell variation within a single experimental condition, is compatible with time-resolved sampling, and can be used to study essential genes.…”

Section: Discussionmentioning

confidence: 99%

“…Several high-throughput approaches have been applied to screen all 20,000 human genes for interactions with flaviviruses, including knockdown screens based on RNA interference (Sessions et al 2009;Kwon et al 2014;Le Sommer et al 2012) , knockout screens via haploid cell lines or CRISPR libraries (Marceau et al 2016;Lin et al 2017) , and bulk transcriptomics via microarrays or RNA-Seq (Sessions et al 2013;Moreno-Altamirano et al 2004;Fink et al 2007;Conceição et al 2010;Becerra et al 2009;Liew and Chow 2006) . While these approaches have provided important insights, our understanding of infection-triggered cellular responses is far from complete.…”

Section: Introductionmentioning

confidence: 99%

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Single-cell transcriptional dynamics of flavivirus infection

Zanini

Bekerman

et al. 2017

Preprint

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Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Single-cell transcriptional dynamics of flavivirus infection

Zanini

Bekerman

et al. 2017

Preprint

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“…Lastly, single-cell transcriptomic studies have also been used to globally investigate virus-host interactions and identify novel candidate genes for host-targeted therapeutics (31). Knockdown screens or knockout studies can only probe a subset of nonessential host genes, limiting their scope (37–42). However, virus-inclusive scRNA-seq is a powerful platform for the discovery of novel proviral and antiviral candidate genes in an unbiased manner as recently highlighted by Zanini and colleagues with DENV and ZIKV (31).…”

Section: Introductionmentioning

confidence: 99%

West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells

O’Neal

Upadhyay

Wolabaugh

et al. 2018

Preprint

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232 words; Importance: 122 words; and Text: 4,850 words. 22 ABSTRACT 23West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. 24Neuroinvasive WNV infection results in encephalitis and can lead to prolonged 25 neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting 26 antiviral defenses through the induction of antiviral effectors, which function to restrict 27 viral replication and spread. However, our understanding of the antiviral response to 28 WNV infection is mostly derived from analysis of bulk cell populations. It is becoming 29 increasingly apparent that substantial heterogeneity in cellular processes exists among 30 individual cells, even within a seemingly homogenous cell population. Here, we present 31 WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the 32 transcriptional variation and viral RNA burden across single cells. We observed that 33 only a few cells within the bulk population displayed robust transcription of IFN-β mRNA, 34 and this did not appear to depend on viral RNA abundance within the same cell. 35 Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I 36 response, with genes displaying high unimodal and bimodal expression patterns. 37 Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, 38 corresponding with a precipitous decline in expression in cells with high viral RNA levels. 39 Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a 40 high-throughput technique for single-cell transcriptomics and WNV RNA detection. This 41 approach can be implemented in other models to provide insights into the cellular 42 features of protective immunity and identify novel therapeutic targets. 43 44 45 IMPORTANCE 46 West Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent 47 epidemics of neuroinvasive disease. Type I interferon is essential for promoting an 48 antiviral response against WNV infection; however, it is unclear how heterogeneity in 49 the antiviral response at the single-cell level impacts viral control. Specifically, 50 conventional approaches lack the ability to distinguish differences across cells with 51 varying viral abundance. The significance of our research is to demonstrate a new 52 technique for studying WNV infection at the single-cell level. We discovered extensive 53 variation in antiviral gene expression and viral abundance across cells. This protocol 54 can be applied to primary cells or in vivo models to better understand the underlying 55 cellular heterogeneity following WNV infection for the development of targeted 56 therapeutic strategies. 57 58 59 60 61

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“…Pro-tein kinases are key regulators in signal transduction and control a wide variety of cellular processes. Thus, assessing their relevance for virus replication can provide a broad perspective on factors and pathways relevant for SARS-CoV replication, as illustrated by previous studies identifying cellular kinases as host factors influencing various stages of the replicative cycle of other ϩRNA viruses (5,10,11,39,40).…”

mentioning

confidence: 99%

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

Wilde

Wannee

Scholte

et al. 2015

J Virol

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To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the ␤2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARSCoV replication. The inventory of pro-and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCEReplication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although substantial progress has been made in dissecting the coronavirus replicative cycle, our understanding of the host factors that stimulate (proviral factors) or restrict (antiviral factors) infection remains far from complete. To study the role of host proteins in SARS-CoV infection, we set out to systematically identify kinase-regulated processes that influence virus replication. Protein kinases are key regulators in signal transduction, controlling a wide variety of cellular processes, and many of them are targets of approved drugs and other compounds. Our screen identified a variety of hits and will form the basis for more detailed follow-up studies that should contribute to a better understanding of SARS-CoV replication and coronavirus-host interactions in general. The identified factors could be interesting targets for the development of host-directed antiviral therapy to treat infections with SARS-CoV or other pathogenic coronaviruses.

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Kinome siRNA screen identifies novel cell-type specific dengue host target genes

Cited by 20 publications

References 26 publications

Single-cell transcriptional dynamics of flavivirus infection

Single-cell transcriptional dynamics of flavivirus infection

West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

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