Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

Abstract

The human papillomavirus (HPV) E2 protein is a key regulator of viral transcription and replication. In this study, we demonstrate that the nonreceptor tyrosine kinase Pyk2 phosphorylates tyrosine 131 in the E2 transactivation domain. Both depletion of Pyk2 and treatment with a Pyk2 kinase inhibitor increased viral DNA content in keratinocytes that maintain viral episomes. The tyrosine-to-glutamic acid (E) mutant Y131E, which may mimic phosphotyrosine, failed to stimulate transient DNA replication, and genomes with this mutation were unable to establish stable episomes in keratinocytes. Using coimmunoprecipitation assays, we demonstrate that the Y131E is defective for binding to the C-terminal motif (CTM) of Bromodomain-containing protein 4 (Brd4). These data imply that HPV replication depends on E2 Y131 interaction with the pTEFb binding domain of Brd4.IMPORTANCE Human papillomaviruses are the major causative agents of cervical, oral, and anal cancers. The present study demonstrates that the Pyk2 tyrosine kinase phosphorylates E2 at tyrosine 131, interfering with genome replication. We provide evidence that phosphorylation of E2 prevents binding to the Brd4-CTM. Our findings add to the understanding of molecular pathways utilized by the virus during its vegetative life cycle and offers insights into the host-virus interactome.

Keywords: E2; HPV; replication; tyrosine phosphorylation.

FIG 1

HPV E2 interacts with Pyk2. (A) HEK293TT cells were transfected with GFP-Pyk2 WT (wild type) or GFP-Pyk2 KD (kinase dead) and FLAG-HPV-31 E2. FLAG-HPV-31 E2 was immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with M2 (FLAG) and Pyk2 antibodies. (B) HEK293TT cells were transfected with GFP-Pyk2 WT and FLAG-HPV-16 E2. FLAG-HPV-16 E2 was immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with M2 (FLAG) and Pyk2 antibodies. (C) HPV-16 E2 in W12 cells was immunoprecipitated using sheep anti-HPV-16 E2 serum or normal sheep IgG. Complexes were blotted with Pyk2 and HPV-16 E2 (TVG-261) antibodies.

FIG 2

Pyk2 interacts with the TAD of E2. (A) Illustration of HPV-31/-16 E2 fragments. (B) HEK293TT cells were transfected with GFP-Pyk2 and FLAG-HPV-31 E2 or HPV-31 E2 fragments (aa 107 to 372, 176 to 372, and 205 to 372) and immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with M2 (FLAG) and Pyk2 antibodies. (C) HEK293TT cells were transfected with GFP-Pyk2 and FLAG-HPV-31 E2 or HPV-31 E2 with the β-sheet region of the E2 TAD deleted (aa 1 to 106 and aa 176 to 372) and immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with M2 (FLAG) and Pyk2 antibodies. (D) HEK293TT cells were transfected with GFP-Pyk2 and FLAG-HPV-16 E2 or HPV-16 E2 fragments (aa 1 to 104, 202 to 365, 105 to 365, and 202 to 365). After immunoprecipitation with M2 (FLAG) antibodies, complexes were blotted with M2 (FLAG) and Pyk2 antibodies.

FIG 3

Pyk2 phosphorylates the E2. (A) HEK293TT cells were transfected with GFP-Pyk2 WT or GFP-Pyk2 KD and FLAG-HPV-31 or FLAG-HPV-16 E2. FLAG-HPV-E2 was immunoprecipitated with M2 (FLAG) antibodies, and complexes were blotted with pTyr-1000, M2 (FLAG), and Pyk2 antibodies. (B) HEK293TT cells were transfected with FLAG-Pyk2 WT or FLAG-Pyk2 KD and FLAG-HPV-31 E2 fragments (aa 1 to 106, 176 to 372, 107 to 372, and 205 to 372). FLAG-HPV-31 E2 and FLAG-Pyk2 were immunoprecipitated with M2 (FLAG) antibodies, and complexes were blotted with pTyr-1000 and M2 (FLAG) antibodies. Phospho-E2 fragments are identified by an asterisk (*).

FIG 4

Pyk2 induced tyrosine phosphorylation of HPV-31 E2 Y to F mutants. HEK293TT cells were transfected with GFP-Pyk2 and FLAG-HPV-31 E2 phenylalanine F mutants (A) or with GFP-Pyk2 WT or GFP-Pyk2 KD and FLAG-HPV-31 F mutants (B). Lysates were immunoprecipitated with M2 (FLAG), and bound proteins were blotted with Pyk2, pTyr-1000, and M2 (FLAG) antibodies. (C) Quantification of phosphotyrosine levels of Y131F. Values are means ± the SEM (n = 3). *, P < 0.05 (two-way t test).

FIG 5

Nuclear localization of HPV E2 and Pyk2. (A) CV-1 cells were transfected with GFP-Pyk2 WT and FLAG-HPV-31 or -16 E2 constructs. Immunofluorescence staining for HPV-E2 proteins with M2 (FLAG) antibodies and DAPI (blue) was performed. Cells were visualized under ×60 magnification using confocal microscopy. Pyk2 (Green), E2 (red), and DAPI (blue) are indicated. (B) HeLa cells were transfected with FLAG-HPV-31 E2 WT or Y131 mutants. Immunofluorescence staining was done with M2 (FLAG, red) antibodies or DAPI (blue). Magnification, ×60.

