Session Chairs: Mandy Muller and Ben Gewurz
Oral Talk #20
A Genome-Wide Screen of EBV Proteins that Modulate Host
SUMOylation Identifies a SUMO E3 Ligase Conserved in
Herpesviruses
Carlos F. De La Cruz Herrera1, Kathy Shire1, Lori Frappier1
1Dept of Molecular Genetics, University of Toronto
Modification by the small ubiquitin-like modifier (SUMO) controls the activity of many proteins central to antiviral responses and oncogenesis. Therefore to modulate cellular processes, some viruses encode SUMO E3 ligases that induce SUMOylation or SUMO- targeted ubiquitin ligases that degrade SUMOylated proteins. EBV has not been reported to encode either, however the cellular impact of most EBV proteins has not been investigated.
We screened 51 EBV proteins for the ability to globally alter SUMO1 and SUMO2 modifications of cellular proteins in three different cellular systems, including nasopharyngeal carcinoma. One EBV protein (BRLF1; Rta) induced the loss of SUMOylated proteins, in a proteasome-dependent manner, as well as the loss of the SUMO-associated promeylocytic leukemia nuclear bodies, suggesting that, like its homologue in KSHV, it is a SUMO-targeted ubiquitin ligase.
Four EBV proteins consistently upregulated the levels of SUMO-conjugated proteins, including the EBV SM protein. SM homologues exist in herpes simplex virus (UL54 or ICP27) and cytomegalovirus (UL69), prompting us to examine the effects of these proteins on cellular SUMOylation. Like SM, HSV-1 UL54 and CMV UL69 induced global SUMOylation. All three viral proteins were purified and tested in an in vitro assay for SUMOylation using p53 as a substrate, revealing that all three proteins had titratable SUMO E3 ligase activity. Consistent with this activity, all three viral homologues bound SUMO and the SUMO E2, Ubc9. Interestingly, the homologues had different specificities for SUMO1 and SUMO2, with SM and UL69 preferentially binding SUMO1 and inducing SUMO1 modifications, and UL54 preferentially binding SUMO2 and inducing SUMO2 modifications. The results provide new insights into the function of this family of conserved herpesvirus proteins. These are the first SUMO E3 ligases discovered for EBV, HSV-1 and CMV and the conservation of this activity across diverse herpesviruses suggests the importance of this activity for herpesvirus infections.
A Genome-Wide Screen of EBV Proteins that Modulate Host
SUMOylation Identifies a SUMO E3 Ligase Conserved in
Herpesviruses
Carlos F. De La Cruz Herrera1, Kathy Shire1, Lori Frappier1
1Dept of Molecular Genetics, University of Toronto
Modification by the small ubiquitin-like modifier (SUMO) controls the activity of many proteins central to antiviral responses and oncogenesis. Therefore to modulate cellular processes, some viruses encode SUMO E3 ligases that induce SUMOylation or SUMO- targeted ubiquitin ligases that degrade SUMOylated proteins. EBV has not been reported to encode either, however the cellular impact of most EBV proteins has not been investigated.
We screened 51 EBV proteins for the ability to globally alter SUMO1 and SUMO2 modifications of cellular proteins in three different cellular systems, including nasopharyngeal carcinoma. One EBV protein (BRLF1; Rta) induced the loss of SUMOylated proteins, in a proteasome-dependent manner, as well as the loss of the SUMO-associated promeylocytic leukemia nuclear bodies, suggesting that, like its homologue in KSHV, it is a SUMO-targeted ubiquitin ligase.
Four EBV proteins consistently upregulated the levels of SUMO-conjugated proteins, including the EBV SM protein. SM homologues exist in herpes simplex virus (UL54 or ICP27) and cytomegalovirus (UL69), prompting us to examine the effects of these proteins on cellular SUMOylation. Like SM, HSV-1 UL54 and CMV UL69 induced global SUMOylation. All three viral proteins were purified and tested in an in vitro assay for SUMOylation using p53 as a substrate, revealing that all three proteins had titratable SUMO E3 ligase activity. Consistent with this activity, all three viral homologues bound SUMO and the SUMO E2, Ubc9. Interestingly, the homologues had different specificities for SUMO1 and SUMO2, with SM and UL69 preferentially binding SUMO1 and inducing SUMO1 modifications, and UL54 preferentially binding SUMO2 and inducing SUMO2 modifications. The results provide new insights into the function of this family of conserved herpesvirus proteins. These are the first SUMO E3 ligases discovered for EBV, HSV-1 and CMV and the conservation of this activity across diverse herpesviruses suggests the importance of this activity for herpesvirus infections.
