CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication

Zhao, Changzhi; Liu, Hailong; Xiao, Tianhe; Wang, Zichang; Nie, Xiongwei; Li, Xinyun; Qian, Ping; Qin, Liuxing; Han, Xiaosong; Zhang, Jinfu; Ruan, Jinxue; Zhu, Mengjin; Miao, Yi‐Liang; Zuo, Bo; Yang, Kui; Xie, Shengsong; Zhao, Shuhong

doi:10.1038/s41467-020-18936-1

Cited by 53 publications

(44 citation statements)

References 57 publications

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“…PAPS serves as an important substrate for sulfation of several proteins as CCR5 [ 41 ] and all members of glycosaminoglycans (GAGs) such as chondroitin [ 42 ] or heparan sulfate [ 43 ] in the Golgi compartment. It is therefore not surprising that SLC35B2 frequently appears in genome-wide screens as a top hit [ 25 , 30 , 41 , 44 ]. Thus, it cannot be excluded that other important host factors for LCMV infection are also affected, as might be indicated by reduced infection of high affinity VSV-GP variants in 293T Δ SLC35B2 cells.…”

Section: Discussionmentioning

confidence: 99%

Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants

et al. 2021

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Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).

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Section: Discussionmentioning

confidence: 99%

Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants

et al. 2021

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“…Williams and Goddard-Borger ( 144 ) suggested that the S protein of SARS-CoV-2 is also likely folded by CNX and proposed alpha-glucosidase inhibitors as antivirals for evaluation. Finally, Zhao et al, using a CRISPR screening of a porcine single guide RNA (sgRNA) library, identified CRT as a major host factor for JEV replication ( 145 ).…”

Section: Other Er Chaperonesmentioning

confidence: 99%

Endoplasmic Reticulum Chaperones in Viral Infection: Therapeutic Perspectives

Kohli

Causse

Baverel

et al. 2021

Microbiol Mol Biol Rev

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Viruses are intracellular parasites that subvert the functions of their host cells to accomplish their infection cycle. The endoplasmic reticulum (ER)-residing chaperone proteins are central for the achievement of different steps of the viral cycle, from entry and replication to assembly and exit. The most abundant ER chaperones are GRP78 (78-kDa glucose-regulated protein), GRP94 (94-kDa glucose-regulated protein), the carbohydrate or lectin-like chaperones calnexin (CNX) and calreticulin (CRT), the protein disulfide isomerases (PDIs) and the DNAJ chaperones.

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“…In our first screens, we used wildtype PrV-Ka for selection, but no cells survived. The same was true when we infected the cells with the highly attenuated vaccine strain Bartha [ 68 ]. This is in line with the perception that PrV is very flexible, can adapt to a number of different settings and can use multiple host cell proteins for successful propagation [ 4 ].…”

Section: Discussionmentioning

confidence: 81%

A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gD–Pass

Hölper

Grey

Baillie

et al. 2021

Viruses

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Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD–Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD–Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD–Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD–Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked.

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CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication

Cited by 53 publications

References 57 publications

Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants

Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants

Endoplasmic Reticulum Chaperones in Viral Infection: Therapeutic Perspectives

A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gD–Pass

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