Human papillomaviruses activate the ATM DNA damage pathway for viral genome amplification upon differentiation

Abstract

Human papillomaviruses (HPV) are the causative agents of cervical cancers. The infectious HPV life cycle is closely linked to the differentiation state of the host epithelia, with viral genome amplification, late gene expression and virion production restricted to suprabasal cells. The E6 and E7 proteins provide an environment conducive to DNA synthesis upon differentiation, but little is known concerning the mechanisms that regulate productive viral genome amplification. Using keratinocytes that stably maintain HPV-31 episomes, and chemical inhibitors, we demonstrate that viral proteins activate the ATM DNA damage response in differentiating cells, as indicated by phosphorylation of CHK2, BRCA1 and NBS1. This activation is necessary for viral genome amplification, as well as for formation of viral replication foci. In contrast, inhibition of ATM kinase activity in undifferentiated keratinocytes had no effect on the stable maintenance of viral genomes. Previous studies have shown that HPVs induce low levels of caspase 3/7 activation upon differentiation and that this is important for cleavage of the E1 replication protein and genome amplification. Our studies demonstrate that caspase cleavage is induced upon differentiation of HPV positive cells through the action of the DNA damage protein kinase CHK2, which may be activated as a result of E7 binding to the ATM kinase. These findings identify a major regulatory mechanism responsible for productive HPV replication in differentiating cells. Our results have potential implications for the development of anti-viral therapies to treat HPV infections.

Figure 1. HPV induces a cellular DNA damage response in infected cells.

(A) DNA was harvested from undifferentiated (T0) HPV positive cells, as well as after 48 and 96 hours of differentiation in high calcium. Southern blot analysis was performed to examine amplification of viral genomes, represented by supercoiled (episomal) viral DNA. Standards of copies of HPV genomes per cell are indicated to the left of the gel. (B) Lysates harvested from undifferentiated (T0) HPV-31 positive HFKs (HFK-31) and from normal HFKs, as well as cells differentiated in high calcium medium for 48 and 96 hours, were analyzed by Western blot analysis for levels of the differentiation markers keratin 10 (K10) and involucrin. (C) Lysates were harvested from undifferentiated HFK-31 and from normal HFKs, as well as cells differentiated in high calcium medium for 48 and 96 hours. Western blot analysis was performed using the indicated phospho-specific antibodies, as well as antibodies to detect total protein levels. (D) HFK-31 cells were treated with DMSO as a vehicle control, or the indicated amounts of the ATM inhibitor KU-55933 for 48 and 96 in high calcium medium. Lysates were harvested at the indicated times and analyzed by Western blot analysis for the activation of CHK2 (Thr68), CHK1 (Ser317), and ATM (Ser1981), as well as antibodies to detect total protein levels. GAPDH served as a loading control. Ca = calcium.

Figure 2. HPV does not affect the levels of the components of the MRN complex.

Lysates were harvested from undifferentiated HFK-31 cells and normal HFKs, as well as from cells induced to differentiate in high calcium for 48 and 96 hours. Western blot analysis was performed using antibodies to NBS1, MRE11 and RAD50. Western blots were stripped and re-probed with an antibody to GAPDH as a loading control. Ca = calcium.

Figure 3. HPV induces ATM activation and the accumulation of DNA repair proteins in nuclear foci.

HFK-31 cells, as well as normal HFKs were harvested, fixed and permeabilized at either time 0 (undifferentiated cells), or after 48 hours of differentiation in high calcium medium containing DMSO, or 10 uM of the ATM inhibitor KU-55933. Samples were stained with (A) anti-pATM Ser1981 (red), anti-H2AX Ser139 (γ-H2AX) (green) antibodies; (B) anti-pCHK2 Thr68 (green), anti-γ-H2AX (red) antibodies; or (C) an anti-K10 antibody as a marker of differentiation. Samples were subsequently analyzed by confocal laser scanning microscopy using either a 60× objective lens (panels A and B), or a 40× objective lens (panel C). Cellular DNA was counterstained with DAPI. (D) Sections from organotypic raft cultures generated from HFK-31 cells, or normal HFKs were stained with an antibody to detect pCHK2-Thr68 (green). Cellular DNA was counterstained with DAPI. Images were visualized using confocal laser scanning microscopy. Ca = calcium.