FIG 6

Pyk2 siRNA increases HPV DNA content. (A) CIN612-9E cells were transfected with a scrambled control, Pyk2, or EGFR siRNA. After 72 h, lysates were immunoblotted with Pyk2, EGFR, or β-actin antibodies. CIN612-9E cells were transfected with a control, Pyk2, or EGFR siRNA. Real-time PCR was performed for the HPV-31 long control region (LCR) and normalized to the level of β-actin. Values are expressed as means ± the SEM (n = 6). *, P < 0.05. (B) W12 cells were transfected with a control, Pyk2, or EGFR siRNA. After 72 h, the lysates were immunoblotted with Pyk2, EGFR, and β-actin antibodies. W12 cells were transfected with a control, Pyk2, or EGFR siRNA. DNA was isolated for real-time PCR for the HPV16 E6 DNA region and normalized to the levels of β-actin. Values are expressed as means ± the SEM (n = 12). *, P < 0.05.

FIG 7

Pyk2 inhibition increases HPV DNA content by decreasing E2 tyrosine phosphorylation. (A) CIN612-9E and W12 cells were treated with the Pyk2 inhibitor PF-431396 for 24 h, and cell viability was quantified by an MTS cell proliferation assay. Values are expressed as means ± the SEM (n = 8). *, P < 0.05. (B) CIN612-9E cells were incubated with 100 nM PF-431396, and W12 cells were incubated with 10 or 100 nM PF-431396 for 72 h. DNA was isolated, and real-time-PCR was performed for HPV-31 or HPV-16 DNA region near the LCR and normalized to the levels of β-actin. Values are expressed as means ± the SEM (n = 6). *, P < 0.05. (C) CIN612-9E cells were treated with 10 or 100 nM PF-431396 for 72 h. RNA was isolated and converted to cDNA using reverse transcription-PCR. Real-time PCR was carried out with primers to HPV-31 E1̂E4 and HPV-31 E2̂E8 mRNA and normalized to 18S transcripts. Values are means ± the SEM (n = 3). *, P < 0.05. (D) HEK293TT cells were transfected with GFP-Pyk2 and FLAG-HPV-31 E2. Cells were treated with 10 or 100 nM PF-431396 for 24 h. E2 was immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with pTyr-1000, Pyk2, and M2 (FLAG) antibodies.

FIG 8

Pyk2 knockout increases HPV genome integration. (A) CIN612-9E cells infected with pLentiCRISPR v2 (control) or pLentiCRISPR v2-Pyk2 guide constructs. Lysates were immunoblotted with Pyk2 and β-actin antibodies. (B) DNA from control and Pyk2 knockout (KO) CIN612-9E cell lines were analyzed by qPCR for the HPV-31 LCR and normalized to β-actin. Values are expressed as means ± the SEM (n = 9). (C) DNA from control and Pyk2 KO CIN612-9E cell lines were subjected to exonuclease V digestion. Resistant HPV-31 DNA was quantified by qPCR. Actin DNA was used as a positive control for digestion, and mitochondrial DNA was used as a resistant control. Values are expressed as means ± the SEM (n = 9) *, P < 0.05. (D). Real-time PCR was performed using primers for HPV-31 E1̂E4, E8̂E2, and E6 transcripts and normalized to actin transcripts. Values are means ± the SEM (n = 3). *, P < 0.05.

FIG 9

E2 Y131 is important for episomal maintenance. (A) NIKS cells were infected with HPV-31 WT and mutant quasiviruses and selected with G418 for 2 weeks. Quantification of viral HPV-31 DNA was carried out using qPCR in NIKS cells infected with HPV-31 WT and Y131F quasiviruses. Values are expressed as means ± the SEM (n = 3). (B) CIN612 episomal and integrated cell lines served as controls in the exonuclease V digestion assay. HPV-31 DNA from NIKS WT and Y131F quasiviruses was quantified using real-time PCR after exonuclease V digestion for actin and mitochondrial DNA. Values are means of the percent exonuclease V resistance ± the SEM (n = 3). (C) Percent exonuclease V resistance for HPV-31 DNA in CIN612 and NIKS WT and Y131F cell lines. Values are means ± the SEM (n = 3) *, P < 0.05 (compared to CIN612 cells by one-way t test). (D) NIKS cells were infected with HPV-31 WT and mutant quasiviruses and selected with G418 for 28 days. Quantification of HPV-31 DNA using qPCR in NIKS cells infected with HPV-31 WT and Y131 mutant quasiviruses. Values expressed as means ± the SEM (n = 3).

FIG 10

Y131E is unable to stimulate transient replication and is defective for Brd4 CTM binding. (A) C33A cells were transfected with HPV-31 E1 and HPV-31 Y131 mutants, along with pFLORI31 and pRL constructs. After 72 h later, the firefly and Renilla luciferase levels were measured using Dual-Glo luciferase reagent. The firefly luciferase levels were normalized to the Renilla luciferase levels. Values are expressed as means ± the SEM (n = 8). (B) HEK293TT cells transfected with HA-HPV-31 E1 and FLAG-HPV-31 E2 Y131 mutants. HPV-31 E2 was immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with E1 and M2 (FLAG) antibodies. (C) HEK293TT cells were transfected with FLAG-ChlR1 and FLAG-HPV-31 E2 Y131 mutants. Immunoprecipitations were performed with goat ChlR1 antibodies and immunoblotted with M2 (FLAG) antibodies. (D) HEK293TT cells transfected with FLAG-Brd4 full-length and FLAG-HPV-31 E2 Y131 mutants. Immunoprecipitations were performed with Brd4 antibodies and immunoblotted with M2 (FLAG) antibodies. (E) HEK293TT cells transfected with GST-CTM and FLAG-HPV-31 E2 Y131 mutants. HPV-31 E2 was immunoprecipitated with M2 (FLAG) antibodies. Complexes were blotted with GST and M2 antibodies.