A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gD
| Autor: | Finn Grey, Lisa Wendt, Martin Schwemmle, Katrin Pannhorst, Thomas C. Mettenleiter, Thiprampai Thamamongood, Julia E. Hölper, J K Baillie, Nicholas J. Parkinson, Dirk Höper, Barbara G. Klupp, Tim Regan |
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| Rok vydání: | 2021 |
| Předmět: |
Swine
animal diseases viruses Mutant Pseudorabies Transferases (Other Substituted Phosphate Groups) Genome Viral Kidney Virus Replication Microbiology Virus Article Cell Line herpesvirus Virology Sphingomyelin synthase CRISPR Animals SMS1 Gene Infectivity Gene Editing gD–Pass sgms1 biology Host Microbial Interactions Wild type virus diseases biochemical phenomena metabolism and nutrition biology.organism_classification pseudorabies virus Herpesvirus 1 Suid QR1-502 PrV Infectious Diseases Mutation biology.protein CRISPR-Cas Systems CRISPR/Cas9 gene editing sphingomyelin synthase |
| Zdroj: | Viruses Hölpe, J E R, Grey, F, Baillie, J K, Regan, T, Parkinson, N, Hoper, D, Thamamongood, T, Schwemmle, M, Pannhorst, K, Wendt, L, Mettenleiter, T C & Klupp, B G 2021, ' A genome-wide CRISPR/Cas9 screen reveals the requirement of host sphingomyelin synthase 1 for infection with Pseudorabies virus mutant gD–Pass ', Viruses, vol. 13, no. 8, 1574 . https://doi.org/10.3390/v13081574 Volume 13 Issue 8 Viruses, Vol 13, Iss 1574, p 1574 (2021) |
| ISSN: | 1999-4915 |
| DOI: | 10.3390/v13081574 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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