Human Papillomavirus 16 E6 and E7 Synergistically Repress Innate Immune Gene Transcription

ABSTRACT Human papillomaviruses (HPV) are causative agents in 5% of all cancers, including the majority of anogenital and oropharyngeal cancers. Downregulation of innate immune genes (IIGs) by HPV to promote the viral life cycle is well documented; E6 and E7 are known repressors of these genes. More recently, we demonstrated that E2 could also repress IIGs. These studies have been carried out in cells overexpressing the viral proteins, and to further investigate the role of individual viral proteins in this repression, we introduced stop codons into E6 and/or E7 in the entire HPV16 genome and generated N/Tert-1 cells stably maintaining the HPV16 genomes. We demonstrate that E6 or E7 individually is not sufficient to repress IIG expression in the context of the entire HPV16 genome; both are required for a synergistic repression. The DNA damage response (DDR) is activated by HPV16 irrespective of E6 and E7 expression, presumably due to viral replication; E1 is a known activator of the DDR. In addition, replication stress was apparent in HPV16-positive cells lacking E6 and E7, manifested by attenuated cellular growth and activation of replication stress genes. These studies led us to the following model. Viral replication per se can activate the DDR following infection, and this activation is a known inducer of IIG expression, which may induce cellular senescence. To combat this, E6 and E7 synergistically combine to manipulate the DDR and actively repress innate immune gene expression promoting cellular growth; neither protein by itself is able to do this. IMPORTANCE The role of human papillomavirus 16 (HPV16) in human cancers is well established; however, to date there are no antiviral therapeutics that are available for combatting these cancers. To identify such targets, we must enhance the understanding of the viral life cycle. Innate immune genes (IIGs) are repressed by HPV16, and we have reported that this repression persists through to cancer. Reversal of this repression would boost the immune response to HPV16-positive tumors, an area that is becoming more important given the advances in immunotherapy. This report demonstrates that E6 and E7 synergistically repress IIG expression in the context of the entire HPV16 genome. Removal of either protein activates the expression of IIGs by HPV16. Therefore, gaining a precise understanding of how the viral oncogenes repress IIG expression represents an opportunity to reverse this repression and boost the immune response to HPV16 infections for therapeutic gain.

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Human papillomavirus 16 E6 and E7 synergistically repress innate immune gene transcription

10.1101/841007 ◽

2019 ◽

Author(s):

Claire D. James ◽

Christian T. Fontan ◽

Raymonde Otoa ◽

Dipon Das ◽

Apurva T. Prabhakar ◽

...

Keyword(s):

Viral Proteins ◽

Innate Immune ◽

Cellular Growth ◽

Immune Gene ◽

Immune Genes ◽

Hpv16 Genome ◽

E6 And E7 ◽

Innate Immune Genes ◽

Innate Immune Gene

AbstractHuman papillomaviruses are causative agents in 5% of all cancers, including the majority of anogenital and oropharyngeal cancers. Downregulation of innate immune genes (IIGs) by HPV to promote the viral life cycle is well documented; E6 and E7 are known repressors of these genes. More recently we demonstrated that E2 could also repress IIGs. These studies have been carried out in cells over-expressing the viral proteins and to further investigate the role of individual viral proteins in this repression we introduced stop codons into E6 and/or E7 in the entire HPV16 genome and generated N/Tert-1 cells stably maintaining the HPV16 genomes. We demonstrate that E6 or E7 individually are not sufficient to repress IIG expression in the context of the entire HPV16 genome, both are required for a synergistic repression. The DNA damage response (DDR) is activated by HPV16 irrespective of E6 and E7 expression, presumably due to viral replication; E1 is a known activator of the DDR. In addition, replication stress was apparent in the HPV16 positive cells lacking E6 and E7, manifested by attenuated cellular growth and activation of replication stress genes. These studies lead us to the following model. Viral replication per se can activate the DDR following infection, and this activation is a known inducer of IIG expression which could induce cellular senescence. To combat this, E6 and E7 synergistically combine to manipulate the DDR and actively repress innate immune gene expression promoting cellular growth; neither protein by itself is able to do this.ImportanceThe role of HPV16 in human cancers is well established; however, to date there are no anti-viral therapeutics that are available for combatting these cancers. To identify such targets, we must enhance understanding of the viral life cycle. Innate immune genes (IIGs) are repressed by HPV16, and we have reported that this repression persists through to cancer. Reversal of this repression would boost the immune response to HPV16 positive tumors, an area that is becoming more important given the advances in immunotherapy. This report demonstrates that E6 and E7 synergistically repress IIG expression in the context of the entire HPV16 genome. Removal of either protein activates the expression of IIGs by HPV16. Therefore, gaining a precise understanding of how the viral oncogenes repress IIG expression represents an opportunity to reverse this repression and boost the immune response to HPV16 infections for therapeutic gain.

