Interferon-γ-induced GBP1 is an inhibitor of human papillomavirus 18

Abstract

Background: Human papillomavirus (HPV) infection is an important factor leading to cervical cell abnormalities. 90% of cervical cancers are closely associated with persistent infection of high-risk HPV, with the highest correlation with HPV16 and 18. Currently available vaccines and antivirals have limited effectiveness and coverage. Guanylate binding protein 1 (GBP1) was induced by interferon gamma and involved in many important cellular processes such as clearance of various microbial pathogens. However, whether GBP1 can inhibit human papillomavirus infection is unclear.

Results: In this study, we found that GBP1 can effectively degrade HPV18 E6, possibly through its GTPase activity or other pathways, and E6 protein degrades GBP1 through the ubiquitin-proteasome pathway to achieve immune escape.

Conclusion: Therefore, GBP1 is an effector of IFN-γ anti-HPV activity. Our findings provided new insights into the treatment of HPV 18 infections.

Keywords: Guanylate-binding protein 1; Human papillomavirus 18; Interferon gamma.

Fig. 1

Expression levels of HPV protein and mRNA in Hela cells transfected with pcDNA3.1-GBP1-EGFP. (a) Western blot. (-) blank control group, (mock) 1.5 µg pcDNA3.1 vector was transfected, (1) 0.5 µg pcDNA3.1-GBP1-EGFP and 1 µg pcDNA3.1 vector were transfected, (2) 1.5 µg pcDNA3.1-GBP1-EGFP was transfected. (b) HPV18 E6 mRNA levels in Hela cells after transfected. Data were shown as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01

Fig. 2

GBP1 inhibits HPV by degrading the E6 protein. Co-transfected pcDNA3.1-GBP1-EGFP and pcDNA3.1-E6-RFP into HEK293T cells in a 3:1 ratio. After 48 h transfected, the fluorescence signals from pcDNA3.1-GBP1-EGFP or pcDNA3.1-E6-RFP were observed under invert fluorescence microscope (a). The intensities of fluorescence from pcDNA3.1-GBP1-EGFP or pcDNA3.1-E6-RFP were detected by multifunctional microplate reader and expressed as relative fluorescence units (RFU) compare to the control group (b). (c) cells were harvested and the protein levels of GBP1 and E6 were determined by Western Blot using GAPDH as an internal reference. blank control (-), (mock) 1.5 µg pcDNA3.1 vector and 0.5 µg pcDNA3.1-E6-RFP were transfected, (control) 1.5µg pcDNA3.1 vector and 0.5µg pcDNA3.1-RFP or 1.5µg pcDNA3.1-EGFP and 0.5µg pcDNA3.1 vector were transfected, (1) 1.5 µg pcDNA3.1-GBP1-EGFP and 0.5 µg pcDNA3.1-E6-RFP were transfected. Data were shown as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01

Fig. 3

Identification of the relationship between GBP1 and E6. Using inhibitor to screen the way of GBP1 degrade HPV E6. Co-transfected pcDNA3.1-GBP1-EGFP and pcDNA3.1-E6-RFP into HEK293T cells at a 3:1 ratio, and the control group was transfected with pcDNA3.1-RFP or pcDNA3.1 vector (mock), and added the proteasome inhibitor Mg132 and autophagy inhibitor BAF-A1 at a ratio of 1:1000 after 36 h transfected respectively, DMSO was used as a control group. After 12 h of dosing, the fluorescence signals from pcDNA3.1-E6-RFP were observed under invert fluorescence microscope (a). The intensities of fluorescence from pcDNA3.1-E6-RFP were detected (b). (c) cells were harvested and the expression of GBP1 and E6 proteins were detected by Western Blot. (1,3,5) 1.5 µg pcDNA3.1 vector and 0.5 µg pcDNA3.1-E6-RFP were transfected. (2,4,6) 1.5 µg pcDNA3.1-GBP1-EGFP and 0.5 µg pcDNA3.1-E6-RFP were transfected. Data were shown as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01

Fig. 4

HPV E6 protein degrades GBP1 via the proteasome pathway. pcDNA3.1-GBP1-EGFP and pcDNA3.1-E6-RFP were co-transfected into HEK293T cells at a ratio of 1:3, and the control group was transfected with pcDNA3.1-EGFP or pcDNA3.1 vector (mock). 36 h after transfection, Mg132 (1:1000) and BAF-A1 (1:1000) were added, and the control group was added with an equal ratio of DMSO. After 48 h of transfection, the fluorescence signals from pcDNA3.1-GBP1-EGFP were observed (a). The intensities of fluorescence from pcDNA3.1-GBP1-EGFP were detected (b). (c) The expression of GBP1 and E6 protein was detected by Western Blot. (1,3,5) 1.5 µg pcDNA3.1 vector and 0.5 µg pcDNA3.1-GBP1-EGFP were transfected. (2,4,6) 1.5 µg pcDNA3.1-E6-RFP and 0.5 µg pcDNA3.1-GBP1-EGFP were transfected. Data were shown as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01