Genome-wide CRISPR knockout screen reveals membrane tethering complexes EARP and GARP important for Bovine Herpes Virus Type 1 replication

Abstract

11We produced a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in 12 the cattle genome and used it to identify host genes important for Bovine Herpes Virus Type 1 (BHV-1) 13 replication. By infecting library transduced MDBK cells with a GFP tagged BHV-1 virus and FACS sorting 14 them based on their GFP intensity, we identified a list of pro-viral and anti-viral candidate host genes that might 15 affect various aspects of the virus biology, such as cell entry, RNA transcription and viral protein trafficking. 16Among them were VPS51, VPS52 and VPS53 that encode for subunits of two membrane tethering complexes 17 EARP and GARP. Simultaneous loss of both complexes in MDBKs resulted in a significant reduction in the 18 production of infectious cell free BHV-1 virions, suggesting the vital roles they play during capsid re-19 envelopment with endocytosed membrane tubules prior to endosomal recycling mediated cellular egress. We 20 also observed potential capsid retention and aggregation in the nuclei of these cells, indicating that they might 21 also indirectly affect capsid egress from the nucleus. The btCRISPRko.v1 library generated here greatly 22 expanded our capability in BHV-1 related host gene discovery; we hope it will facilitate efforts intended to 23 study interactions between the host and other pathogens in cattle and also basic host cell biology. 24 25 26 27 28 29 30Bovine Herpes Virus Type 1(BHV-1) causes major economic loss through multiple disease manifestations such 31 as infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis, balanoposthitis and abortions in 32 cows 1 . The virus is endemic in Ireland and the UK, with 80% of herds seropositive for infections 2-4 . Following 33 successful clearance of acute infection, BHV-1 establishes lifelong latency in sensory neurons via neuronal 34 retrograde transport. Stressful life events such as a pregnancy, co-mingling and transporting during extreme 35 weather can trigger reactivation to induce clinical diseases. Vaccination can be used to control IBR; however, a 36 recent study showed that strains isolated from respiratory cases and aborted fetuses corresponded to modified 37 live vaccine strains 5 . Improved knowledge in host-pathogen interactions is needed to develop better disease 38 control and resistance strategies. 39Like other alpha-herpesvirus, BHV-1 replicates its dsDNA genome in the host nucleus but matures into 40 infectious particles in the cytoplasm. To initiate a lytic cycle of infection, envelope glycoprotein gC of the virus 41 interacts with cell surface Heparan Sulfate proteoglycans 6,7 , enabling close contact and binding between other 42 glycoproteins and cell surface proteins. The binding of gD to the poliovirus receptor (PVR) and nectin-1 8-10 and 43 interaction between gB or gH/gL with putative receptor paired immunoglobulin-like type 2 receptor α or 44 PILRα 11-14 brings the virus closer to the cellular membrane and catalyzes cell penetrance by membrane fusion or 45 endocytosi...

Host cell factors important for BHV-1 cell entry revealed by genome-wide CRISPR knockout screen

“…Taken together, these results confirm that both PVR and PVRL2 can serve as receptors for BHV-1 and they work cooperatively for the virus. Our data also consistently suggest that PVRL2 mediates more efficient entry than PVR in MDBK cells; guides targeting PVRL2 were more enriched in the CRISPRko screen 49 and its loss lead to a more severer impact on viral replication manifested by greater loss of viral titre and spread ( Fig. 2A,B ) as well as bigger reduction and delay in viral protein synthesis ( Fig.…”

“…Guide RNA copy number changes obtained from the CRISPRko screen by comparing GFP Negative cells to GFP High cells 49 were plotted. Each dot represents one of the 21,216 protein coding genes targeted by the btCRISPRko.v1 library, with its genomic location plotted against the x-axis and −log10(p-value) based on MAGeCK 79 analysis against the y-axis; the sizes of dots represent −log2FoldChange values.…”

“…While PVRL2 has been hypothesised as an entry receptor for BHV-1 its candidacy has never been properly tested. We first generated biallelic KO clones lacking this gene by transfecting in vitro transcribed sgRNA targeting PVRL2 into an MDBK cell line expressing Cas9 (Cas9+/+) 49 . By plaquing directly on these KO cells, we detected up to 1.9-fold decrease in virus titre and 2.7-fold reduction in plaque size compared to the parental Cas9+/+ cells ( Fig.…”

“…Using the same targeting and genotyping strategies employed to make the Cas9 expressing cell lines for the screen ( Fig. 6C ) 49 , we created a homozygous dCas9+/+ cell line for CRISPRi which was confirmed by genotyping PCR ( Fig. 6D , second lane).…”

“…In a previous genome-wide CRISPR knockout study, we screened all protein coding genes in cattle to identify those that influence BHV-1 replication 49 . Having compiled a list of host genes with potential involvement in the viral replicative cycle, here we set about further evaluation of the 41 genes most likely to be involved in viral entry.…”