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. 2019 Aug 13;93(17):e00915-19.
doi: 10.1128/JVI.00915-19. Print 2019 Sep 1.

Tissue-Specific Gene Expression during Productive Human Papillomavirus 16 Infection of Cervical, Foreskin, and Tonsil Epithelium

Sreejata Chatterjee  1 , Sa Do Kang  1 , Samina Alam  1 , Anna C Salzberg  2 , Janice Milici  1 , Sjoerd H van der Burg  3 , Willard Freeman  4 , Craig Meyers  5
Affiliations

Tissue-Specific Gene Expression during Productive Human Papillomavirus 16 Infection of Cervical, Foreskin, and Tonsil Epithelium

Sreejata Chatterjee et al. J Virol. .

Abstract

Epidemiological data confirm a much higher incidence of high-risk human papillomavirus 16 (HPV16)-mediated carcinogenesis of the cervical epithelium than for other target sites. In order to elucidate tissue-specific responses to virus infection, we compared gene expression changes induced by productive HPV16 infection of cervical, foreskin, and tonsil organotypic rafts. These rafts closely mimic persistent HPV16 infection, long before carcinogenesis sets in. The total number of gene expression changes varied considerably across the tissue types, with only 32 genes being regulated in common. Among them, we confirmed the Kelch-like family protein KLHL35 and the laminin-5 complex to be upregulated and downregulated, respectively, in all the three tissues. HPV16 infection induces upregulation of genes involved in cell cycle control, cell division, mitosis, DNA replication, and DNA damage repair in all the three tissues, indicative of a hyperproliferative environment. In the cervical and tonsil epithelium, we observe significant downregulation of genes involved in epidermis development, keratinocyte differentiation, and extracellular matrix organization. On the other hand, in HPV16-positive foreskin (HPV16 foreskin) tissue, several genes involved in interferon-mediated innate immunity, cytokine signaling, and cellular defenses were downregulated. Furthermore, pathway analysis and experimental validations identified important cellular pathways like STAT1 and transforming growth factor β (TGF-β) to be differentially regulated among the three tissue types. The differential modulation of important cellular pathways like TGF-β1 and STAT1 can explain the sensitivity of tissues to HPV cancer progression.IMPORTANCE Although the high-risk human papillomavirus 16 infects anogenital and oropharyngeal sites, the cervical epithelium has a unique vulnerability to progression of cancer. Host responses during persistent infection and preneoplastic stages can shape the outcome of cancer progression in a tissue-dependent manner. Our study for the first time reports differential regulation of critical cellular functions and signaling pathways during productive HPV16 infection of cervical, foreskin, and tonsil tissues. While the virus induces hyperproliferation in infected cells, it downregulates epithelial differentiation, epidermal development, and innate immune responses, according to the tissue type. Modulation of these biological functions can determine virus fitness and pathogenesis and illuminate key cellular mechanisms that the virus employs to establish persistence and finally initiate disease progression.

Keywords: HPV16; epithelial differentiation; gene expression; human papillomavirus; immune response; tissue specific.

