{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T13:13:29Z","timestamp":1789132409948,"version":"build-2803163510"},"posted":{"date-parts":[[2026,9,8]]},"group-title":"Microbiology","reference-count":115,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Nuclear delivery of human papillomavirus (HPV) requires infected cells to undergo mitosis. Incoming HPV DNA is protected by a transport vesicle structure, which becomes unstable post-mitosis. We have previously shown that HPV genome egress takes on average 4 hours post-mitosis completion; thus, we propose that an enzymatic process is involved in degrading this transport vesicle to allow genome egress. Several phospholipase C (PLC) isoforms, including PLC\u03b43, PLC\u03b61, and PLCL1, have been previously implicated in HPV infection in large-scale siRNA screens, and the parvovirus VP1 capsid protein has phospholipase enzymatic activity. Therefore, we hypothesized that cellular phospholipases residing in the nucleus mediate egress of the HPV genome from transport vesicles. We utilized siRNA-mediated knockdown and CRISPR\/Cas9 knockout techniques to target specific PLC isoforms in both HeLa and HaCaT cell lines. We found that targeting PLC isoforms PLC\u03b21, \u03b24, \u03b43, \u03b44, and \u03b31 significantly decreased HPV infection in both HeLa and HaCaT cells, as measured using a luciferase-based reporter assay. We also discovered a novel interaction between HPV16 minor protein L2 and PLCs \u03b21, \u03b24, \u03b41, \u03b43, and \u03b44. Furthermore, we observed that knockout of PLC\u03b24 and \u03b44 delayed egress of the HPV genome from nuclear membrane-bound vesicles after infection of HeLa cells. We propose that HPV utilizes phospholipase Cs to achieve genome egress from its protective vesicular structure after nuclear delivery.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>The cellular factors involved in the later stages of HPV entry remain to be elucidated. Previous data gleaned from large-scale siRNA screens implicated the involvement of phospholipases in HPV infection. We expanded upon these findings and discovered that the individual phospholipase C (PLC) isoforms \u03b24 and \u03b44 are important for HPV infectivity. We further linked the loss of infectivity in PLC\u03b24- and PLC\u03b44-deficient cells to a defect in HPV genome release from its transport vesicle, which we demonstrated using our differential staining technique. Furthermore, we discovered a novel interaction between the minor protein L2 of HPV16 and PLC isoforms \u03b21, \u03b24, \u03b41, \u03b43, and \u03b44.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.03.749318","type":"posted-content","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T13:15:14Z","timestamp":1788873314000},"source":"Crossref","is-referenced-by-count":0,"title":["HPV16 Utilizes Phospholipase C(s) for its Genome Egress"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7104-6338","authenticated-orcid":false,"given":"Timothy R","family":"Keiffer","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Center for Applied Immunology and Pathological Processes, Louisiana State University Health Sciences Center, Shreveport, LA 71103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abida","family":"Siddiqa","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Center for Applied Immunology and Pathological Processes, Louisiana State University Health Sciences Center, Shreveport, LA 71103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mireya","family":"Represa-Perez","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Center for Applied Immunology and Pathological Processes, Louisiana State University Health Sciences Center, Shreveport, LA 71103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiba","family":"Zabir","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Center for Applied Immunology and Pathological Processes, Louisiana State University Health Sciences Center, Shreveport, LA 71103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anand","family":"Kushwaha","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Center for Applied Immunology and Pathological Processes, Louisiana State University Health Sciences Center, Shreveport, LA 71103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martin","family":"Sapp","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Center for Applied Immunology and Pathological Processes, Louisiana State University Health Sciences Center, Shreveport, LA 71103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katarzyna","family":"Zwolinska","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Center for Applied Immunology and Pathological Processes, Louisiana State University Health Sciences Center, Shreveport, LA 71103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091106050893000_2026.09.03.749318v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-021-00617-5"},{"key":"2026091106050893000_2026.09.03.749318v1.2","unstructured":"Al Aboud AM , Nigam PK. 2026. 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