Journal of Virology

Proliferative proteome induced by HPV 16E6 and NFX1-123 partnership in longitudinal keratinocyte cultures

ABSTRACT Human papillomaviruses (HPVs) cause cervical, anogenital, and oropharyngeal cancers. Despite the availability of preventive HPV vaccines, their poor uptake leaves individuals at risk for these cancers, many of which remain common globally and others that are increasing domestically. The HPV 16 E6 (16E6) protein plays a significant role in inducing and maintaining cellular transformation of its infected host cell; however, 16E6 itself has no enzymatic activity and executes its functions through partnerships with host proteins. Previously, we demonstrated that 16E6 binds to the host protein NFX1-123, and NFX1-123 expression is increased in HPV 16-positive cervical cancer cell lines and primary cancers compared to normal tissues. In this study, we quantify growth patterns of 16E6-expressing keratinocytes with endogenous or overexpressed NFX1-123 (16E6/vec and 16E6/FN123, respectively) levels and interrogate proteome expression changes early and late in longitudinal growth cultures. Early-passage 16E6/vec and 16E6/FN123 cells showed similar growth rates; however, late-passage 16E6/FN123 cells had accelerated growth, greater population doublings, increased telomerase activity, and a dysplastic phenotype when compared to 16E6/vec cells. Matching this, mass spectrometry revealed only 22 differentially expressed proteins at early passage; meanwhile, late-passaged cells had 827 differentially expressed proteins among 16E6/FN123 and 16E6/vec cells. In that, DNA maintenance and repair, proteins, namely, MCM2 and MCM4, were highly expressed in the 16E6/FN123 compared to 16E6/vec cells. These findings indicate that the 16E6 and NFX1-123 partnership, especially with sustained higher levels of NFX1-123, alters the longitudinal cellular environment in a manner that may initiate a preneoplastic phenotype. IMPORTANCE The high-risk HPV 16E6 protein plays a significant role in inducing and maintaining cellular transformation of its infected host cell; however, 16E6 has no enzymatic activity and carries out most of its functions through partnerships with host endogenous proteins, one being NFX1-123. That NFX1-123 expression is increased in HPV 16-positive cervical cancer and primary cancers compared to normal tissues, and NFX1-123 binds directly to 16E6. This study leverages proteomics to reinforce the synergistic actions of HPV16E6 and NFX1-123 and identifies cellular pathways impacted by this protein partnership over time. These findings encourage further investigation of NFX1-123 as a potential therapeutic target of HPV 16-positive tissues.

Identification and Complete Validation of Prognostic Gene Signatures for Human Papillomavirus-Associated Cancers: Integrated Approach Covering Different Anatomical Locations

Persistent infection with high-risk HPV interferes with cell function regulation and causes cell mutations, which accumulate over the long term and eventually develop into cancer. Results of pathway enrichment analysis presumably showed this accumulation of intracellular damage during the chronic HPV-infected state.

Repression of Memo1, a Novel Target of Human Papillomavirus Type 16 E7, Increases Cell Proliferation in Cervical Cancer Cells

Although numerous targets have been reported to interact with the HPV E7 oncoprotein, the mechanisms involved in HPV-induced carcinogenesis and the maintenance of cell transformation are still lacking. Here, through pulldown assays using a peptide encompassing the C-terminal region of HPV16 E7, we report Memo1 as a novel E7 interactor.

Regulation of HPV E7 Stability by E6-Associated Protein (E6AP)

This is the first demonstration that E6AP can directly help stabilize the HPV E7 oncoprotein, in a manner similar to that observed with HPV E6. This redefines how E6 and E7 can cooperate and potentially modulate each other’s activity and further highlights the essential role played by E6AP in the viral life cycle and malignancy.

