The Human Papillomavirus (HPV) E1 protein regulates the expression of cellular genes involved in immune response

Abstract

The Human Papillomavirus (HPV) E1 protein is the only viral protein with enzymatic activity. The main known function of this protein is the regulation of the viral DNA replication. Nevertheless, it has been demonstrated that the ablation of HPV18 E1 mRNA in HeLa cells promotes a deregulation of several genes, particularly those involved in host defense mechanisms against viral infections; however, the specific contribution of E1 protein in HPV-independent context has not been studied. The aim of this work was to determine the effect of the HPV E1 protein in the regulation of cellular gene expression profiles evaluated through RNA-seq. We found that E1 proteins from HPV16 and 18 induced an overexpression of different set of genes associated with proliferation and differentiation processes, as well as downregulation of immune response genes, including IFNβ1 and IFNλ1 and Interferon-stimulated gene (ISG), which are important components involved in the antiviral immune response. Together, our results indicate that HR-(High-Risk) and LR-(Low-Risk) HPV E1 proteins play an important role in inhibiting the anti-viral immune response.

Figure 1

HA-HPV E1 expression in HaCaT cells. (A) HPV16, 18 and 11 E1 mRNA expression. HaCaT cells transfected with the pCA control vector and the HPV E1 expressing plasmids were collected 24 h after transfection. Then RNA was extracted, cDNA synthesized, and PCR performed to evaluate E1 mRNA expression. Amplification of a fragment of the E1 mRNA indicates that all three plasmids were successfully expressed in HaCaT cells. (B) HA-tagged HPV E1 protein expression. HaCaT transfected cells were collected and protein expression ascertained by western blot using anti-HA antibody. HPV16, 18 and 11 E1 HA tagged proteins were detected at 72 KDa. Additional bands are part of the E1 protein processing. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control (37 KDa).

Figure 2

Genes differentially regulated by HPV E1 proteins. Heatmap showing genes differentially expressed in presence of HPV16, 18 and 11 E1 proteins based on the p-adj value (≤0.05). The scale of Log 2-fold change indicates the expression level of the genes. Red indicates overexpressed genes (expression levels over the median), green indicates under-expressed genes (expression levels under the median; see legend), and grey indicates unchanged genes.

Figure 3

Immune response, cell-substrate junction assembly and cell proliferation networks are altered by the HPV16 E1 protein. HPV16 E1 significantly regulated genes were analyzed by ClueGO. (A) Gene ontology (GO) pathway terms obtained for up-regulated genes. (B) GO pathway terms obtained for down-regulated genes. The number of associated genes is depicted on the right of each bar and above is presented the percentage of altered genes. Significance (p ≤ 0.05) is represented by red asterisks. Clustered networks for up-regulated genes (Red color) (C) and downregulated genes (Green color) (D) in presence of HPV-16 E1 protein. The major term per group is displayed. Node size exposes the degree of enrichment. Organic layout algorithm introduced in Cytoscape supplied the obtained networks. Genes showing Padj changes with a significance of p < 0.05 were analyzed.

Figure 4

Immune response and response to corticosterone networks are altered by the HPV18 E1 protein. HPV18 E1 significantly regulated genes were analyzed by ClueGO. (A) Gene ontology (GO) pathway terms obtained for up-regulated genes. (B) GO pathway terms obtained for down-regulated genes. The number of associated genes is depicted on the right of each bar and above is presented the percentage of altered genes. Significance (p ≤ 0.05) is represented by red asterisks. Clustered networks for up-regulated genes (Red color) (C) and downregulated genes (Green color) (D) in presence of HPV-18 E1 protein. The major term per group is displayed. Node size exposes the degree of enrichment. Organic layout algorithm introduced in Cytoscape supplied the obtained networks. Genes showing Padj changes with a significance of p < 0.05 were analyzed.

Figure 5

Immune response network is altered by the HPV11 E1 protein. HPV11 E1 significantly regulated genes were analyzed by ClueGO. (A) Gene ontology (GO) pathway terms obtained for down-regulated genes. The number of associated genes is depicted on the right of each bar and above is presented the percentage of altered genes. Significance (p ≤ 0.05) is represented by red asterisks. (B) GO pathway terms obtained for down-regulated genes. Clustered networks for downregulated genes (Green color) in presence of HPV-11 E1 protein. The major term per group is displayed. Node size exposes the degree of enrichment. Organic layout algorithm introduced in Cytoscape supplied the obtained networks. Genes showing Padj changes with a significance of p < 0.05 were analyzed.

