Focal Adhesion Kinase Binds to the HPV E2 Protein to Regulate Initial Replication after Infection

Abstract

Human papillomaviruses are small DNA tumor viruses that infect cutaneous and mucosal epithelia. The viral lifecycle is linked to the differentiation status of the epithelium. During initial viral infection, the genomes replicate at a low copy number but the mechanism(s) the virus uses to control the copy number during this stage is not known. In this study, we demonstrate that the tyrosine kinase focal adhesion kinase (FAK) binds to and phosphorylates the high-risk viral E2 protein, the key regulator of HPV replication. The depletion of FAK with a specific PROTAC had no effect on viral DNA content in keratinocytes that already maintain HPV-16 and HPV-31 episomes. In contrast, the depletion of FAK significantly increased HPV-16 DNA content in keratinocytes infected with HPV-16 quasiviruses. These data imply that FAK prevents the over-replication of the HPV genome after infection through the interaction and phosphorylation of the E2 protein.

Keywords: HPV; focal adhesion kinase; replication.

Figure 1

FAK interacts with and phosphorylates the PV E2 protein. (A) HEK293TT cells were transfected with GFP-Pyk2, GFP-Pyk2 KD (kinase dead, Y402F), GFP-FAK, and FLAG-HPV-31 E2 constructs. FLAG-HPV-31 E2 was immunoprecipitated with FLAG (M2) antibodies and complexes/inputs were blotted with FLAG (HPV-31 E2), GFP (Pyk2, FAK), and pTyr-1000 (pE2) antibodies. (B) HEK293TT cells were transfected with FLAG-HPV-31 E2 and GFP-FAK constructs and FLAG-HPV-31 E2 was immunoprecipitated with FLAG (M2) antibodies. Complexes were blotted with FLAG (HPV-31 E2), GFP (FAK), and pTyr-1000 (pFAK, pE2) antibodies. (C) HEK293TT cells were transfected with FLAG-HPV-16 E2 and GFP-FAK constructs and FLAG-HPV-16 E2 was immunoprecipitated with FLAG (M2) antibodies. Complexes were blotted with FLAG (HPV-16 E2), GFP (FAK), and (D) pTyr-1000 (pFAK, pE2) antibodies. (E) W12 cells were lysed and endogenous E2 immunoprecipitated with E2 antibodies (sheep anti-16E2). Proteins were blotted with FAK and HPV-16 E2 (TVG-261) antibodies. IgG sheep was used a negative control.

Figure 2

FAK protein is expressed in the nucleus of W12 cells. W12 cells (clone 20850) were stained for immunofluorescence with FAK and Pyk2 antibodies. Green (488), FAK (B,E) or Pyk2 (C,F); blue DAPI. Magnification ×40. Secondary antibodies alone were used as a negative control to determine fluorescence background (A,D).

Figure 3

FAK protein degradation has no effect on HPV-16 viral replication in W12 cells. (A) Episomal status was confirmed in W12 cells (clone 20850). DNA from episomal and integrated W12 cells were subjected to exonuclease V digestion. Resistant HPV-16 DNA was quantified using PCR. Actin DNA served as a positive control for ExoV digestion. Values are means ± the SEM (n = 9). (B) BI-0319 was treated on episomal W12 cells as various concentrations (250 nM, 500 nM, 1 µM) for 48 h. Lysates were immunoblotted with FAK, Pyk2, and β-actin antibodies. (C) W12 episomal cells were treated with 1 µM BI-0319 (FAK PROTAC) for 72 h. Percent (%) cell viability, measured using MTT assay, was completed 72 h after treatment (n = 24). DNA from episomal W12 cells treated with control (DMSO) or 1 µM BI-0319 (FAK PROTAC) for 48 h (D) and 96 h (E) were analyzed with primers against the HPV-16 LCR and normalized to β-actin. Values are means ± the SEM (n = 8, n = 6).

Figure 4

FAK protein degradation has no effect on HPV-31 DNA content in proliferating or differentiated CIN612 cells. (A) Episomal and differentiating CIN612 cells were treated with 1 µM BI-0319 for 72 h. Differentiation was induced with 2 mM CaCl₂ for 72 h along with FAK PROTAC treatment. Lysates were immunoblotted with FAK and β-actin antibodies. (B) CIN612 cells were treated with 1 µM BI-0319 for 72 h. Lysates were immunoblotted with Pyk2 and β-actin antibodies. Blots were quantified using Image Lab. FAK PROTAC groups were normalized to DMSO groups. Values are means ±, n = 3. DNA from proliferating CIN612 proliferating (C) or differentiating (D) cells were treated with control (DMSO) or 1 µM BI-0319 (FAK PROTAC) for 72 h and analyzed with primers against the HPV-31 LCR and normalized to β-actin. Values are means ± the SEM (n = 9).

Figure 5

HPV-16 L1 and L2 PsV demonstrate robust infection but HPV-16 L1 and L2 capsids (VLPs) inefficiently deliver externally attached DNA. HEK293TT cells either transfected with (1) 16pSheLL and mCherry DNA and benzonase was added at virus maturation (Pseudovirus), (2) 16pSheLL DNA and mCherry DNA with benzonase was added at virus maturation, (3) 16pSheLL DNA and mCherry DNA was added at virus maturation, or (4) 16pSheLL DNA and benzonase was added at virus maturation. VLPs were added to SiHa or HEK293TT cells and mCherry was visualized 72 h post infection.

Figure 6

FAK protein degradation increases HPV-16 DNA copy number after initial viral infection. (A) NIKS cells were infected with mCherry PsV and treated with 1 µM FAK PROTAC (BI-0319) at infection for 48 h. (B) Lysates were immunoblotted with FAK and β-actin antibodies. (C) DNA from NIKS cells infected with HPV-16neo quasiviruses and treated with control (DMSO) or 1 µM BI-0319 (FAK PROTAC) for 72 h were analyzed with primers against the HPV-16 LCR and normalized to β-actin. Values are means ± the SEM (n = 9). * p < 0.05.