Next Article in Journal
The Multifaceted Role of Macrophages in Oncolytic Virotherapy
Next Article in Special Issue
Efficient Inhibition of HIV Using CRISPR/Cas13d Nuclease System
Previous Article in Journal
Variable In Vivo Hepatitis D Virus (HDV) RNA Editing Rates According to the HDV Genotype
Previous Article in Special Issue
An MHV-68 Mutator Phenotype Mutant Virus, Confirmed by CRISPR/Cas9-Mediated Gene Editing of the Viral DNA Polymerase Gene, Shows Reduced Viral Fitness
 
 
Article

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

1
Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald, Insel Riems, Germany
2
The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK
3
Intensive Care Unit, Royal Infirmary of Edinburgh, Edinburgh EH25 9RG, UK
4
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, 17493 Greifswald, Insel Riems, Germany
5
Institute of Virology, Medical Center-University of Freiburg, 79110 Freiburg, Germany
6
Spemann Graduate School of Biology and Medicine, University of Freiburg, 79110 Freiburg, Germany
7
Faculty of Biology, University of Freiburg, 79110 Freiburg, Germany
*
Author to whom correspondence should be addressed.
Academic Editors: Jiri Hejnar and Ben Berkhout
Viruses 2021, 13(8), 1574; https://doi.org/10.3390/v13081574
Received: 6 July 2021 / Revised: 26 July 2021 / Accepted: 2 August 2021 / Published: 9 August 2021
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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-gDPass) 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-gDPass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gDPass 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-gDPass. 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. View Full-Text
Keywords: herpesvirus; pseudorabies virus; PrV; gDPass; CRISPR/Cas9 gene editing; sphingomyelin synthase; SMS1; sgms1 herpesvirus; pseudorabies virus; PrV; gDPass; CRISPR/Cas9 gene editing; sphingomyelin synthase; SMS1; sgms1
Show Figures

Figure 1

MDPI and ACS Style

Hölper, J.E.; Grey, F.; Baillie, J.K.; Regan, T.; Parkinson, N.J.; Höper, D.; Thamamongood, T.; Schwemmle, M.; Pannhorst, K.; Wendt, L.; Mettenleiter, T.C.; Klupp, B.G. A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gDPass. Viruses 2021, 13, 1574. https://doi.org/10.3390/v13081574

AMA Style

Hölper JE, Grey F, Baillie JK, Regan T, Parkinson NJ, Höper D, Thamamongood T, Schwemmle M, Pannhorst K, Wendt L, Mettenleiter TC, Klupp BG. A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gDPass. Viruses. 2021; 13(8):1574. https://doi.org/10.3390/v13081574

Chicago/Turabian Style

Hölper, Julia E., Finn Grey, John Kenneth Baillie, Tim Regan, Nicholas J. Parkinson, Dirk Höper, Thiprampai Thamamongood, Martin Schwemmle, Katrin Pannhorst, Lisa Wendt, Thomas C. Mettenleiter, and Barbara G. Klupp. 2021. "A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gDPass" Viruses 13, no. 8: 1574. https://doi.org/10.3390/v13081574

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop