126. Identification of Human Papillomavirus Entry Receptors with CRISPR-Cas9 Library

Abstract

Human papillomaviruses (HPVs) are a family of small nonenveloped viruses that induce mostly benign papillomas. However, infection of several high-risk HPV types, most prominently HPV 16 and 18, can cause cervical cancer and other epithelial tumors. The cell biology of HPV entry is still a subject of scientific debate, and in particular, the cellular receptors for HPV infection have not been firmly determined. The present study was designed to use CRISPR-Cas9 gene knock-out library to identify cellular proteins that may be used by HPV for entry. HEK293 cells were initially transduced with CRISPR-Cas9 knockout screening libraries, and then selected with puromycin for two weeks. The cells were then transduced with HPV-16 pseudoviruses containing the GFP marker gene, and GFP-negative cells were subsequently selected by flow cytometry. This process was repeated multiple rounds to enrich the GFP− cell population, and the percentage of GFP+ cells went down from over 90% to less than 10% during this enrichment process. A combination of PCR and DNA sequencing was then used to determine the genes that had been knocked out from this cell population. Our data show that solute carrier family 35 (adenosine 3’-phospho 5’-phosphosulfate transporter) member B2 (SLC35B2), malectin (MLEC), xylosylprotein beta 1,4-galactosyltransferase and transmembrane protein 8C (TMEM8C) were the most frequently knocked out genes in these cells. We then validated the involvement of one of these genes, SLC35B2 in HPV entry, by either deleting genes from fresh HEK293 cells or by reintroducing the gene back to the cells that had it knocked out. Interestingly, SLC35B2 knockout was found to affect entry of some other viruses such as herpes simplex viruses. In conclusions, CRISPR-Cas9 knockout screening system is a useful tool for identifying cellular receptors for viruses such as HPV, in which the entry mechanism has not been fully elucidated.