GARP and EARP are required for efficient BoHV-1 replication as identified by a genome wide CRISPR knockout screen

The advances in gene editing bring unprecedented opportunities in high throughput functional genomics to animal research. Here we describe a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in the cattle genome. Using it, we conducted genome wide screens during Bovine Herpes Virus type 1 (BoHV-1) replication and compiled a list of pro-viral and anti-viral candidates. These candidates might influence multiple aspects of BoHV-1 biology such as viral entry, genome replication and transcription, viral protein trafficking and virion maturation in the cytoplasm. Some of the most intriguing examples are VPS51, VPS52 and VPS53 that code for subunits of two membrane tethering complexes, the endosome-associated recycling protein (EARP) complex and the Golgi-associated retrograde protein (GARP) complex. These complexes mediate endosomal recycling and retrograde trafficking to the trans Golgi Network (TGN). Simultaneous loss of both complexes in MDBKs resulted in greatly reduced production of infectious BoHV-1 virions. We also found that viruses released by these deficient cells severely lack VP8, the most abundant tegument protein of BoHV-1 that are crucial for its virulence. In combination with previous reports, our data suggest vital roles GARP and EARP play during viral protein packaging and capsid re-envelopment in the cytoplasm. It also contributes to evidence that both the TGN and the recycling endosomes are recruited in this process, mediated by these complexes. The btCRISPRko.v1 library generated here has been controlled for quality and shown to be effective in host gene discovery. We hope it will facilitate efforts in the study of other pathogens and various aspects of cell biology in cattle. Bovine Herpes Virus Type I (BoHV-1) causes significant pathology in cows and huge economic losses to cattle farmers worldwide. To aid efforts in controlling BoHV-1 infection, we set out to better understand the interplay between the virus and host cell factors. To this end, we created a large CRISPR knockout (KO) library that is designed to disrupt every protein coding gene in the cow. We then conducted genome wide KO screens during BoHV-1 infection in cells using this library. By doing so we compiled a list of more than 200 genes that, when deactivated by the CRISPRs, lead to less or more efficient replication of the virus. Combining these data with literature searches, we extrapolated candidate complexes or pathways that might facilitate or conversely restrict the virus. We further found that cells deficient in two complexes, the Golgi-associated retrograde protein (GARP) complex and the endosome-associated recycling protein (EARP) complex, released mostly defective viruses that greatly lacked the tegument protein VP8, which is crucial for the virus to establish new infection. Our results suggest important roles these complexes play, along with the membrane vesicles they reside on i.e. recycling endosomes and the previously contended Trans Golgi Network, during cytoplasmic viral protein packaging and envelopment for this virus. The CRISPR KO library generated here was proven effective and should be useful resource in the study of other viruses and many aspects of biology in the cow.