NFX1-123 Increases hTERT Expression and Telomerase Activity Posttranscriptionally in Human Papillomavirus Type 16 E6 Keratinocytes

Abstract

High-risk human papillomavirus (HPV) E6 protein induces telomerase activity through transcriptional activation of hTERT, the catalytic subunit of telomerase. HPV type 16 (HPV16) E6 interacts with two splice variants of NFX1 to increase hTERT expression. NFX1-91 is a transcriptional repressor of hTERT that is polyubiquitinated and targeted for degradation by HPV16 E6 in concert with E6-associated protein. We previously showed that NFX1-123 augments hTERT expression through binding to cytoplasmic poly(A) binding proteins (PABPCs). In this study, we determined that unlike NFX1-91, NFX1-123 is a cytoplasmic protein that colocalized with PABPCs but does not shuttle with PABPCs between the nucleus and cytoplasm. NFX1-123 requires both its PAM2 motif, with which it binds PABPCs, and its R3H domain, which has putative nucleic acid binding capabilities, to increase hTERT mRNA levels and telomerase activity in keratinocytes expressing HPV16 E6. In keratinocytes expressing HPV16 E6 and overexpressing NFX1-123, there was increased protein expression from in vitro-transcribed RNA fused with the 5 untranslated region (5 UTR) of hTERT. This posttranscriptional increase in expression required the PAM2 motif and R3H domain of NFX1-123 as well as the coexpression of HPV16 E6. NFX1-123 bound endogenous hTERT mRNA and increased its stability in HPV16 E6-expressing human foreskin keratinocytes, and NFX1-123 increased the stability of in vitro-transcribed RNA fused with the 5 UTR of hTERT. Together, these studies describe the first evidence of posttranscriptional regulation of hTERT, through the direct interaction of the cytoplasmic protein NFX1-123 with hTERT mRNA, in HPV16 E6-expressing keratinocytes.Human papillomavirus (HPV) is a double-stranded DNA tumor virus, and persistent high-risk HPV (HR HPV) infections are associated with cervical and other anogenital cancers (3, 8-10, 19, 41). Dividing epithelial cells are needed for HPV DNA replication and amplification, but differentiated cells are needed for full HPV genome expression. Therefore, HPV drives the upper layers of stratified squamous epithelium to continue to divide as they differentiate, and the HR E6 and E7 proteins are critical to the programmatic disruption of normal epithelium, cellular immortalization in culture, and cervical cancer in mouse models (48, 52). The HR HPV E7 protein drives cells to continue through S phase by targeting pocket proteins for degradation (22, 66), thus allowing the E2F transcriptional factor to activate S-phase genes for DNA replication. The HR HPV E6 protein has several targets in epithelial cells. HR HPV E6 targets p53 for degradation (50), blocking apoptotic and cellular senescence signals due to DNA damage. HR HPV E6 also affects PDZ domain-containing proteins, such as MUPP-1, Scribble, MAGI-1, -2, and -3, PTPN3, and hDlg (17,24,30,36,44,56). Finally, HR HPV E6 blocks cellular senescence signals by activating telomerase (31, 58). The degradation of retinoblastoma protein by E7 and the activation of telomerase by E6 are two critical ste...

Characterization of a cluster of oncogenic mutations in E6 of a human papillomavirus 83 variant isolated from a high-grade squamous intraepithelial lesion

“…Transcriptional control of the catalytic subunit of human telomerase reverse transcriptase (hTERT) plays a major role in the regulation of telomerase activity in many human cancers (Ducrest et al, 2002;Horikawa & Barrett, 2003). In normal somatic cells, E6 of HR-HPV induces the hTERT promoter (Liu et al, 2009;Veldman et al, 2001) through degradation of the transcriptional repressor NFX1-91 (Gewin et al, 2004;Katzenellenbogen et al, 2007Katzenellenbogen et al, , 2009 or through its binding to Myc (Liu et al, 2008;Veldman et al, 2003). Indeed, E6 from HR-HPV can increase cell proliferation through the upregulation of telomerase activity (Veldman et al, 2001).…”

HPV E6 protein interacts physically and functionally with the cellular telomerase complex

“…However, hTERT transactivation by E6 is dependent upon its binding the cellular ubiquitin ligase, E6AP (13,14,(17)(18)(19)(20). There appear to be two critical targets for E6/E6AP on the hTERT promoter, Myc (12,15,16) and NFX-1 (13,18,21), which transactivate and transrepress the promoter respectively. The model for E6 regulation of the hTERT promoter is likely to be quite complex and it is anticipated that there might be additional targets for E6 on this promoter.…”

NFX1-123 and Human Papillomavirus 16E6 Increase Notch Expression in Keratinocytes

“…E6-associated protein (E6AP) is an E3 ubiquitin ligase that is the most well-studied protein partner of HR E6 (13-19). However, HR E6 interacts with other cellular proteins, such as NFX1 splice variants, and these are important in oncogenesis as well (20,21).In human epithelial cells, NFX1 is a gene expressed as two splice variants, . NFX1-91 has been shown to transcriptionally repress telomerase expression in epithelial cells, and HR HPV 16E6 with E6AP targets NFX1-91 for degradation (22,23).…”