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Human Papillomavirus 16 E2 Regulates Keratinocyte Gene Expression Relevant to Cancer and the Viral Life Cycle

Journal of Virology ◽

10.1128/jvi.01941-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 4

Author(s):

Michael R. Evans ◽

Claire D. James ◽

Molly L. Bristol ◽

Tara J. Nulton ◽

Xu Wang ◽

...

Keyword(s):

Gene Expression ◽

Radiation Therapy ◽

Life Cycle ◽

Innate Immune ◽

Host Gene ◽

Host Gene Expression ◽

Human Papillomavirus 16 ◽

Immune Genes ◽

E6 And E7 ◽

Innate Immune Genes

ABSTRACTHuman papillomaviruses (HPVs) are causative agents in ano-genital and oropharyngeal cancers. The virus must reprogram host gene expression to promote infection, and E6 and E7 contribute to this via the targeting of cellular transcription factors, including p53 and pRb, respectively. The HPV16 E2 protein regulates host gene expression in U2OS cells, and in this study, we extend these observations into telomerase reverse transcriptase (TERT) immortalized oral keratinocytes (NOKs) that are capable of supporting late stages of the HPV16 life cycle. We observed repression of innate immune genes by E2 that are also repressed by the intact HPV16 genome in NOKs. Transcriptome sequencing (RNA-seq) data identified 167 up- and 395 downregulated genes by E2; there was a highly significant overlap of the E2-regulated genes with those regulated by the intact HPV16 genome in the same cell type. Small interfering RNA (siRNA) targeting of E2 reversed the repression of E2-targeted genes. The ability of E2 to repress innate immune genes was confirmed in an ano-genital immortalized keratinocyte cell line, N/Tert-1. We present the analysis of data from The Cancer Genome Atlas (TCGA) for HPV16-positive and -negative head and neck cancers (HNC) suggesting that E2 plays a role in the regulation of the host genome in cancers. Patients with HPV16-positive HNC with a loss of E2 expression exhibited a worse clinical outcome, and we discuss how this could, at least partially, be related to the loss of E2 host gene regulation.IMPORTANCEHuman papillomavirus 16 (HPV16)-positive tumors that retain expression of E2 have a better clinical outcome than those that have lost E2 expression. It has been suggested that this is due to a loss of E2 repression of E6 and E7 expression, but this is not supported by data from tumors where there is not more E6 and E7 expression in the absence of E2. Here we report that E2 regulates host gene expression and place this regulation in the context of the HPV16 life cycle and HPV16-positive head and neck cancers (the majority of which retain E2 expression). We propose that this E2 function may play an important part in the increased response of HPV16-positive cancers to radiation therapy. Therefore, host gene regulation by E2 may be important for promotion of the HPV16 life cycle and also for the response of HPV16-positive tumors to radiation therapy.

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Flagellin induces innate immune genes in bronchial epithelial cells in vivo: Role of TET2

Scandinavian Journal of Immunology ◽

10.1111/sji.13046 ◽

2021 ◽

Author(s):

Wanhai Qin ◽

Xanthe Brands ◽

Cornelis Veer ◽

Alex F. Vos ◽

Brendon P. Scicluna ◽

...

