The Deacetylase SIRT1 Regulates the Replication Properties of Human Papillomavirus 16 E1 and E2

Abstract

Human papillomaviruses (HPV) replicate their genomes in differentiating epithelium using the viral proteins E1 and E2 in association with host proteins. While the roles of E1 and E2 in this process are understood, the host factors involved and how they interact with and regulate E1-E2 are not. Our previous work identified the host replication and repair factor TopBP1 as an E2 partner protein essential for optimal E1-E2 replication and for the viral life cycle. The role of TopBP1 in host DNA replication is regulated by the class III deacetylase SIRT1; activation of the DNA damage response prevents SIRT1 deacetylation of TopBP1, resulting in a switch from DNA replication to repair functions for this protein and cell cycle arrest. Others have demonstrated an essential role for SIRT1 in regulation of the HPV31 life cycle; here, we report that SIRT1 can directly regulate HPV16 E1-E2-mediated DNA replication. SIRT1 is part of the E1-E2 DNA replication complex and is recruited to the viral origin of replication in an E1-E2-dependent manner. CRISPR/Cas9 was used to generate C33a clones with undetectable SIRT1 expression and lack of SIRT1 elevated E1-E2 DNA replication, in part due to increased acetylation and stabilization of the E2 protein in the absence of SIRT1. The results demonstrate that SIRT1 is a member of, and can regulate, the HPV16 replication complex. We discuss the potential role of this protein in the viral life cycle. HPV are causative agents in a number of human diseases, and currently only the symptoms of these diseases are treated. To identify novel therapeutic approaches for combating these diseases, the viral life cycle must be understood in more detail. This report demonstrates that a cellular enzyme, SIRT1, is part of the HPV16 DNA replication complex and is brought to the viral genome by the viral proteins E1 and E2. Using gene editing technology (CRISPR/Cas9), the SIRT1 gene was removed from cervical cancer cells. The consequence of this was that viral replication was elevated, probably due to a stabilization of the viral replication factor E2. The overall results demonstrate that an enzyme with known inhibitors, SIRT1, plays an important role in controlling how HPV16 makes copies of itself. Targeting this enzyme could be a new therapeutic approach for combating HPV spread and disease.

Werner helicase control of human papillomavirus 16 E1-E2 DNA replication is regulated by SIRT1 deacetylation

“…S1a). This restoration, combined with SIRT1 over expression restoring wild type E1-E2 replication levels in the SIRT1 CRISPR/Cas9 knock out cells (41), demonstrates that the replication effects following SIRT1 depletion are not due to off target effects of the CRISPR/Cas9 targeting sequences.…”

“…The reduction in the WRN recruitment is statistically significant as indicated with * with a p-value less than 0.05, standard error bars are shown. E) There is no significant difference in E1 and E2 recruitment to the replicating DNA in the absence of SIRT1, although E2 levels to trend higher, presumably due to the elevated levels of E2 in the absence of SIRT1 (41). Results represent a summary of at least 3 independent experiments, and standard error bars are shown.…”

“…The control for this figure is provided in Fig S1d. There is no difference in the levels of E1 binding to the replicating DNA between the two cell types while there is an increase in the E2 levels, perhaps due to the elevated levels of E2 in the absence of SIRT1 (41). This is the first report of WRN being involved in E1-E2 DNA replication; WRN is a known substrate of SIRT1 (57, 60, 68) and a DDR protein (61, 62, 64, 68).…”

“…SIRT1 can also regulate the acetylation status of other proteins involved in DNA replication initiation (40), therefore we postulated that SIRT1 may be able to regulate E1-E2 DNA replication. We demonstrated that SIRT1 interacts with both E1 and E2 and is recruited to E1-E2 replicating DNA and that CRISPR/Cas9 editing of SIRT1 resulted in elevated E1-E2 replication, perhaps via increased acetylation of E2 (41). SIRT1 has been shown to play a similar role in mammalian DNA replication (42); phosphorylation of SIRT1 on threonine 530 promotes SIRT1 association with replication origins and facilitates replication fork elongation and is required to maintain genome integrity following replication stress.…”

“…Exploiting HR to maintain the fidelity of E1-E2 replication would therefore promote generation of successful viral progeny. The E1-E2 interacting factor SIRT1 (41) plays a role in HR. NBS1, a member of the MRN complex, is a SIRT1 substrate and deacetylation of NBS1 by SIRT1 is required for ATM phosphorylation of NBS1 promoting the formation of the MRN complex (53).…”