Figure 4. ATM activity is required for differentiation-dependent viral genome amplification, but not episomal maintenance.

(A) DNA was isolated at the indicated passages from undifferentiated HPV-31 positive CIN612 cells grown in the presence of DMSO, or the ATM inhibitor KU-55933 and analyzed by Southern blot analysis for the presence of episomal (supercoiled) HPV DNA. (B, C) DNA was harvested from undifferentiated (0 hour) CIN612 cells, as well as cells differentiated in high calcium (48 and 96 hr) in the presence of DMSO, (B) 5 or 10 uM KU-55933, or (C) 5 uM CHK2 inhibitor (CHK2i) and analyzed for amplification of viral genomes by Southern blot analysis. (D) Lysates were harvested from undifferentiated (0 hour) CIN612 cells, as well as from CIN612 cells induced to differentiate in high calcium in the presence or absence of 5 or 10 uM KU-55933. Western blot analysis was performed using an antibody to keratin 10 (K10) and involucrin. (E) Lysates were harvested from undifferentiated (T0) HPV-31 positive cells, as well as from cells induced to differentiate in high calcium for 48 and 96 hours in the presence or absence of DMSO. Western blot analysis was performed using an antibody to K10. Blots were stripped and probed for GAPDH as a loading control. Ca = calcium.

Figure 5. Formation of replication foci is impaired in the absence of ATM activity.

FISH analysis was performed using undifferentiated (T0), as well as differentiated (48 hr high calcium) HPV-31 positive HFKs using a HPV-31 nick translated probe (green). FISH analysis was also performed on HPV positive cells grown in high calcium for 48 hr in the presence of 10 uM KU-55933. Cellular DNA was counterstained with DAPI, and images were visualized using confocal laser scanning microscopy. Ca = calcium.

Figure 6. HPV-mediated caspase activation requires CHK2 kinase activity.

Lysates were harvested from undifferentiated (0 hour) HPV-31 positive CIN612 cells, as well as from HPV-31 cells induced to differentiate in high calcium in the presence of DMSO or 5 uM of the CHK2 inhibitor (CHK2i) for the indicated times. Western blot analysis was performed using antibodies to detect the cleaved (active) form of caspase-7, as well as an antibody to detect pro-caspase-7. GAPDH served as a loading control. Ca = calcium.

Figure 7. E7 induces CHK2 phosphorylation upon differentiation and interacts with the phosphorylated form of ATM.

(A) Lysates were harvested from HFKs transduced with the retroviral vector pLXSN alone, or pLXSN-HPV-31 E7 at 0 hr (undifferentiated cells), as well as after differentiation in high calcium for 48 and 96 hours. Western blot analysis was performed using a phospho-specific antibody to pCHK2 Thr68, as well as an antibody to total CHK2. GAPDH served as a loading control. Ca = calcium. (B) Lysates were prepared from U2OS cells transiently transfected with vectors expressing HA, wild-type HA-31E7, HA-31E7 ΔLHCYE or HA-31E7 L67R. Immunoprecipitations were performed using an antibody to endogenous ATM, followed by Western blot analysis using an antibody to HA. (C) Immunoprecipitations were performed on lysates harvested from U2OS cells transfected with HA alone, HA-E7, HA-E7 DLHCYE or HA-E7 L67R using an antibody to HA followed by Western blot analysis to detect precipitated pATM Ser1981. (D) Lysates were prepared from transfected U20S cells treated with DMSO or 10 uM KU-55933 for 12 hours. Immunoprecipitatations were performed using an antibody to ATM, followed by Western blot analysis using an antibody to HA. IP = Immunoprecipitation, IB = Immunoblot. No Ab = No antibody (control).