Keyword(s):

Epithelial Cells ◽

Bronchial Epithelial Cells ◽

Innate Immune ◽

Bronchial Epithelial ◽

Immune Genes ◽

Innate Immune Genes

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The role of innate immune genes in Alzheimer's disease

Current Opinion in Neurology ◽

10.1097/wco.0000000000000911 ◽

2021 ◽

Vol Publish Ahead of Print ◽

Author(s):

Ana Griciuc ◽

Rudolph E. Tanzi

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Innate Immune ◽

Immune Genes ◽

Innate Immune Genes

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The role of coagulation factors VII, VIII, and IX on the tropism and inflammatory effect of Adenovector on liver and breast cell lines

10.21203/rs.3.rs-458048/v1 ◽

2021 ◽

Author(s):

Rozita Ghojoghi ◽

Fatemeh Nekooei ◽

Fatemeh Vatanparast ◽

Jamal Srv ◽

Ali-Mohammad Tamaddon ◽

...

Keyword(s):

Cell Lines ◽

Fluorescent Microscopy ◽

Coagulation Factors ◽

Innate Immune ◽

P Value ◽

Target Cells ◽

Potential Measurement ◽

Immune Genes ◽

Innate Immune Genes

Abstract Introduction: Adenovectors are promising vectors for gene delivery to the target cells. During gene therapy, AdV interact with plasma components particularly vitamin K-dependent coagulation factors. In this study, we analyzed the comparison between cell entrance, inflammatory patterns, and innate immune induction which induced by the Adenoventor and Adenovector coated by FVII, FVIII, and FIX on two cell-lines; HepG2, MCF7. Methods: The Adenovector expressing GFP (AdVGFP) was prepared and then loading of AdV by coagulation factors were analyzed by zeta potential measurement. The non-toxic MOI of vector employed alone or in complex with coagulation factors VII, FVIII, and FIX applied on HepG2 and MCF7 cell lines. The transduction rates of complexes were analyzed by fluorescent microscopy and flow cytometry. The expression levels of innate immune genes (PKR, STING, IRF-3 and MX-1) were measured by Real-time PCR. Also the level of IL-6 and IL-1β were evaluated by using ELISA assay. Results: The loading of Adenovector by FVII and FIX decreased the zeta charge of the complex particles and enhanced the entry rate in both HepG2 (FVII/AdV: 38.3% and FIX/AdV: 61.9%) and MCF7 (FVII/AdV: 31.2% and FIX/AdV: 36.6%). The expression of IL-6 cytokine enhanced when AdV exposed to FVII (P value: 0.005) in MCF7 and also FVII (P value: 0.01) and FIX (P value: 0.009) in HepG2. Adenovector coated by FVII and FIX could significantly alter the expression pattern of innate immune genes. Conclusion: The findings are highlighted the role of FVII and in particular FIX in facilitating the entry of vector into the cells; also they are enhanced the inflammation and innate immune responses. Interestingly, FVIII had no effect or even adverse effect on entry, inflammation, and innate immune induction.

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Human papillomavirus 16 E2 regulates keratinocyte gene expression relevant to cancer and the viral life cycle

10.1101/461715 ◽

2018 ◽

Cited By ~ 2

Author(s):

Michael R Evans ◽

Claire D James ◽

Molly L Bristol ◽

Tara J Nulton ◽

Xu Wang ◽

...

