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Principal Investigator: Katarzyna Zwolinska
Organization: LOUISIANA STATE UNIV HSC SHREVEPORT
Fiscal Year: 2024
Award: $367,373
Funding agency: National Cancer Institute
Little is known about the immediate early events of the human papillomavirus (HPV) lifecycle. This is mostly due
to the lack of a cell culture model allowing efficient infection of primary keratinocytes and at the same time a
genetic analysis of viral factors. We have recently described such a system using 293TT-derived HPV16
quasivirions and extracellular matrix (ECM)-to-cell transfer that achieves highly efficient infection of primary
keratinocytes. Infected cells support the complete viral lifecycle when subjected to growth in organotypic raft
cultures and the system is amenable to genetic screening of viral factors. Current models suggest that the human
papillomavirus undergoes three modes of replication during a complete life cycle. During the establishment
phase, incoming viral genome is amplified to reach up to several hundred episomal genome copies. During
maintenance, genome copy number is maintained. Mechanistically, post-transcriptional and -translational
regulation of the viral E1 replication function as well as the E8^E2 repressor have been suggested to be important
for genome maintenance. Lastly, differentiation-induced genome amplification in the late stages of the viral life
cycle results in amplification of viral genome to thousands of copies. Rather than using a bidirectional mode of
replication, a rolling circle mode of replication was suggested. Using an infection model combined with highly
sensitive in situ detection of viral genome, which we developed in the last funding period, we have recently made
unexpected discoveries: (i) HPV16 and HPV31 viral genome is amplified in each S phase during the maintenance
stage in monolayer cell culture; (ii) a significant fraction of viral genome is lost to the cytosol during mitosis by
failing to tether to mitotic chromosomes; lost nuclear viral genome is replenished in the next S phase; (iii)
cytosolic viral genome is also detected in organotypic raft cultures; (iv) cytosolic viral genome is degraded during
G1 and S phase by a lysosomal pathway likely utilizing (micro)autophagy; (v) E7 protein is required for efficient
degradation of the cytosolic viral genome; (vi) cytosolic viral genome fails to induce cGAS/STING signaling
despite a functioning cGAS/STING signaling pathway in HPV harboring keratinocytes. Our findings suggest that
genome maintenance is regulated at the tethering level. Based on our findings, we hypothesize that genome
copy number maintenance is regulated through restricted genome tethering to host cell chromosomes during
mitosis rather than being regulated by replication initiation. The degradation of cytosolic genomes resets genome
copy number after each mitosis. This immediately opens questions that regard the factor(s) that are limiting viral
genomes tethering to mitotic chromosomes, the mechanism by which degradation of cytosolic viral genome is
occurring, and why cytosolic viral genome does not induce innate immune signaling. We propose three specific
aims to test the hypothesis. We will identify and characterize the mechanism by which genome retention is
restricted; we will delineate the pathway by which viral genome is degraded and define E7 protein functions
essential for this process; and define the mechanism by which cGAS/STING pathway activation is prevented.
Terms: <Apoptosis><Apoptosis Pathway><Autophagocytosis><Basal Layer><Cell Body><Cell Communication and Signaling><Cell Culture Techniques><Cell Cycle><Cell Division Cycle><Cell Line><Cell Signaling><Cell division><Cell-Extracellular Matrix><CellLine><Cells><Cervical Carcinoma><Cervix Carcinoma><Cervix Uteri Carcinoma><Chromosomes><Cytosol><DNA><DNA Damage><DNA Injury><DNA Replication Initiation><Deoxyribonucleic Acid><Detection><ECM><Event><Extracellular Matrix><Funding><G2 Phase><G2 period><Gap Phase 2><Generalized Growth><Genetic Alteration><Genetic Change><Genetic Screening><Genetic analyses><Genetic defect><Genome><Growth><HPV><HPV 16><HPV 31><HPV-16><HPV16><HPV31><High Risk HPV31><Human Papilloma Virus><Human Papillomavirus><Human papilloma virus 31><Human papilloma virus type 16><Human papillomavirus 16><Human papillomavirus 31><Human papillomavirus type 16><Immune signaling><In Situ><Infection><Infectious Human Wart Virus><Intracellular Communication and Signaling><Knock-out><Knockout><Knowledge><Licensing Factor><Life Cycle><Life Cycle Stages><M Phase><Maintenance><Mitosis><Mitosis Stage><Mitotic Chromosome><Modeling><Mucosa><Mucosal Tissue><Mucous Membrane><Mutation><Nuclear><Oncogene Products><Oncogene Proteins><Oncoproteins><Pathway interactions><Phase><Post-Translational Regulation><Posttranslational Regulation><Preparation><Process><Programmed Cell Death><Proteins><Proteolytic Clipping><Proteolytic Processing><RNA Splicing><Replication Initiation><Rete Malpighii><Role><S Period><S phase><Second Gap Phase><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Skin><Splicing><Stimulator of Interferon Genes><Strains Cell Lines><Stratum Basale><Stratum Germinativum><Supporting Cell><Synthesis Period><Synthesis Phase><System><Testing><Textbooks><Time><Tissue Growth><Transcription Regulation><Transcriptional Control><Transcriptional Regulation><Translational Regulation><Uterine Cervix Carcinoma><Viral><Viral Genome><Virion><Virus><Virus Particle><Virus Replication><autophagy><biological signal transduction><cGAMP STING><cGAMP-STING><cGAMP/STING><cGAS/STING><cell culture><cell cultures><cell immortalization><cultured cell line><cyclic GMP-AMP synthase/STING><genetic analysis><genome mutation><human papilloma virus 16><human papilloma virus type 31><human papillomavirus type 31><keratinocyte><life course><monolayer><ontogeny><pathway><posttranscriptional><preparations><prevent><preventing><protein function><response><social role><tumor><type 16 Human papilloma virus><type 16 Human papillomavirus><viral detection><viral multiplication><viral replication><virus detection><virus genome><virus multiplication><wart virus>