Brittany Forte Abstracts
Cancer Biology GIDP
Molecular Biology of DNA Tumor Viruses Conference (DNA Tumor Virus Meeting)
July 31-Aug 4, 2018
Oncogenic human papillomaviruses (HPVs) infect and replicate in differentiating mucosal epithelium, causing 5% of cancers worldwide and essentially all cervical cancers. During infection, HPV must traffic viral genome (vDNA) to the nuclei of basal keratinocytes. Minor capsid protein L2 facilitates intracellular transport of the subviral L2-vDNA complex to the Golgi; upon mitosis, the L2-vDNA complex penetrates limiting membranes and localizes to mitotic chromosomes, ensuring infection of both daughter cells.
Cytosolic DNA sensor cGAS recognizes cytosolic dsDNA as a pathogen-associated molecular pattern and produces the second messenger cGAMP; cGAMP causes activation and relocalization of ER- resident STING to the Golgi, where it recruits TBK1 to phosphorylate and activate IRF3, initiating a type-I IFN response. The cGAS/STING pathway is assumed, though never demonstrated, to be inactive during mitosis to avoid detecting self-DNA. Since the Golgi serves as a platform for STING/TBK1/IRF3 recruitment and activation, we hypothesize natural Golgi dispersal deactivates the pathway during open mitosis. Further, we hypothesize HPV has specifically evolved to traffic to and translocate from the mitotic Golgi as an immunoevasive tactic to avoid detection by cGAS/STING during mitosis.
HaCaTs, a human keratinocyte line, were transfected with DNA or infected with HPV pseudovirions and analyzed for cGAS/STING activation. DNA transfection resulted in IRF3 phosphorylation and nuclear translocation, and STING translocation to the Golgi, indicating cGAS/STING activity. Strikingly, chemical disruption of the Golgi ribbon potently blocked IRF3 activation in response to foreign DNA, suggesting Golgi morphology might modulate cGAS/STING activity during mitosis. In accordance, DNA-dependent IRF3 phosphorylation was transiently reduced in synchronized mitotic cells, but chemical impairment of mitotic Golgi vesiculation enabled cGAS/STING activation, even without foreign DNA transfection. Further, HPV infection resulted in minimal IRF3 phosphorylation, indicating HPV can efficiently evade detection during initial infection.
To determine if HPV’s unique trafficking enables cGAS/STING evasion, we used cationic liposomes to permit premature virion translocation across limiting membranes. Treatment with cationic liposomes renders a non-infectious, translocation-defective mutant HPV infectious, yet susceptible to cGAS/STING sensing. Overall, the cGAS/STING pathway may be inactivated by mitotic Golgi dispersal, allowing incoming HPV to evade detection while translocating from the Golgi to the cytosol/nucleus during open mitosis.
Abstract for Lay Audience
Human Papillomavirus (HPV) is the most common sexually transmitted infection, and causes nearly 5% of all cancers worldwide, including essentially all cervical cancers. HPV infections are mostly eliminated by the body’s immune system, but long-lasting, persistent HPV infections can cause deadly head, neck, and genital cancers in both men and women. Further, these viruses can also cause the formation of warts, from common hand and foot warts to less-desirable oral and genital warts in affected individuals. Unfortunately, the current HPV vaccines do not provide complete protection against all of the cancer-causing HPV types, nor do they protect against all types that cause warts. Therefore, it is essential to continue to study HPV so we can better understand HPV infection, and the formation of warts and cancer, so we can improve human health.
To meet these needs, I have the goal of better understanding how the virus avoids setting off our body’s innate “alarm system,” which is meant to detect and eliminate viruses like HPV before they have a chance to harm us. I speculate HPV’s unique means of infection might enable these viruses sneak past our body’s immune system; specifically, evasion of foreign DNA sensors, our body’s alarm system intended to detect foreign genetic material, such as the DNA of Human Papillomavirus. Although normally active and able to detect other viruses like Herpesvirus or HIV, we believe these sensors are inactive during a special, short window called cell division, when our own DNA, or genes, are no longer protected from the sensors. It is logical to hypothesize that the DNA sensors are off during cell division in order to prevent an autoimmune response against our own genetic material every time our cells divide, which occurs at least once a day. Furthermore, a structure called the Golgi drastically changes shape during cell division; namely, the structure breaks into tiny pieces. Our alarm system relies on the Golgi as a physical “landing dock” to assemble and activate, so I think an intact Golgi is required for proper detection of foreign DNA, and that a broken Golgi during cell division is not sufficient to activate the immune system against HPV infection.
I study HPV infection in a human skin cell line to best mimic natural HPV infection without experimenting on humans. Overall, the results from my studies support my hypothesis: the immune sensors are indeed inactive during cellular division, due to the breaking apart of the Golgi. Further, I have demonstrated that the immune sensors do not detect HPV infection because it uniquely times its infection when the alarm system is inactive, unlike other viruses such as Herpes or HIV that set off these alarms.
Overall, my data demonstrates the possibility of a unique, early evasion tactic used by only HPV that may contribute to long-lasting viral infection, and ultimately, cancer. The findings in my work advance the fields of cell biology and immunology because this is the first time anyone has ever demonstrated how cell division regulates our immune system. Further, this work significantly advances the field of virology because it is the first time anyone has demonstrated how HPV can so uniquely evade detection, unlike other similar viruses. Lastly, my work may have implications in preventing persistent, long-lasting HPV infections, as researchers may be able to use drugs to activate the immune system to “force” HPV to be sensed by the immune system and efficiently eliminated from our bodies before the infection progresses to wart formation, or worse, to cancer.