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Figures

FIG 1
FIG 1
Venn diagram showing total numbers of gene expression changes and overlapping changes in HPV16-positive cervix, foreskin, and tonsil. For microarray analysis of each tissue type, three primary cell lines and three HPV16-transformed cell lines were used to grow rafts. Each of these experiments was repeated at separate times, representing a total of six individually grown raft tissue for primary (n = 6) and HPV16-positive (n = 6) cell lines. Only gene expression changes of more than 1.5-fold and with a P value of <0.05 were considered significant for the analysis.
FIG 2
FIG 2
Common genes regulated during productive HPV16 infection of cervical, foreskin, and tonsil epithelium. (a) Histograms representing gene expression fold changes of genes commonly regulated. We observe genes belonging to biological functions of the cell cycle, DNA damage repair, and cell division to be upregulated in all the tissues during productive infection. (b to d) Volcano plots representing log2 fold changes (x axis) plotted against the −log10 P value (y axis), obtained from CLC Genomics Workbench 4.8 package, in HPV16-positive cervical, foreskin, and tonsil rafts. The black scatter points represent all the significantly regulated genes in each tissue type. The commonly regulated genes are color-coded according to their biological functions, as indicated in the key.
FIG 3
FIG 3
Productive HPV16 infection results in upregulation of KLHL35 and downregulation of the laminin-5 complex in cervical, foreskin, and tonsil rafts. (a) Protein-protein network of commonly regulated genes with productive HPV16 infection in cervix, foreskin, and tonsil tissue. Highlighted are the p53-DREAM pathway targets, KHLH35, and laminin-5 subunits. The STRING database and Cytoscape were used to derive the interacting network. (b) Immunofluorescence reveals increased expression of KLHL35 (green) in HPV16-infected cervical, foreskin, and raft tissues harvested at day 10 and day 20. DAPI (4′,6-diamidino-2-phenylindole) (blue) was used to stain the nuclei. (c) Immunofluorescence reveals decreased expression of the laminin-5 complex (green) in the basal layer cells of HPV16-infected cervical, foreskin, and tonsil rafts harvested at day 10 and day 20. DAPI (blue) was used to stain the nuclei. Images were acquired using a Nikon Eclipse 80i microscope and NIS Elements (v4.4) software at a ×200 magnification.
FIG 4
FIG 4
(a) Gene ontology analysis reveals significant upregulation of gene categories related to increased cell proliferation in HPV16 cervical, foreskin, and tonsil epithelium. (b) Among the downregulated genes, the GO terms related to epidermis development and ECM organization were enriched in HPV16 cervical and tonsil tissues. Several GO terms related to the type I interferon response and immune responses were downregulated in HPV16 foreskin. The histograms represent the negative log10 P value obtained from GOrilla analysis for the related GO category (biological function).
FIG 5
FIG 5
The protein interaction networks of upregulated genes depict dense interconnectivity and high representation of proteins related to mitosis and cell division in HPV16 cervical (a), foreskin (b), and tonsil (c) epithelium. The CLUSTER function (STRING database) was used to identify the most relevant protein interactions among the upregulated targets. Cytoscape software (3.6.0) was used to construct the networks, using yFiles hierarchic layout.
FIG 6
FIG 6
Protein interactome of genes downregulated upon productive HPV16 infection of epithelial tissue types. The CLUSTER function (STRING database) was used to identify the most relevant protein interactions among the upregulated targets. Cytoscape software (3.6.0) was used to construct the networks, using yFiles hierarchic layout. (a to c) In HPV16 cervical tissue, clusters representing proteins involved in epithelial differentiation and ECM organization were identified. (d and e) Several immune response-related proteins, including type I interferon signaling pathway candidates, formed the major clusters in HPV16 foreskin. Owing to a minimum number of gene expression changes, we obtain a single cluster in HPV16 tonsil that comprises proteins involved in ECM organization and hemidesmosome assembly (c).
FIG 7
FIG 7
(a, d, and g) RT-PCR validation of selected genes in HPV16-infected cervical (a), foreskin (d), and tonsil (g) rafts harvested at day 10. RNA expression levels of KRT14, KRT19, KRT23, GPER, KLK8, and RPTN were measured and normalized to GAPDH levels. The expression levels in the uninfected rafts were normalized to a value of 1. Histograms indicate average means and standard errors of the means (SEM) from three experiments. * indicates a P value of <0.05 (two-tailed t test). Western blot analysis was performed on day 10 rafts to determine protein levels of KRT14, KRT19, KRT23, and GPER. GAPDH was used as a housekeeping control. (b, e, and h) Representative blots showing protein expression of selected targets in HPV16-infected cervical (b), foreskin (e), and tonsil (h) rafts. Images were acquired with a Bio-Rad ChemiDoc MP imaging system and Image Lab (v6.0.0) software. Histograms represent the average ratios of protein levels normalized with the loading control GAPDH (means and SEM). (c, f, and i) Densitometry analysis using ImageJ software was used to calculate normalized protein levels in the cervical (c), foreskin (f), and tonsil (i) tissues. * represents a P value of <0.05, and ** represents a P value of <0.01. Rafts obtained from a new set of two uninfected and two HPV16-infected cell lines, different from those used for microarray analysis, were used for RT-PCR and Western blot analysis.
FIG 8
FIG 8
Heat maps depicting expression levels of the genes regulated by the upstream regulators TGF-β and STAT1 in HPV16 foreskin, cervix, and tonsil, compared to their uninfected controls. The online tool CIMminer (NCI) was used to create the heat maps. The one-matrix method using the Euclidean distances and average linkage cluster algorithm was utilized. Heat maps represent STAT1-regulated interferon signaling genes (a), TGF-β-regulated cell cycle-related genes (b), and TGF-β regulated genes with function in ECM organization and assembly (c). IPA (Qiagen) was used to identify the target genes regulated by STAT1 and TGF-β from the data set.
FIG 9
FIG 9
Expression levels of total STAT1, pSTAT1, total SMAD3, and pSMAD3 in uninfected and HPV16-infected rafts. (a) Representative blots indicating expression levels of total STAT1, pSTAT1, total SMAD3, and pSMAD3 in HPV16-infected cervical, foreskin, and tonsil rafts harvested at day 10. GAPDH was used as a loading control. (b to d) Histograms representing average ratios of protein expression levels normalized to GAPDH levels (means and SEM) in cervical (b), foreskin (c), and tonsillar (d) tissues. * indicates a P value of <0.05.
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