Loss of the E6AP Ubiquitin Ligase Induces p53-Dependent Phosphorylation of Human Papillomavirus 18 E6 in Cells Derived from Cervical Cancer

This study demonstrates that the knockdown of E6AP from cervical cancer-derived cells leads to an increase in phosphorylation of the E6 oncoprotein. We show that this phosphorylation of E6 requires p53 transcriptional activity and the enzyme DNA PK. This study therefore defines a feedback loop whereby activation of p53 can induce phosphorylation of E6 and which in turn can inhibit p53 transcriptional activity independently of E6’s ability to target p53 for degradation.

Human Papillomavirus 58 E7 T20I/G63S Variant Isolated from an East Asian Population Possesses High Oncogenicity

Epidemiological studies have revealed that a wild-type variant of HPV58 carrying an E7 variation, T20I/G63S (V1), is associated with a higher risk of cervical cancer. We previously reported that this increased oncogenicity could be the result of the virus’s greater ability to degrade pRB, thereby leading to an increased ability to grow in an anchorage-independent manner. In addition to this, this report further showed that this HPV variant induced activation of the AKT and K-Ras/ERK signaling pathways, thereby explaining its genuine oncogenicity in promoting cell proliferation, migration, invasion, and formation of tumors, all to a greater extent than the prototype HPV58 and other common variants.

Nuclear proinflammatory cytokine S100A9 enhances expression of human papillomavirus oncogenes via transcription factor TEAD1

ABSTRACT Transcription of the human papillomavirus (HPV) oncogenes, E6 and E7 , is regulated by the long control region (LCR) of the viral genome. Although various transcription factors have been reported to bind to the LCR, little is known about the transcriptional cofactors that modulate HPV oncogene expression in association with these transcription factors. Here, we performed in vitro DNA-pulldown purification of nuclear proteins in cervical cancer cells, followed by proteomic analyses to identify transcriptional cofactors that bind to the HPV16 LCR via the transcription factor TEAD1. We detected the proinflammatory cytokine S100A9 that localized to the nucleus of cervical cancer cells and associated with the LCR via direct interaction with TEAD1. Nuclear S100A9 levels and its association with the LCR were increased in cervical cancer cells by treatment with a proinflammatory phorbol ester. Knockdown of S100A9 decreased HPV oncogene expression and reduced the growth of cervical cancer cells and their susceptibility to cisplatin, whereas forced nuclear expression of S100A9 using nuclear localization signals exerted opposite effects. Thus, we conclude that nuclear S100A9 binds to the HPV LCR via TEAD1 and enhances viral oncogene expression by acting as a transcriptional coactivator. IMPORTANCE Human papillomavirus (HPV) infection is the primary cause of cervical cancer, and the viral oncogenes E6 and E7 play crucial roles in carcinogenesis. Although cervical inflammation contributes to the development of cervical cancer, the molecular mechanisms underlying the role of these inflammatory responses in HPV carcinogenesis are not fully understood. Our study shows that S100A9, a proinflammatory cytokine, is induced in the nucleus of cervical cancer cells by inflammatory stimuli, and it enhances HPV oncogene expression by acting as a transcriptional coactivator of TEAD1. These findings provide new molecular insights into the relationship between inflammation and viral carcinogenesis.

The Transcriptional Cofactor VGLL1 Drives Transcription of Human Papillomavirus Early Genes via TEAD1

Although a number of transcription factors have been reported to be involved in HPV gene expression, little is known about the cofactors that support HPV transcription. In this study, we demonstrate that the transcriptional cofactor VGLL1 plays a prominent role in HPV early gene expression, dependent on its association with the transcription factor TEAD1. Whereas TEAD1 is ubiquitously expressed in a variety of tissues, VGLL1 displays tissue-specific expression and is implicated in the development and differentiation of epithelial lineage tissues, where HPV gene expression occurs. Our results suggest that VGLL1 may contribute to the epithelial specificity of HPV gene expression, providing new insights into the mechanisms that regulate HPV infection. Further, VGLL1 is also critical for the growth of cervical cancer cells and may represent a novel therapeutic target for HPV-associated cancers.