Oral Talk #21
Computational Analysis of Ribonomics Datasets Identifies
Long Non-Coding RNA Targets of γ-herpesviral miRNAs
Sunantha Sethuraman1, Lauren Appleby Gay1, Rolf Renne1,2,3
1Department of Molecular Genetics and Microbiology, University of Florida, 2UF Health Cancer Center, University of Florida, 3UF Genetics Institute, University of Florida
Ribonomics experiments involving crosslinking and immuno-precipitation (CLIP) of Ago proteins have expanded the understanding of the miRNA targetome of several organisms.These techniques, collectively referred to as CLIP-Seq, have been applied to identifying the mRNA targets of miRNAs expressed by Kaposi’s Sarcoma Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV). However, these studies focused on identifying only those RNA targets of KSHV and EBV miRNAs that are known to encode proteins. Recent studies have demonstrated that long non-coding RNAs (lncRNAs) are also targeted by miRNAs. In this study, we performed a systematic re-analysis of published datasets from KSHV- and EBV-driven cancers. We used CLIP-Seq data from lymphoma cells or EBV- transformed B cells, and a crosslinking, ligation and sequencing of hybrids (CLASH) dataset from KSHV-infected endothelial cells, to identify novel lncRNA targets of viral miRNAs. Here, we catalog the lncRNA targetome of KSHV and EBV miRNAs, and provide a detailed in silico analysis of lncRNA-miRNA binding interactions. Viral miRNAs target several hundred lncRNAs, including a subset previously shown to be aberrantly expressed in human malignancies. In addition, we identified thousands of lncRNAs to be putative targets of human miRNAs, suggesting that miRNA-lncRNA interactions broadly contribute to the regulation of gene expression.
Computational Analysis of Ribonomics Datasets Identifies
Long Non-Coding RNA Targets of γ-herpesviral miRNAs
Sunantha Sethuraman1, Lauren Appleby Gay1, Rolf Renne1,2,3
1Department of Molecular Genetics and Microbiology, University of Florida, 2UF Health Cancer Center, University of Florida, 3UF Genetics Institute, University of Florida
Ribonomics experiments involving crosslinking and immuno-precipitation (CLIP) of Ago proteins have expanded the understanding of the miRNA targetome of several organisms.These techniques, collectively referred to as CLIP-Seq, have been applied to identifying the mRNA targets of miRNAs expressed by Kaposi’s Sarcoma Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV). However, these studies focused on identifying only those RNA targets of KSHV and EBV miRNAs that are known to encode proteins. Recent studies have demonstrated that long non-coding RNAs (lncRNAs) are also targeted by miRNAs. In this study, we performed a systematic re-analysis of published datasets from KSHV- and EBV-driven cancers. We used CLIP-Seq data from lymphoma cells or EBV- transformed B cells, and a crosslinking, ligation and sequencing of hybrids (CLASH) dataset from KSHV-infected endothelial cells, to identify novel lncRNA targets of viral miRNAs. Here, we catalog the lncRNA targetome of KSHV and EBV miRNAs, and provide a detailed in silico analysis of lncRNA-miRNA binding interactions. Viral miRNAs target several hundred lncRNAs, including a subset previously shown to be aberrantly expressed in human malignancies. In addition, we identified thousands of lncRNAs to be putative targets of human miRNAs, suggesting that miRNA-lncRNA interactions broadly contribute to the regulation of gene expression.
