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

Abstract

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.

A Nectin1 Mutant Mouse Model Is Resistant to Pseudorabies Virus Infection

“…Third, the recently developed CRISPR library can effectively screen host genes essential for viral infection [ 37 , 38 ]. A genome-wide screen has been performed for some alphaherpesviruses including PRV and Bovine Herpes Virus Type 1 (BHV-1) and revealed more potential gene targets, which are promising resources facilitating antiviral study [ 39 , 40 ].…”

The Epidemiology and Variation in Pseudorabies Virus: A Continuing Challenge to Pigs and Humans

“…When SMS1 was knocked out in the cells, the infection efficiency of PRV mutants without the gD gene decreased by 90%. This indicates that SMS1 plays a crucial role in PRV infection when the gD-mediated invasion pathway is blocked [ 97 ].…”

Advances in CRISPR-Based Functional Genomics and Nucleic Acid Detection in Pigs

“…related to Japanese encephalitis virus (JEV) infection ( Zhao et al, 2020 ). Several reports have identified multiple host factors required for the entry of other viruses and toxins in pigs and humans by using the CRISPR screening strategy ( Hölper et al, 2021 ; Luo et al, 2021 ; Sun et al, 2021 ; Yu et al, 2021 ; Zhou et al, 2021 ).…”

“…Adopting the same strategy, the Golgi apparatus complex protein (COG8) was identified as a pivotal regulator of influenza virus infection ( Zhou et al, 2021 ). Host sphingomyelin synthase 1 (SMS1) was also found to be involved in pseudorabies virus (PRV) infection when the gD-mediated entry pathway was blocked ( Hölper et al, 2021 ). In addition, HBEGF (heparin-binding EGF-like growth factor), DPH1-5 (diphthamide biosynthesis 1–5), DNAJC24 (Hsp40 member C24), and ZBTB17 were determined as diphtheria toxin (DT) receptors ( Yu et al, 2021 ).…”