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. 2022 Oct 20;19(1):165.
doi: 10.1186/s12985-022-01899-8.

HPV 16 E7 alters translesion synthesis signaling

Sebastian O Wendel  1 , Avanelle Stoltz  1 , Xuan Xu  1 , Jazmine A Snow  1 , Nicholas Wallace  2
Affiliations

HPV 16 E7 alters translesion synthesis signaling

Sebastian O Wendel et al. Virol J. .

Abstract

A subset of human papillomaviruses (HPVs) are the cause of virtually every cervical cancer. These so-called "high-risk" HPVs encode two major oncogenes (HPV E6 and E7) that are necessary for transformation. Among "high-risk" HPVs, HPV16 causes most cervical cancers and is often used as a representative model for oncogenic HPVs. The HPV16 E7 oncogene facilitates the HPV16 lifecycle by binding and destabilizing RB, which ensures the virus has access to cellular replication machinery. RB destabilization increases E2F1-responsive gene expression and causes replication stress. While HPV16 E6 mitigates some of the deleterious effects associated with this replication stress by degrading p53, cells undergo separate adaptations to tolerate the stress. Here, we demonstrate that this includes the activation of the translesion synthesis (TLS) pathway, which prevents replication stress from causing replication fork collapse. We show that significantly elevated TLS gene expression is more common in cervical cancers than 15 out of the 16 the other cancer types that we analyzed. In addition to increased TLS protein abundance, HPV16 E7 expressing cells have a reduced ability to induct a critical TLS factor (POLη) in response to replication stress-inducing agents. Finally, we show that increased expression of at least one TLS gene is associated with improved survival for women with cervical cancer.

Keywords: Cervical cancer; Human papillomavirus; Replication stress; Translesion synthesis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Translesion synthesis gene expression is frequently elevated in cervical cancers compared to other cancer types. A Overall frequency with which at least tumors have elevated (z > 2) expression of at least one TLS gene (left). The frequency with which tumors have elevated (z > 2) expression of individual TLS genes (right). Unbiased cluster analysis was used to group individual TLS genes by similarities of gene expression changes. B Gene ontology analysis of cervical cancers (CaCx) and cell lines with elevated E2F1 expression (Increased E2F1) showing enrichment of genes involved in the indicated cellular processes. The size of the circle indicates the relative breadth of the cellular process, with large circles indicating broad categories and smaller circles indicating more specific cellular responses. p-values are indicated by the color of the circle with darker greens denoting lower p-values than lighter colored circles. C This table shows (i) the extent that expression of individual TLS genes correlates with E2F1 expression, (ii) the nature of that correlation, and (iii) how the expression of TLS genes changes with cervical cancer progression. “ − “ indicates that a correlation does not exist or did not reach statistical significance. “ + ”,” +  + ”,” +  +  + ” indicate increasing magnitudes of correlation with E2F1 expression. “POS” indicates the correlation between the expression of the indicated TLS gene and E2F1 is positive. “NEG” indicates the correlation is negative. “Up” indicates that expression of the indicated TLS gene increases as cervical cancers progress from early to later stages of the disease. “Neutral” indicates that these changes are inconsistent (both up and down). “N/A” indicates that this gene was not included in the analyzed data set. “Down” indicates that expression of the indicated gene decreased with disease progression
Fig. 2
Fig. 2
Elevated TLS gene expression is frequently the result of copy number increases. A The extent that expression of individual TLS genes significantly correlates with the indicated variable (Copy number, promoter methylations, KDM6A expression, KDM6B expression, and RRM2 expression), and whether this correlation is negative or positive is shown. B Bar graphs show the frequency that increases in copy number (red) occur in tumors with elevated expression of the indicated TLS gene
Fig. 3
Fig. 3
Exogenous nucleoside supplementation decreases replication stress and TLS activation in cervical cancer cell lines. Representative immunoblots of cervical cancer cell lines (HeLa and SiHa) probed for A a marker of replication stress (p-ATR) and TLS pathway activation (ub-PCNA) along with total levels of each of these proteins and B representative TLS proteins. Whether the cell lines were grown in media supplemented with exogenous nucleosides is indicated below the immunoblot image with “ + ” indicating nucleosides were added and “−“ indicating that they were not. GAPDH and Nucleolin were used as loading controls
Fig. 4
Fig. 4
HPV16 E7 increases TLS pathway activation by binding RB. Representative immunoblot of vector control (LXSN) and HPV16 E7 wild type and mutant E7 expressing HFKs probed for A replication stress-responsive proteins and TLS proteins and B densitometry of these proteins calculated from three individual immunoblots. The bars represent the mean of densitometry from at least three independent repeats. As a result, they may not perfectly match the representative blot shown in A. Error bars denote standard errors of the mean. # denote a statistical difference between HPV16 E7 wild type and HPV16 E7 mutant (#− p < 0.05, ##− 0.01). *denotes that there is a statistical difference between LXSN HFKs and the indicated cell line (*− p < 0.05, **− 0.01, ***− 0.001). GAPDH was used as a loading control
Fig. 5
Fig. 5
HPV16 E7 alters POLη and TopBP1 induction in response to replication stress-inducing agents. Representative immunoblots of vector control (LXSN) and HPV16 E7 wild type and mutant E7 expressing HFKs probed for TopBP1 (A–C) or POLη (D–F). Whether these cells were exposed to the indicated replication stress-inducing agent (Hydroxyurea or HU, UV, and Cisplatin or CISP) is indicated by “−“ (not exposed) and “ + ” (exposed). GAPDH was used as a loading control
Fig. 6
Fig. 6
Increased expression of at least one TLS gene is associated with longer cervical cancer survival. Kaplan Meyer analysis of cervical cancers with increased expression of at least one TLS gene (red) compared to cervical cancers without expression of a TLS gene (blue). The median survival for each group is shown below the graph. P-value was calculated by Log Rank Test
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