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Radiation Therapy ◽

Life Cycle ◽

Innate Immune ◽

Host Gene ◽

Host Gene Expression ◽

Immune Genes ◽

E6 And E7 ◽

Innate Immune Genes

AbstractHuman papillomaviruses (HPV) are causative agents in ano-genital and oropharyngeal cancers. The virus must reprogram host gene expression to promote infection, and E6 and E7 contribute to this via targeting of cellular transcription factors including p53 and pRb, respectively. The HPV16 E2 protein regulates host gene expression in U2OS cells and in this study we extend these observations into TERT immortalized oral keratinocytes (NOKs) that are capable of supporting late stages of the HPV16 life cycle. We observed repression of innate immune genes by E2 that are also repressed by the intact HPV16 genome in NOKs. RNA-seq data identified 167 up and 395 downregulated genes by E2; there was a highly significant overlap of the E2 regulated genes with those regulated by the intact HPV16 genome in the same cell type. siRNA targeting of E2 reversed repression of E2 targeted genes. The ability of E2 to repress innate immune genes was confirmed in an ano-genital immortalized keratinocyte cell line, N/Tert-1. We present analysis of data from The Cancer Genome Atlas (TCGA) for HPV16 positive and negative head and neck cancers (HNC) suggesting that E2 plays a role in regulation of the host genome in cancers. Patients with HPV16 positive HNC with a loss of E2 expression exhibit a worse clinical outcome and we discuss how this could, at least partially, be related to the loss of E2 host gene regulation.ImportanceHPV16 positive tumors that retain expression of E2 have a better clinical outcome than those that have lost E2 expression. It has been suggested that this is due to a loss of E2 repression of E6 and E7 expression but this is not supported by data from tumors where there is not more E6 and E7 expression in the absence of E2. Here we report that E2 regulates host gene expression and place this regulation in context of the HPV16 life cycle and HPV16 positive head and neck cancers (the majority of which retain E2 expression). We propose that this E2 function may play an important part in the increased response of HPV16 positive cancers to radiation therapy. Therefore, host gene regulation by E2 may be important for promotion of the HPV16 life cycle, and also for the response of HPV16 positive tumors to radiation therapy.

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Global transcriptomics analyses reveal specialized roles for splicing regulatory proteins in the macrophage innate immune response

10.1101/2020.12.06.413690 ◽

2020 ◽

Author(s):

KO West ◽

AR Wagner ◽

HM Scott ◽

KJ Vail ◽

K Carter ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

Innate Immune Response ◽

Innate Immune ◽

Regulatory Proteins ◽

Raw 264.7 ◽

Immune Gene ◽

Post Translational Modification ◽

Immune Genes ◽

Innate Immune Genes

ABSTRACTWhile the signaling cascades and transcription factors that activate gene expression in macrophages following pattern recognition receptor engagement are well known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Recent phosphoproteomics analyses revealed that members of the SR and hnRNP families of splicing regulatory proteins undergo dynamic post-translational modification in infected macrophages. To begin to test if these splicing factors play a privileged role in controlling the innate immune transcriptome, we analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We observed that thousands of genes were up or downregulated in SR/hnRNP knockdown cells and differentially expressed genes (DEGs) varied significantly depending on the SR/hnRNP examined. We discovered that a subset of critical innate immune genes (Nos2, Mx1, Il1a) rely heavily on SR/hnRNPs for proper induction and/or repression, while others (Tnf, Il1b) are generally unaffected by splicing factor knockdown. We also discovered that many key immune sensors and signaling molecules are subject to regulation by alternative splicing. While our data does not provide evidence for positive correlation between a transcripts’ reliance of SR/hnRNPs for proper expression and the gene’s induction level, length, or intron/exon architecture, we found that many rapidly induced primary response genes are repressed by SR/hnRNPs. Consistent with SR/hnRNP proteins contributing to innate immune outcomes, knockdown of hnRNP K and U significantly enhanced the ability of RAW 264.7 macrophages to control viral replication. Based on these collective findings, we conclude that many innate immune genes have evolved to rely on one or more splicing regulatory factors to ensure the proper timing and magnitude of their induction, supporting a model wherein pre-splicing is a critical regulatory node in the innate immune response.

