Interplay Between Human Papillomavirus and cGAS/STING

Abstract

The germline-encoded innate immune system is the human body’s initial defense system against foreign pathogens. Essential to this system are pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs). A key PRR system, the cGAS/STING pathway, recognizes foreign DNA in the cytoplasm of a cell. Upon binding foreign DNA, cGAS creates second messenger 2’-3’-cGAMP which activates STING at the endoplasmic reticulum. Once activated, STING translocates to the Golgi, where it recruits the kinase TBK1 and transcription factor IRF3. TBK1 phosphorylates IRF3, enabling the transcription factor to translocate to the nucleus and induce the transcription of type-I interferon (IFN-I) genes. Mechanisms that regulate the cGAS/STING pathway are needed to ensure that an IFN-I response is mounted against only foreign or harmful material at the correct place and time. While cGAS typically only mounts a response to DNA in the cytoplasm of a cell, nuclear and cytoplasmic contents are mixed during mitosis, therefore giving cGAS access to genomic DNA. It is unknown whether cGAS mounts an IFN-I response to exogenous DNA during mitosis, and if not, what mechanisms are in place to ensure the pathway is inactive during cellular division. Human papillomavirus (HPV) causes one of the most commonly transmitted sexual infections and is attributed to nearly 5% of all human cancers. HPV infects the basal cells of differentiated cutaneous and mucosal epithelium. Work from our lab and others has shown that the viral genome hides within cellular organelles until the onset of mitosis. This trafficking mechanism is unique, but the evolutionary rationale for such vesicular, mitosis- dependent egress is unknown. Further, it is unknown how HPV evades detection during both initial infection and genome maintenance. Overall, we hypothesize that HPV evades detection by cGAS/STING during both initial infection and viral persistence to support the viral lifecycle. We speculate HPV uses a vesicular trafficking mechanism, carefully timed with mitosis when we propose cGAS/STING to be inactive, to evade detection during initial infection. Further, we hypothesize HPV antagonizes the cGAS/STING pathway during the genome maintenance phase. Using biochemical inhibitors and cell synchronization methods in a spontaneously immortalized cell line, we show that mitotic Golgi vesiculation attenuates cGAS/STING activity at the level of STING. Further, we use HPV pseudovirions to show that the virus evades detection by cGAS/STING. By alternatively delivering those virions via cationic liposomes, we show this evasion during initial infection is due to the virus’ vesicular, mitosis-dependent trafficking mechanism. Lastly, we show that persistent HPV18 infection in human foreskin keratinocytes suppresses IFN genes and further inhibits cGAS/STING responses to exogenous foreign DNA. Overall, this dissertation provides significant contributions to the fields of innate immunity and virology. Specifically, the findings herein provide insights into a novel regulatory mechanism of the innate immune system. Additionally, this work uncovers unique immunoevasive tactics that enable HPV to evade and antagonize the cGAS/STING system to achieve infection and viral persistence. Ultimately, understanding how HPV takes advantage of cGAS/STING regulation and further inhibits its function is essential to learning how HPV achieves viral persistence, as persistent infections cause cancer.