Comparative RNAi Screening Reveals Host Factors Involved in Enterovirus Infection of Polarized Endothelial Monolayers

Coyne, Carolyn B.; Bozym, Rebecca A.; Morosky, Stefanie; Hanna, Sheri L.; Mukherjee, Amitava; Tudor, Matthew; Kim, Kwang Sik; Cherry, Sara

doi:10.1016/j.chom.2011.01.001

Cited by 64 publications

(69 citation statements)

References 37 publications

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“…The identification of validated genes associated with the ERK1/2-and CREB-dependent pathways is consistent with previous findings indicating that these pathways interfere with poliovirus and coxsackievirus B3 replication in human brain microvascular endothelial cells (25). Polioviruses are also known to interfere with the CREB-dependent pathway through cleavage of CREB by the viral 3C protease, resulting in shutdown of RNA polymerase II-mediated gene transcription (25,26).…”

Section: Discussionsupporting

confidence: 74%

“…Polioviruses are also known to interfere with the CREB-dependent pathway through cleavage of CREB by the viral 3C protease, resulting in shutdown of RNA polymerase II-mediated gene transcription (25,26). In this study, TAF1L, which has a role in assembly of the RNA polymerase II complex (27), was validated as a gene whose silencing enhances poliovirus replication (see Table S3 in the supplemental material).…”

Section: Discussionmentioning

confidence: 99%

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Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game

Sanden

Wang

Dybdahl‐Sissoko

et al. 2016

J Virol

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Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccinepreventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine. IMPORTANCEUsing a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases. Vaccines are important defenses in the fight against infectious disease. Currently, a complex set of factors such as population dynamics and bioproduction costs limit the ability to provide adequate immunization coverage to many economically distressed countries. The current efforts to eradicate poliovirus exemplify these challenges. The oral polio vaccine (OPV), consisting of attenuated Sabin strains, has reduced the total number of poliomyelitis cases by Ͼ99% since the late 1980s. However, live attenuated strains carry the risk of phenotypic reversion to a neurovirulent "vaccine-derived poliovirus" (VDPV) capable of inducing vaccine-associated paralytic poliomyelitis (VAPP) (1, 2). To prevent the emergence and circulation of VDPVs during the polio eradication effort and beyond, OPV must be replaced by the inactivated poliovirus vaccine (IPV) (3). The cost of IPV, which is approximately $3.00 per dose compared to approximately $0.20 for OPV, is a major o...

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Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game

Sanden

Wang

Dybdahl‐Sissoko

et al. 2016

J Virol

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“…HEK293T, HeLa, and U2OS cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and penicillin-streptomycin. Experiments were performed with coxsackievirus B3 (CVB3)-RD or PV (Sabin type I), expanded as described previously (19,20). Echovirus 11 (E11 [Gregory strain]) and enterovirus 71 (EV71 [BrCr]) were purchased from the ATCC.…”

Section: Cells and Virusesmentioning

confidence: 99%

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

Morosky

Lennemann

Coyne

2016

J Virol

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Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. IMPORTANCEEnterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression correlates with pronounced defects in the secretory pathway and greatly reduces the replication of CVB, PV, and EV71. Our results thus identify a novel host cell therapeutic target whose function could be targeted to alter enterovirus replication.T ransport of cargo from the endoplasmic reticulum (ER) to the Golgi complex requires a highly controlled system of proteins that function to regulate a variety of steps along the secretory pathway. These components must not only synchronize the loading of diverse cargo but also navigate the trafficking of this cargo to specific cellular compartments. The Golgi complex functions as a focal point of secretory pathway trafficking as it controls not only the anterograde trafficking of newly synthesized proteins from the ER to the cell membrane but also must facilitate the retrograde trafficking of surface-associated molecules from the plasma membrane back to the ER. Vesicles trafficking along the secretory pathway target their cargo to the Golgi complex via the use of distinct tethering machinery, suc...

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“…CAR is not universally rate-limiting for CVB3 infection-A549 lung adenocarcinoma cells express as much CAR as permissive HeLa cells, but their ability to propagate the virus is restricted by deficient expression of the DAF coreceptor (125). CVB3 permissiveness is also dictated by more than cell-surface receptors, with many intracellular factors contributing positively or negatively to infectivity (41). For example, HeLa cells become non-permissive upon overexpression of the mitochondrial antiviral signaling protein MAVS (126).…”

Section: Discussionmentioning

confidence: 99%

“…For example, CVB3 infection cleaves a negative regulator of Ras, which gives rise to ERK phosphorylation that is important for viral replication (37)(38)(39)(40). Various protein phosphatases are required for early CVB3 infection (41), and CVB3-encoded proteins can also modify host phosphatase activity directly. For example, viral protein 2C forms a complex with PP1 to inhibit IKK␤ phosphorylation and NF-B signaling (42).…”

mentioning

confidence: 99%

Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes

Shah¹,

Smolko²,

Kinicki³

et al. 2017

Molecular & Cellular Proteomics

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, and the nucleus. The extracts contain all major classes of protein phosphatases and catalyze dephosphorylation of plate-bound phosphosubstrates in a microtiter format, with cellular activity quantified at the end point by phosphospecific ELISA. The platform is optimized for six phosphosubstrates (ERK2, JNK1, p38␣, MK2, CREB, and STAT1) and measures specific activities from extracts of fewer than 50,000 cells. The assay was exploited to examine viral and antiviral signaling in AC16 cardiomyocytes, which we show can be engineered to serve as susceptible and permissive hosts for CVB3. Phosphatase responses were profiled in these cells by completing a full-factorial experiment for CVB3 infection and type I/II interferon signaling. Over 850 functional measurements revealed several independent, subcellular changes in specific phosphatase activities. During CVB3 infection, we found that type I interferon signaling increases subcellular JNK1 phosphatase activity, inhibiting nuclear JNK1 activity that otherwise promotes viral protein synthesis in the infected host cell. Our assay provides a high-throughput way to capture perturbations in important negative regulators of intracellular signaltransduction networks. Molecular & Cellular Proteomics 16:

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Comparative RNAi Screening Reveals Host Factors Involved in Enterovirus Infection of Polarized Endothelial Monolayers

Cited by 64 publications

References 37 publications

Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game

Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes

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