Nuclear Accumulation of the Papillomavirus E1 Helicase Blocks S-Phase Progression and Triggers an ATM-Dependent DNA Damage Response

Abstract

Replication of the papillomavirus genome is initiated by the assembly of a complex between the viral E1 and E2 proteins at the origin. The E1 helicase is comprised of a C-terminal ATPase/helicase domain, a central domain that binds to the origin, and an N-terminal regulatory region that contains nuclear import and export signals mediating its nucleocytoplasmic shuttling. We previously reported that nuclear accumulation of E1 has a deleterious effect on cellular proliferation which can be prevented by its nuclear export. Here we have shown that nuclear accumulation of E1 from different papillomavirus types blocks cell cycle progression in early S phase and triggers the activation of a DNA damage response (DDR) and of the ATM pathway in a manner that requires both the origin-binding and ATPase activities of E1. Complex formation with E2 reduces the ability of E1 to induce a DDR but does not prevent cell cycle arrest. Transient viral DNA replication still occurs in S-phase-arrested cells but surprisingly is neither affected by nor dependent on induction of a DDR and of the ATM kinase. Finally, we provide evidence that a DDR is also induced in human papillomavirus type 31 (HPV31)-immortalized keratinocytes expressing a mutant E1 protein defective for nuclear export. We propose that nuclear export of E1 prevents cell cycle arrest and the induction of a DDR during the episomal maintenance phase of the viral life cycle and that complex formation with E2 further safeguards undifferentiated cells from undergoing a DDR when E1 is in the nucleus.Human papillomaviruses (HPVs) are small double-stranded DNA viruses that infect the differentiating epithelium of the skin or mucosa (reviewed in references 10 and 91). About 25 types infect the anogenital tract (6, 19), characterized either as low-risk or high-risk types according to their association with benign or malignant hyperproliferative lesions. Clinically, low-risk HPV types cause benign warts while high-risk types are associated with lesions that can progress to cancer (28,56,63,86).The HPV life cycle is dependent on the differentiation program that keratinocytes undergo within a stratified epithelium. Viral DNA replication is required during the three distinct phases of the viral life cycle (reviewed in references 30 and 36). Upon infection of cells from the basal layer of the epithelium, the viral genome is established as a nuclear episome and is replicated by up to 50 to 100 copies (reviewed in reference 30). These episomes are then maintained at a constant copy number by low levels of replication in the lower layers of the epithelium. During this maintenance phase, viral DNA replication is thought to occur only once per cell cycle, during S phase, and in synchrony with replication of the host DNA (32). Finally, as the infected keratinocytes reach the uppermost differentiated layers of the epithelium, the copy number of the viral episome is amplified to very high levels (reviewed in reference 30), presumably through multiple rounds of replication in S-phase-arrested c...

Human Papillomavirus Episome Stability Is Reduced by Aphidicolin and Controlled by DNA Damage Response Pathways

“…In this way, ssDNA may be generated, stimulating activation of the ATR/ Chk1 pathway. Interestingly, transfected papillomavirus E1 alone or with E2 is sufficient for activation of both ATR and ATM (32,34). In light of the present results, it is conceivable that aphidicolin uncouples pol␣ and E1, resulting in a stalled, unstable replication fork.…”

“…Acting downstream of ATM and ATR are the Chk2 and Chk1 kinases, respectively, which phosphorylate a host of substrates to coordinate the DDR (30). Elements of both the ATM and ATR pathways are activated in HPV-positive cells, and a role for ATM activation has been implicated in productive HPV DNA replication (31)(32)(33)(34).…”

The E1 Protein of Human Papillomavirus Type 16 Is Dispensable for Maintenance Replication of the Viral Genome

“…In the maintenance phase, minimal expression of viral proteins in host cells with low copy numbers of viral genomes would allow HPV to evade cellular immune surveillance. Moreover, as recent studies indicate, a high level of E1 expression in basal-layer cells could activate an ATM-dependent damage response and cause growth suppression (10,32).…”

Intratypic changes of the E1 gene and the long control region affect ori function of human papillomavirus type 18 variants

“…Therefore, an HPV variant promoting an increase in viral replication could have a diminished transforming potential. Furthermore, Fradet-Turcotte et al (2011) demonstrated that the nuclear export of E1 is required for the maintenance of the viral genome in undifferentiated keratinocytes but not for its amplification in differentiated cells, suggesting that the increased levels of E1 in the nucleus could create a favourable environment for viral DNA amplification by blocking cells in the S-phase. This blockage would induce DNA damage response by ATM-dependent pathway, promoting DNA repair or leading to apoptosis.…”