Oral Talk #22
The Receptor or Coreceptors in Epstein-Barr Virus Infection of
Nasopharyngeal Epithelial Cells
Hua Zhang1, Hong-Bo Wang1, Yan Li1, Qian Zhong1, Yi-Xin Zeng1, Elliott Kieff2, Benjamin Gewurz2, Bo Zhao2, Mu-sheng Zeng1
1Sun Yat-sen University Cancer Center, 2 Brigham and Women’s Hospital, Harvard University
Epstein-Barr virus (EBV) is implicated as an etiological factor in B lymphomas and undifferentiated nasopharyngeal carcinoma (NPC), a malignant epithelial cancer occurring frequently in South China and Southeast Asia. However, the mechanisms of cell-free EBV infection of nasopharyngeal epithelial cells (NEPCs) remain elusive, mainly due to the deficiency of highly susceptible NPECs model. We focus on establishing the highly susceptible NPECs model and identifying cellular factors responsible for EBV infection. We found EGF-treated NPECs and premalignant NPECs-Bmi1 cells grown as sphere-like cells (SLCs) significantly enhanced EBV infection. We use cDNA array, siRNA library and pull- down assay followed by liquid chromatography-tandem MS to screen the key cellular factors involved in EBV infection of epithelial cells. We found that non-muscle myosin heavy chain IIA (NMHCIIA) interacts with EBV gH/gL and mediates EBV attachment to the epithelial cells; EphA2 interacts with both gH/gL and gB and mediates EBV fusion with the cellular membrane, while neuropilin 1 (NRP1) interacts with EBV gB and triggers the activation of EGFR/RAS/ERK signaling induced by EBV. Taken together, NMHCIIA, EphA2 and NRP1 are identified as the receptor or coreceptors in EBV infection of NPECs, indicative of their capacity to serve as targets for blocking EBV infection.
The Receptor or Coreceptors in Epstein-Barr Virus Infection of
Nasopharyngeal Epithelial Cells
Hua Zhang1, Hong-Bo Wang1, Yan Li1, Qian Zhong1, Yi-Xin Zeng1, Elliott Kieff2, Benjamin Gewurz2, Bo Zhao2, Mu-sheng Zeng1
1Sun Yat-sen University Cancer Center, 2 Brigham and Women’s Hospital, Harvard University
Epstein-Barr virus (EBV) is implicated as an etiological factor in B lymphomas and undifferentiated nasopharyngeal carcinoma (NPC), a malignant epithelial cancer occurring frequently in South China and Southeast Asia. However, the mechanisms of cell-free EBV infection of nasopharyngeal epithelial cells (NEPCs) remain elusive, mainly due to the deficiency of highly susceptible NPECs model. We focus on establishing the highly susceptible NPECs model and identifying cellular factors responsible for EBV infection. We found EGF-treated NPECs and premalignant NPECs-Bmi1 cells grown as sphere-like cells (SLCs) significantly enhanced EBV infection. We use cDNA array, siRNA library and pull- down assay followed by liquid chromatography-tandem MS to screen the key cellular factors involved in EBV infection of epithelial cells. We found that non-muscle myosin heavy chain IIA (NMHCIIA) interacts with EBV gH/gL and mediates EBV attachment to the epithelial cells; EphA2 interacts with both gH/gL and gB and mediates EBV fusion with the cellular membrane, while neuropilin 1 (NRP1) interacts with EBV gB and triggers the activation of EGFR/RAS/ERK signaling induced by EBV. Taken together, NMHCIIA, EphA2 and NRP1 are identified as the receptor or coreceptors in EBV infection of NPECs, indicative of their capacity to serve as targets for blocking EBV infection.