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Innate immune gene expression in Acropora palmata is consistent despite variance in yearly disease events

10.1101/2020.01.20.912410 ◽

2020 ◽

Cited By ~ 1

Author(s):

Benjamin Young ◽

Xaymara M. Serrano ◽

Stephanie Rosales ◽

Margaret W. Miller ◽

Dana Williams ◽

...

Keyword(s):

Amino Acid ◽

Innate Immune ◽

Acid Oxidase ◽

Immune Gene ◽

Acropora Palmata ◽

Amino Acid Oxidase ◽

Transcriptomic Response ◽

Immune Genes ◽

Innate Immune Genes ◽

Disease Exposure

AbstractCoral disease outbreaks are expected to increase in prevalence, frequency and severity due to climate change and other anthropogenic stressors. This is especially worrying for the Caribbean branching Acropora palmata which has already seen an 80% decrease in its coral cover, with this primarily due to disease. Despite the importance of this species, there has yet to be a characterization of its transcriptomic response to disease exposure. In this study we provide the first transcriptomic analysis of 12 A. palmata genotypes, and their symbiont Symbiodiniaceae, exposed to disease in 2016 and 2017. Year was the primary driver of sample variance for A. palmata and the Symbiodiniaceae. Lower expression of ribosomal genes in the coral, and higher expression of transmembrane ion transport genes in the Symbiodiniaceae indicate that the increased virulence in 2017 may have been due to a dysbiosis between the coral and Symbiodiniaceae. We also identified a conserved suite of innate immune genes responding to the disease challenge that was activated in both years. This included genes from the Toll-like receptor and lectin pathways, and antimicrobial peptides. Co-expression analysis identified a module positively correlated to disease exposure rich in innate immune genes, with D-amino acid oxidase, a gene implicated in phagocytosis and microbiome homeostasis, as the hub gene. The role of D-amino acid oxidase in coral immunity has not been characterized but holds potential as an important enzyme for responding to disease. Our results indicate that A. palmata mounts a similar immune response to disease exposure as other coral species previously studied, but with unique features that may be critical to the survival of this keystone Caribbean species.

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Evidence of Pathogen-Induced Immunogenetic Selection across the Large Geographic Range of a Wild Seabird

Molecular Biology and Evolution ◽

10.1093/molbev/msaa040 ◽

2020 ◽

Vol 37 (6) ◽

pp. 1708-1726

Author(s):

Hila Levy ◽

Steven R Fiddaman ◽

Juliana A Vianna ◽

Daly Noll ◽

Gemma V Clucas ◽

...

Keyword(s):

Geographic Range ◽

Gentoo Penguin ◽

Innate Immune ◽

Evolutionary Time ◽

Immune Genes ◽

Changing Environments ◽

Pathogen Challenge ◽

Innate Immune Genes ◽

Latitudinal Range

Abstract Over evolutionary time, pathogen challenge shapes the immune phenotype of the host to better respond to an incipient threat. The extent and direction of this selection pressure depend on the local pathogen composition, which is in turn determined by biotic and abiotic features of the environment. However, little is known about adaptation to local pathogen threats in wild animals. The Gentoo penguin (Pygoscelis papua) is a species complex that lends itself to the study of immune adaptation because of its circumpolar distribution over a large latitudinal range, with little or no admixture between different clades. In this study, we examine the diversity in a key family of innate immune genes—the Toll-like receptors (TLRs)—across the range of the Gentoo penguin. The three TLRs that we investigated present varying levels of diversity, with TLR4 and TLR5 greatly exceeding the diversity of TLR7. We present evidence of positive selection in TLR4 and TLR5, which points to pathogen-driven adaptation to the local pathogen milieu. Finally, we demonstrate that two positively selected cosegregating sites in TLR5 are sufficient to alter the responsiveness of the receptor to its bacterial ligand, flagellin. Taken together, these results suggest that Gentoo penguins have experienced distinct pathogen-driven selection pressures in different environments, which may be important given the role of the Gentoo penguin as a sentinel species in some of the world’s most rapidly changing environments.

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