Human papillomaviruses (HPV) infect stratified squamous epithelia and link their productive life cycles to the differentiation of the host cell. HPV replication is regulated by cellular factors that control cell cycle progression, differentiation and DNA damage repair pathways.Many of these factors are regulated by post-translational modifications such as acetylation and phosphorylation. During infections viral proteins often control the levels and activities of epigenetic modifiers to regulate these processes. This application examines the role of the nuclear and cytoplasmic sirtuin decetylases in regulating the differentiation-dependent life cycle of human papillomaviruses. The sirtuin proteins (SIRT1 – SIRT7) are deacetylases that utilize NAD+ as a cofactor and regulate the deacetylation of a variety of cellular proteins such as histones and DNA damage repair factors. Our recent studies have shown that sirtuin 1 (SIRT1) levels are increased in HPV positive cells relative to normal keratinocytes and are retained at high levels throughout differentiation. Knockdown of SIRT1 in HPV positive cells with shRNAs was found to impair viral genome maintenance, amplification and late gene transcription, with minimal effects on cellular proliferation ability. The differentiation-dependent replication of high-risk HPVs requires activation of ATM DNA damage factors and we found that SIRT1 controls the recruitment of two members of this pathway, NBS1 and Rad51, to the viral genomes. These observations demonstrate that SIRT1 is a critical regulator of multiple aspects of the high-risk HPV life cycle. Our recent preliminary studies have also identified important roles for the cytoplasmic sirtuin SIRT2 in regulating the viral life cycle. SIRT2 deactylates a different group of factors than SIRT1 and we found that its expression is suppressed in undifferentiated HPV positive cells as compared to normal keratinocytes. In contrast, upon differentiation the levels of SIRT2 levels are increased several fold higher than matched normal keratinocytes. This suggests that SIRT2 may have different functions in undifferentiated as compared to differentiated cells. Our preliminary studies show that the reduced levels of SIRT2 in undifferentiating HPV positive cells still provide important functions as knockdown with shRNAs dramatically reduced stable HPV genome maintenance. A second sirtuin whose levels we found to be increased in HPV positive cells is SIRT6. SIRT6 is a nuclear protein that has deacetylase activity that regulates the DNA damage response as well as mono-ribosylation, which controls the activity of PARP1, which is a critical regulator of the DNA damage response. We will address the following questions: Aim 1:What is the role of SIRT2 in the HPV life cycle in undifferentiated cells? Do SIRT1 and SIRT2 cooperate to regulate viral functions? Aim 2: How does SIRT6 act in the viral life cycle? What are its targets in HPV positive cells? Does it act cooperatively with SIRT1 or SIRT2?
|Effective start/end date||7/15/17 → 6/30/20|
- National Institute of Allergy and Infectious Diseases (1R21AI133984-01)