Oral Talk #23
Examination of the Effects of Rhesus Macaque Rhadinovirus
(RRV) Infection and Viral CD200 (vCD200) on Cellular Gene
Expression Profiles During in vivo Infection
Ryan D. Estep1, Helen Li1, Scott W. Wong1,2,3
1Vaccine and Gene Therapy Institute, Oregon Health & Science University West Campus, 2Division of Pathobiology and Immunology, Oregon National Primate Research Center, 3Department of Molecular Microbiology and Immunology, Oregon Health & Science University
Rhesus macaque rhadinovirus (RRV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) both encode viral CD200 (vCD200) molecules that are functional homologues of cellular CD200, an immunoregulatory molecule that inhibits the activation of cells expressing CD200 receptor (CD200R). Previous infection studies in rhesus macaques (RM) utilizing a recombinant RRV lacking expression of vCD200 (vCD200ns) indicated that vCD200 is immunosuppressive in vivo, and is capable of inhibiting T cell activation at early times post-infection. RRV vCD200 also demonstrates unanticipated negative effects on viral replication levels in infected RM, with higher viral loads being detected in blood and lymph node (LN) tissues of RM infected with RRV vCD200ns. In order to assess the overall effects of RRV infection on cellular gene expression patterns in vivo, and in an attempt to understand the effects of vCD200 on viral replication, RNA samples from LN biopsies obtained pre- and post-WT or vCD200ns RRV infection were subjected to RNA-Seq analysis. From this investigation, we have determined that RRV infection has measurable effects on the expression of a variety of cellular genes in LN tissue, and that vCD200 can affect the expression of cellular genes during infection. Specific genes of interest that displayed 2-fold or greater changes in expression, and which were confirmed to be significantly up-regulated in LN after RRV infection, include those associated with immunity and oncogenesis, such as the B cell-associated activation-induced cytidine deaminase (AICDA), chemokine receptors (e.g. CX3CR1), and a cancer biomarker (Glypican-1), while significantly downregulated genes include a negative regulator of cell growth and proliferation (RasD1). Thioredoxin interacting protein (TXNIP), a multifunction protein that can regulate a variety of processes, including proliferation, apoptosis, and tumor suppression, was identified as a gene that is differentially expressed between WT and vCD200ns RRV-infected RM. TXNIP is now being investigated for its potential role in RRV replication in vivo.
Examination of the Effects of Rhesus Macaque Rhadinovirus
(RRV) Infection and Viral CD200 (vCD200) on Cellular Gene
Expression Profiles During in vivo Infection
Ryan D. Estep1, Helen Li1, Scott W. Wong1,2,3
1Vaccine and Gene Therapy Institute, Oregon Health & Science University West Campus, 2Division of Pathobiology and Immunology, Oregon National Primate Research Center, 3Department of Molecular Microbiology and Immunology, Oregon Health & Science University
Rhesus macaque rhadinovirus (RRV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) both encode viral CD200 (vCD200) molecules that are functional homologues of cellular CD200, an immunoregulatory molecule that inhibits the activation of cells expressing CD200 receptor (CD200R). Previous infection studies in rhesus macaques (RM) utilizing a recombinant RRV lacking expression of vCD200 (vCD200ns) indicated that vCD200 is immunosuppressive in vivo, and is capable of inhibiting T cell activation at early times post-infection. RRV vCD200 also demonstrates unanticipated negative effects on viral replication levels in infected RM, with higher viral loads being detected in blood and lymph node (LN) tissues of RM infected with RRV vCD200ns. In order to assess the overall effects of RRV infection on cellular gene expression patterns in vivo, and in an attempt to understand the effects of vCD200 on viral replication, RNA samples from LN biopsies obtained pre- and post-WT or vCD200ns RRV infection were subjected to RNA-Seq analysis. From this investigation, we have determined that RRV infection has measurable effects on the expression of a variety of cellular genes in LN tissue, and that vCD200 can affect the expression of cellular genes during infection. Specific genes of interest that displayed 2-fold or greater changes in expression, and which were confirmed to be significantly up-regulated in LN after RRV infection, include those associated with immunity and oncogenesis, such as the B cell-associated activation-induced cytidine deaminase (AICDA), chemokine receptors (e.g. CX3CR1), and a cancer biomarker (Glypican-1), while significantly downregulated genes include a negative regulator of cell growth and proliferation (RasD1). Thioredoxin interacting protein (TXNIP), a multifunction protein that can regulate a variety of processes, including proliferation, apoptosis, and tumor suppression, was identified as a gene that is differentially expressed between WT and vCD200ns RRV-infected RM. TXNIP is now being investigated for its potential role in RRV replication in vivo.
