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. 2017 Nov 8;12(11):e0187514.
doi: 10.1371/journal.pone.0187514. eCollection 2017.

Human papillomavirus type 16 E6 and NFX1-123 mislocalize immune signaling proteins and downregulate immune gene expression in keratinocytes

Justine Levan  1   2 , Portia A Vliet-Gregg  1 , Kristin L Robinson  1 , Rachel A Katzenellenbogen  1   2   3
Affiliations

Human papillomavirus type 16 E6 and NFX1-123 mislocalize immune signaling proteins and downregulate immune gene expression in keratinocytes

Justine Levan et al. PLoS One. .

Abstract

Human papillomavirus (HPV) is the most prevalent sexually transmitted infection, affecting an estimated 11% of the world's population. The high-risk HPV types (HR HPV) account for approximately 5% of the global burden of cancer and thus cause high morbidity and mortality. Although it is known that persistent infection with HR HPV is the greatest risk factor for developing HPV-associated cancer, and that the HPV early proteins E6 and E7 dysregulate immune detection by its host cells, the mechanisms of immune evasion by HR HPV are not well understood. Previous work in the laboratory identified the endogenous cytoplasmic host protein NFX1-123 as a binding partner of the HR HPV type 16 oncoprotein E6 (16E6). Together NFX1-123 and 16E6 affect cellular growth, differentiation, and immortalization genes and pathways. In a whole genome microarray, human foreskin keratinocytes (HFKs) stably expressing 16E6 and overexpressing NFX1-123 showed a diverse set of innate immune genes downregulated two-fold or more when compared to 16E6 cells with endogenous NFX1-123. We demonstrated that 16E6 and NFX1-123 decreased expression of pro-inflammatory cytokines and interferon-stimulated genes (ISGs) in 16E6 HFKs at the mRNA and protein level. Knock down of NFX1-123 in 16E6 HFKs resulted in a derepression of innate immune genes, pointing to the requirement of NFX1-123 for immune regulation in the context of 16E6. Studies using immunofluorescent microscopy revealed that 16E6 and NFX1-123 disturbed the normal localization of signaling proteins involved in initiating the immune response. This study identifies NFX1-123 as a critical host protein partner through which 16E6 is able to subvert the immune response and in turn permit a long-lived HR HPV infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Microarray analysis of genes downregulated in 16E6 HFKs with increased NFX1-123.
Whole genome expression microarrays were conducted in HFKs stably expressing 16E6 and overexpressing NFX1-123 or with endogenous levels of NFX1-123. (A) Three biologically independent HFKs were transduced with 16E6 and p53 protein levels assessed. (B) 16E6 HFK lines 1, 2, and 3 were transduced with NFX1-123 overexpression construct (FN123) or vector control (control). Levels of NFX1-123 mRNA and protein expression levels were quantified and compared. All qPCRs were normalized to the housekeeping gene 36B4, and all error bars represent 95% confidence intervals from the technical replicates shown (n = 3). GAPDH = loading control for (A and C). (D) Venn diagram of genes whose average expression was decreased at least two-fold in 16E6/FN123 cells compared to 16E6/control. Box indicates 26 genes represented in (D) (E) Heat map with hierarchical clustering of the 26 genes decreased in 16E6/FN123 cells compared to 16E6/control.
Fig 2
Fig 2. Genes and pathways decreased in 16E6/FN123 HFKs.
(A) A subset of the over 200 genes that were decreased in 16E6/FN123 cells compared to 16E6/control. Fold change shown is the average fold change over all 16E6 HFK cell lines in which that gene was decreased. (B) Microarray data were analyzed using GeneSpring GX11.5.1, and the significant Gene Ontology pathways for the collection of genes decreased in two out of three 16E6 HFK cell lines were found. Only pathways with a p-value ≤ 0.001 are shown.
Fig 3
Fig 3. Immune gene expression decreased in 16E6 HFKs with more NFX1-123 or increased in 16E6 HFKs with NFX1-123 knockdown.
Expression levels of innate immune genes were examined in 16E6 HFKs with increased NFX1-123 compared to endogenous levels, or with NFX1-123 decreased via short hairpin RNA. (A) mRNA levels of CXCL1, TNF, OAS1, and OAS2 were quantified by qPCR in 16E6/FN123 cells compared to 16E6/control. 16E6 HFKs with increased NFX1-123 showed a decrease in many innate immune genes. (B) Protein levels of IRF7 in 16E6/control cells or 16E6/NFX1-123 cells. 16E6 HFKs overexpressing NFX1-123 had reduced IRF7 protein. (C) Two biologically independent HFKs were transduced with 16E6 and p53 protein levels assessed. Cells were then transduced with a scramble short hairpin construct (scr) or a short hairpin RNA targeting NFX1-123 overexpression construct (sh1). Levels of NFX1-123 mRNA and protein expression levels were quantified and compared. (D) mRNA levels of CXCL1, TNF, OAS1, and OAS2 were quantified by qPCR in 16E6/sh1 cells compared to 16E6/scr. 16E6 HFKs with decreased NFX1-123 showed a rebound in many innate immune genes. (E) Protein levels of IRF7 in 16E6/scr or 16E6/sh1 cells were assessed. 16E6 HFKs with NFX1-123 knocked down had increased IRF7 protein. Innate immune gene qPCRs were normalized to the housekeeping gene GAPDH, while NFX1-123 qPCRs were normalized to the housekeeping gene 36B4. All error bars represent 95% confidence intervals from the technical replicates shown (n = 3) GAPDH = loading control.
Fig 4
Fig 4. Innate immune signal transduction pathways in keratinocytes.
An example of an immune signaling cascade downstream of pattern recognition receptors is shown. Stimulus from pathogen infection is sensed by PRRs, communicated through the adaptor protein TRIF, and further communicated through complexes formed by signaling proteins shown. These result in translocation of the transcription factors IRF-3 or NFkB to induce expression of interferon-stimulated genes, or proinflammatory cytokines and other mediators.
Fig 5
Fig 5. Total amounts of innate immune signaling pathway proteins are not globally decreased.
Three independent HFK cell lines were serially transduced with 16E6 and then either FN123 or vector control. Whole cell lysates were collected from each, and subsequent protein blots were probed for the immune signaling proteins TRIF, TRAF6, TAK1, TAB1, and TAB2. NFX1-123 overexpression was confirmed in FN123 cells compared to HFK or 16E6/control. Actin = loading control. Data shown are from one representative cell line.
Fig 6
Fig 6. Co-localization and subcellular localization of innate immune signaling pathway proteins are altered by 16E6 and NFX1-123.
Immunofluorescence was performed on HFKs, 16E6/control HFKs, and 16E6/FN123 HFKs grown on cover slips. Cells were fixed, permeabilized, and probed with primary antibodies. Scale bars = 20 micron. (A) Cells were stained for TRIF (red), TRAF6 (green), and TAK1 (blue) and shown as a merged image (top panel). The green channel representing TRAF6 was isolated and displayed as a greyscale image (bottom panel). 16E6/FN123 cells show strong perinuclear localization of TRAF6 compared to HFKs or 16E6/HFKs. (B) Cells were stained for TAB1 (red), TAB2 (green), and TAK1 (blue) and shown as a merged image (top panel). The green channel representing TAB2 was isolated and displayed as a greyscale image (bottom panel). 16E6/FN123 cells contain bright, punctate staining of TAB2 in the nuclear compared to 16E6/control cells.
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