Activation of cap-dependent translation by mucosal human papillomavirus E6 proteins is dependent on the integrity of the LXXLL binding motif

Abstract

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.

Fig 1

Mucosal but not cutaneous HPV E6 proteins increase cap-dependent translation. (A) U2OS cells were transiently cotransfected with the pFR_CrPV_xb bicistronic reporter construct and cytomegalovirus promoter-driven expression vectors for high-risk mucosal HPV E6 proteins (HPV16, HPV18), low-risk mucosal HPV E6 proteins (HPV6b, HPV11), cutaneous HPV E6 proteins (HPV5, HPV8), or empty vector (C) as a control. (B) Primary HFKs were transiently cotransfected with the pFR_CrPV_xb construct and human β-actin promoter-driven E6 expression vectors. Cells were lysed, and Renilla and firefly luciferase activities were measured at 48 h posttransfection. Firefly and Renilla luciferase values were normalized to the values for control vector-transfected cells and are presented as the fold change of normalized firefly luciferase relative to normalized Renilla luciferase (FF/Ren). The bar graphs depict averages and standard deviations of four independent experiments for U2OS and five independent experiments for HFKs. *, statistical significance (P ≤ 0.01 and P < 0.05 for U2OS and HFKs, respectively). (C) Immunoblot analysis of various HPV E6 proteins after transient transfection of U2OS cells with lentiviral expression plasmids. An actin blot is shown as a loading control.

Fig 2

The LXXLL and PDZ binding motifs are important for the HPV16 E6-mediated increase in cap-dependent translation. (A) HFKs were transiently cotransfected with the pFR_CrPV_xb reporter construct and human β-actin promoter-driven expression vectors for wild-type HPV16 E6 (16E6), HPV16E6 Y54D (16E6 Y54D) and HPV16 E6 I128T (16E6 I128T) E6AP binding-defective mutants, HPV16E6 ΔPDZ (16 E6 ΔPDZ) PDZ binding-defective mutant, or empty vector (C) as a control. Cells were lysed, and firefly and Renilla luciferase activities were measured at 48 h posttransfection. Firefly and Renilla luciferase values were normalized to the values for control vector-transfected cells and are presented as the fold change of normalized firefly luciferase relative to normalized Renilla luciferase (FF/Ren). The bar graph depicts averages and standard deviations of three independent experiments *, statistical significance relative to empty vector (P ≤ 0.001); **, statistical significance relative to wild-type HPV16 E6 (P ≤ 0.01).

Fig 3

High- and low-risk HPV E6 proteins increase cap-dependent translation through overlapping mechanisms. (A) Schematic representation of the HPV E6 mutants used in these experiments. The amino acid sequences of the carboxyl-terminal zinc binding domains of HPV16, HPV18, HPV6b, and HPV11 are shown with the positions of the cysteine residues highlighted in yellow. The positions of the various LXXLL protein binding motif mutants are highlighted in green, and the amino acid residues deleted in the HPV16 E6 ΔPDZ mutant are highlighted in red. (B) Western blot analysis of HA immunoprecipitations (IP) from 293T cells transiently transfected with N-terminally Flag-HA-tagged cytomegalovirus (NCMV) promoter-driven E6 expression vectors. (Upper three panels) One percent of input for immunoprecipitations; (lower two panels) E6AP coprecipitated by the various HPV E6 proteins (detected by Flag epitope-specific antibody). (C) Analysis of cap-dependent translation. U2OS cells were transiently cotransfected with the pFR_CrPV_xb bicistronic reporter construct and the human β-actin promoter-driven E6 expression vectors or empty vector as a control. Cells were lysed, and Renilla and firefly luciferase activities were measured at 48 h posttransfection. Firefly and Renilla luciferase values were normalized to those for control vector-transfected cells and are presented as the fold change of normalized firefly luciferase relative to normalized Renilla luciferase (FF/Ren). The bar graph depicts averages and standard deviations from seven independent experiments, *, statistical significance relative to empty vector (P < 0.001); **, statistical significance relative to the corresponding wild-type HPV E6 protein (P ≤ 0.022).

Fig 4

The LXXLL binding motif is important for mTORC1 activation by mucosal HPV E6 proteins. (A) Western blot analysis of S6K T389 phosphorylation in HFKs with pLentiN6.3-based lentiviral vectors expressing wild-type HPV16 E6 (16E6), HPV16 E6 Y54D (16E6 Y54D) and HPV16 E6 I128T (16E6 I128T) E6AP binding-defective mutants, HPV16 E6 ΔPDZ (16E6 ΔPDZ) PDZ binding-defective mutant, or empty vector (C). Blots for total S6K and actin are also shown. Quantifications relative to actin are indicated below each panel. Expression of the corresponding Flag epitope-tagged E6 proteins in this HFK population is also shown. (B) Western blot analysis of S6K T389 phosphorylation in HFKs with pLentiN6.3-based vectors expressing wild-type HPV11 E6, the E6AP binding-defective HPV11 E6 L111Q (11E6 L111Q), HPV11 E6 I127T (11E6 I127T), or HPV11 E6 I129T (11E6 I129T) mutants, or empty vector (C). Blots for total S6K and actin are also shown. Quantifications relative to actin are indicated below each panel. Expression of the corresponding Flag epitope-tagged E6 proteins in this HFK population is also shown.