Figure 6

The HPV16, 18 and 11 E1 proteins alter similar genes. (A) Venn diagram summarizing the shared number of genes overlapping between differentially expressed genes altered by HPV E1 proteins. (B) HaCaT cells transfected with pCA vector control and HPV16 and HPV18 E1 expressing plasmids were collected and RNA extracted 24 h after transfection. Then, cDNA was synthesized and the mRNA levels of FOSB was measured using specific primers. Relative mRNA levels of FOSB showed an increase in presence of E1 from HPV16 and 18. Values are expressed as the difference in double delta-Ct compared with pCA control transfected cells. The expression of the housekeeping gene 18S was used for normalizing. Bars represent the mean ± SD. *p < 0.05 vs control (pCA).

Figure 7

The E1 protein from HPV16, 18 and 11 downregulates IFNβ1 and IFNλ1 expression. HaCaT cells transfected with pCA control vector and HPV E1 expressing plasmids were collected and RNA extracted 24 h after transfection. Then, cDNA was synthesized and the mRNA levels of IFNβ1 and IFNλ1 genes were measured using specific primers (Supplementary Table 1). Relative mRNA levels of (A) IFNβ1 and (B) IFNλ1 genes showing a decrease in presence of the HPV-16, 18 and 11 E1. Values are expressed as the difference in double delta-Ct compared with pCA control transfected cells. The expression of the housekeeping gene 18S was used for normalizing. Bars represent the mean ± SD. ***p < 0.0001 vs control (pCA).

Figure 8

The E1 protein from HPV16, 18 and 11 downregulates interferon-stimulated genes expression. HaCaT cells transfected with pCA vector control and HPV E1 expressing plasmids were collected and RNA extracted 24 h after transfection. Then, cDNA was synthesized and the mRNA levels of CCL5, RSAD2 (Viperin) and IFIT2 genes were measured using specific primers (Supplementary Table 1). Relative mRNA levels of CCL5, RSAD2 (Viperin) and IFIT2 genes showing a decrease in presence of the HPV-16, 18 and 11 E1. Values are expressed as the difference in double delta-Ct compared with pCA control transfected cells. The expression of the housekeeping gene 18 S was used for normalizing. Bars represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs control (pCA).

Figure 9

HPV E1 protein decreases IFNβ1 and IFNλ1 expression in the presence of the Poly I:C agonist. HaCaT cells were transfected using 0.01, 0.1, or 1 μg of Poly I:C and pCA vector to a final amount of 4.5 μg. After 24 h post transfection total RNA was extracted and the IFNβ1 expression was evaluated by pPCR. (A) The Poly I:C increases the IFNβ1 and IFNλ1 expression in HaCaT cells in all concentrations tested. The 0.1 μg concentration was selected for further analyses. Bars represent the mean ± SD. ***p < 0.001 vs control (pCA). (B) IFNβ1 and (C) IFNλ1 expression is repressed in the presence of HPV16, 18 and 11 E1 proteins. After 24 h of transfection with 0.1 μg of Poly I:C and the indicated HPV E1 plasmid, total RNA was extracted, cDNA synthesized, and qPCR performed to evaluate the IFNβ1 and IFNλ1 expression. In presence of the different HPV E1 proteins there is a decrease of IFNβ1 and IFNλ1 expression. Values are expressed as the difference in double delta-Ct compared with cells transfected with the pCA vector. The expression of the housekeeping gene 18S was used for normalizing RNA expression. Bars represent the mean ± SD. ***p < 0.001 vs pCA control. (D) IkBα and p52-NFκB protein levels. HaCaT cells transfected with pCA vector and HPV E1 expressing plasmids were collected and total protein extracted 24 h after transfection. Levels of IkBα and p52-NFκB were ascertained by western blot. Proteins were separated in a 10% SDS-PAGE and membrane was probed with anti-phospho-IκBα (Ser32/36)(5A5) (9246; Cell signaling Technology Europe, B.V) or anti-p52 (NFκB) (Santa Cruz Biotechnology, California) observing decreased levels of IkBα and an increase of p52-NFκB levels in the presence of HPV16, 18 and 11 E1 proteins. GAPDH was used as loading protein control (GAPDH, 37 KDa).