HPV E7 viral oncoprotein disrupts transcriptional regulation of L1Md retrotransposon

Abstract

Murine L1Md-A5 retrotransposon is a redox-inducible element regulated by Nrf-2/JunD and E2F/Rb-binding sites within its promoter (5'-UTR). Because the human papillomavirus (HPV) oncoprotein E7 interacts with retinoblastoma (pRb) and members of the AP1 family, studies were conducted to examine functional interactions between HPV E7, pRb, and histone deacetylase 2 (HDAC2) in the regulation of L1Md-A5. Using a transient heterologous transcription system we found that HPV E7 alone, or in combination with HDAC2, disrupted pRb-mediated L1MdA-5 transactivation. HPV E7 also ablated the transcriptional response of L1Md-A5 to genotoxic stress, but did not interfere with basal activity. We conclude that HPV E7 associates with proteins involved in the assembly of macromolecular complexes that regulate antioxidant and E2F/Rb sites within L1MdA-5 to regulate biological activity.

Fig. 1. The mouse L1MdA5 retroelement promoter contains several EpRE-containing monomers

(A) Schematic structure and monomer organization for the L1MdA5 retrotransposon. A full-length L1MdA5 retroelement (top) is composed of a 5′ untranslated region (UTR) that contains three full-length A-type monomers (A3, A2, and A1) preceded by a 2/3 A-type monomer. Two open reading frames (ORF1 and ORF2), separated by a short intergenic region (IGR), encode the cis-acting proteins ORF1p and ORF2p respectively. The 3′ terminal region is composed of a 3′UTR followed by a poly-A tail. The location and orientation of putative EpRE and E2F DNA binding sites within the A-type monomer promoter are shown (bottom). (B) Simplified map of the luciferase reporter vectors used in the study. pGL3-BASIC (pBASIC) firefly luciferase (top) is a promoterless reporter vector used as the control in the study. The HindIII (H) restriction site within the vector multiple cloning site (MCS) was used to place the L1MdA5 promoter in front of the luciferase reporter gene (bottom). The vector also contains the simian virus 40 (SV 40) late poly adenylation signal (poly-A).

Fig. 2. Transactivation of L1 promoter activity by Rb is ablated by HPV E7 viral oncoprotein

Transient transcription assays in HeLa cells with 500 ng of either the pBASIC-luciferase or the pL1Md-A5-luciferase reporter vectors and the forced overexpression of HPV E7 or pRb alone or in combination are shown. As an internal control for normalization, 10 ng of a second reporter vector, Renilla luciferase (pRL), were cotransfected within each experiment. (A) pBASIC-luciferase reporter activity corrected for pRL after cotransfection with 100 ng of empty vector control, HPV E7, Rb or HPV E7 and Rb together. (B) pL1Md-A5-luciferase reporter activity corrected for pRL after cotransfection with 100 ng of empty vector control, HPV E7, Rb or HPV E7 and Rb together. Cells were transfected for 6 hours, allowed to recover for 30 hours prior to luciferase activity measurements. Results are expressed as arbitrary luciferase units (ALU) and represent the mean ± S.D. of triplicate experiments after normalization to pRL. Statistic analyses were done using ANOVA (*, **, and *** indicate statistically significant differences, p<0.05, p<0.005 and p<0.0005 respectively). Each experiment was repeated at least 2 times.

Fig. 3. Transactivation of L1 promoter activity by Rb and HDAC2 is ablated by HPV E7 viral oncoprotein

Transient transcription assays in HeLa cells with the pBASIC-luciferase or the pL1Md-A5-luciferase reporter vectors and the forced overexpression of HPV E7, pRb, or HDAC2 proteins alone or in combination are shown. As an internal control for normalization, 10 ng of pRL were cotransfected within each experiment. (A) pBASIC-luciferase reporter activity corrected for pRL after cotransfection with 100 ng of empty vector control, or expression vectors indicated in the figure label. (B) pL1Md-A5-luciferase reporter activity corrected for pRL after cotransfection with 100 ng of empty vector control, or expression vectors indicated in the figure label. Data were normalized as indicated in the Y axis. Cells were transfected for 6 hours, allowed to recover for 30 hours prior to luciferase activity measurements. Results are expressed as arbitrary luciferase units (ALU) and represent the mean ± S.D. of triplicate experiments after normalization to pRL. Statistic analyses were done using ANOVA (*, **, and *** indicate statistically significant differences, p<0.05, p<0.005 and p<0.0005 respectively). Each experiment was repeated at least 2 times.

Fig. 4. BaP induction of L1 promoter activity is ablated by HPV E7 viral oncoprotein

Transient transcription assays in HeLa cells with the pBASIC-luciferase (A), or the pL1Md-A5-luciferase (B), reporter vectors showing the effects of 0.06% DMSO or 3 μM BaP with or without HPV E7 oncoprotein forced overexpression. Cells were transfected for 6 hours, allowed to recover for 18 hours and chemically treated for 16 hours. Results are expressed as arbitrary luciferase units (ALU) and represent the mean ± S.D. of triplicate experiments after normalization to pRL. Statistic analyses were done using ANOVA (*, **, and *** indicate statistically significant differences, p<0.05, p<0.005 and p<0.0005 respectively). Each experiment was repeated at least 2 times.

Fig. 5. Hypothetical mechanisms for the role of HPV E7 in control of the cellular response to redox-mediated injury and redox-dependent L1 reactivation

Redox stress alters both cell signaling and transcription programs. Injury might lead either directly or indirectly to pRb/HDAC-dependent repression of factors required for LINE-1 silencing thus allowing for its reactivation. Cells expressing HPV E7 viral oncoprotein inactivate the Rb/HDAC effect, likely through direct interaction with Rb, thus eliminating the redox-mediated retroelement reactivation.