Oral Talk #24
PIAS1 Restricts Epstein-Barr Virus Reactivation and it is
Antagonized by Caspases upon Lytic Induction
Renfeng Li1, Kun Zhang1, Dong-Wen Lv1
1Virginia Commonwealth University
Epstein-Barr virus (EBV) in tumor cells is predominately in latent phase but the virus can undergo lytic reactivation in response to various stimuli. However, the cellular factors that control latency and lytic replication are poorly defined. Using gene-knockout and reconstitution methods, we recently demonstrated that PIAS1 is a cellular restriction factor for EBV. We found that depletion of PIAS1 facilitates, while reconstitution of PIAS1 suppresses, EBV lytic reactivation. Strikingly, we showed that lytic induction by the cross-linking of B cell receptor (BCR) or chemicals triggers caspase-dependent cleavage of PIAS1. We demonstrated that caspase-3, -6 and -8 are major caspases responsible for PIAS1 cleavage at two evolutionarily conserved sites (D100 and D433) and that caspase inhibition abrogates EBV reactivation through preventing PIAS1 cleavage. We further demonstrated that a cleavage-resistant PIAS1 mutant strongly suppresses EBV replication upon lytic induction. Mechanistically, we showed that PIAS1 specifically inhibits multiple viral and cellular factors through protein-protein interaction to suppress EBV lytic gene expression and that caspase-mediated cleavage antagonizes PIAS1-mediated restriction toward EBV reactivation. (這是不是和那令人不解的實驗結果有關?)
Together, our results establish PIAS1 as a critical restriction factor for EBV lytic replication and uncover a previously unappreciated mechanism by which EBV exploits apoptotic caspases to antagonize PIAS1-mediated restriction.
PIAS1 Restricts Epstein-Barr Virus Reactivation and it is
Antagonized by Caspases upon Lytic Induction
Renfeng Li1, Kun Zhang1, Dong-Wen Lv1
1Virginia Commonwealth University
Epstein-Barr virus (EBV) in tumor cells is predominately in latent phase but the virus can undergo lytic reactivation in response to various stimuli. However, the cellular factors that control latency and lytic replication are poorly defined. Using gene-knockout and reconstitution methods, we recently demonstrated that PIAS1 is a cellular restriction factor for EBV. We found that depletion of PIAS1 facilitates, while reconstitution of PIAS1 suppresses, EBV lytic reactivation. Strikingly, we showed that lytic induction by the cross-linking of B cell receptor (BCR) or chemicals triggers caspase-dependent cleavage of PIAS1. We demonstrated that caspase-3, -6 and -8 are major caspases responsible for PIAS1 cleavage at two evolutionarily conserved sites (D100 and D433) and that caspase inhibition abrogates EBV reactivation through preventing PIAS1 cleavage. We further demonstrated that a cleavage-resistant PIAS1 mutant strongly suppresses EBV replication upon lytic induction. Mechanistically, we showed that PIAS1 specifically inhibits multiple viral and cellular factors through protein-protein interaction to suppress EBV lytic gene expression and that caspase-mediated cleavage antagonizes PIAS1-mediated restriction toward EBV reactivation. (這是不是和那令人不解的實驗結果有關?)
Together, our results establish PIAS1 as a critical restriction factor for EBV lytic replication and uncover a previously unappreciated mechanism by which EBV exploits apoptotic caspases to antagonize PIAS1-mediated restriction.
Oral Talk #25
Metabolic Reprogramming of Kaposi’s Sarcoma Associated
Herpes Virus Infected B-Cells in Hypoxia
Rajnish K Singh1, Fengchao Lang1, Yonggang Pei1, Hem C Jha2, Erle S Robertson1 1University of Pennsylvania, 2Indian Institute of Technology
Kaposi’s sarcoma associated herpesvirus (KSHV) infection stabilizes hypoxia inducible factors (HIFs). The interaction between KSHV encoded factors and HIFs plays a critical role in KSHV latency, reactivation and associated disease phenotypes. Besides modulation of large-scale signaling, KSHV infection also reprograms the metabolic activity of infected cells. However, the mechanism and cellular pathways modulated during these changes are poorly understood. We performed comparative RNA sequencing analysis on cells with stabilized hypoxia inducible factor 1 alpha (HIF1α) of KSHV negative or positive background to identify changes in global and metabolic gene expression. Our results show that hypoxia induces glucose dependency of KSHV positive cells with high glucose uptake and high lactate release. We identified the KSHV-encoded vGPCR, as a novel target of HIF1α and one of the main viral antigens of this metabolic reprogramming. Bioinformatics analysis of vGPCR promoter identified 9 distinct hypoxia responsive elements which were activated by HIF1α in-vitro. Expression of vGPCR alone was sufficient for induction of changes in the metabolic phenotype similar to those induced by KSHV under hypoxic conditions. Silencing of HIF1α rescued the hypoxia associated phenotype of KSHV positive cells. Analysis of the host transcriptome identified several common targets of hypoxia as well as KSHV encoded factors and other synergistically activated genes belonging to cellular pathways. These include those involved in carbohydrate, lipid and amino acids metabolism. Further, the DNA methyltranferases, DNMT3A and DNMT3B were found to be regulated by either KSHV, or hypoxia at the transcript and protein levels. These changes were synergistically induced due to KSHV infection in the hypoxic environment. This study showed distinct and common, as well as collaborative effects of HIF1α and KSHV-encoded proteins on metabolic reprogramming of KSHV-infected cells in hypoxia.
Metabolic Reprogramming of Kaposi’s Sarcoma Associated
Herpes Virus Infected B-Cells in Hypoxia
Rajnish K Singh1, Fengchao Lang1, Yonggang Pei1, Hem C Jha2, Erle S Robertson1 1University of Pennsylvania, 2Indian Institute of Technology
Kaposi’s sarcoma associated herpesvirus (KSHV) infection stabilizes hypoxia inducible factors (HIFs). The interaction between KSHV encoded factors and HIFs plays a critical role in KSHV latency, reactivation and associated disease phenotypes. Besides modulation of large-scale signaling, KSHV infection also reprograms the metabolic activity of infected cells. However, the mechanism and cellular pathways modulated during these changes are poorly understood. We performed comparative RNA sequencing analysis on cells with stabilized hypoxia inducible factor 1 alpha (HIF1α) of KSHV negative or positive background to identify changes in global and metabolic gene expression. Our results show that hypoxia induces glucose dependency of KSHV positive cells with high glucose uptake and high lactate release. We identified the KSHV-encoded vGPCR, as a novel target of HIF1α and one of the main viral antigens of this metabolic reprogramming. Bioinformatics analysis of vGPCR promoter identified 9 distinct hypoxia responsive elements which were activated by HIF1α in-vitro. Expression of vGPCR alone was sufficient for induction of changes in the metabolic phenotype similar to those induced by KSHV under hypoxic conditions. Silencing of HIF1α rescued the hypoxia associated phenotype of KSHV positive cells. Analysis of the host transcriptome identified several common targets of hypoxia as well as KSHV encoded factors and other synergistically activated genes belonging to cellular pathways. These include those involved in carbohydrate, lipid and amino acids metabolism. Further, the DNA methyltranferases, DNMT3A and DNMT3B were found to be regulated by either KSHV, or hypoxia at the transcript and protein levels. These changes were synergistically induced due to KSHV infection in the hypoxic environment. This study showed distinct and common, as well as collaborative effects of HIF1α and KSHV-encoded proteins on metabolic reprogramming of KSHV-infected cells in hypoxia.
Oral Talk #26
Caspase 3 Activation Facilitates Lytic Replication of Epstein-
Barr Virus in Epithelial Cells
Dong-Yan Jin1, Tsz-Fung Cheng1, Ting-Hin Ho1, Kit-San Yuen1 1The University of Hong Kong
Epstein-Barr virus (EBV) undergoes lytic replication and also maintains latency states in B cells and epithelial cells. Both states are required for maintenance and oncogenesis of EBV. How lytic replication of EBV is regulated in epithelial cells is not well understood. Particularly, it is unclear how apoptosis and activation of caspase 3 might affect EBV lytic induction. (讚) In this study we found that activation of caspase 3 facilitates productive infection of EBV in epithelial cells.(這是不是和那令人不解的實驗結果有關?) Pro-caspase 3 was identified in a CRISPR activation screen for cellular dependency factors of EBV infection in HEK293 cells. The positive role of caspase 3 activation in de novo infection of EBV in HEK293 cells was verified. Pre-treatment with 5- flurouracil (5FU) and cisplatin was highly capable of sensitizing HEK293 cells to infection with M81 EBV, leading to much higher ratios of GFP+ cells. This phenotype was abrogated completely with the addition of QVD-OPh (QV), a potent and broad-spectrum caspase inhibitor. Furthermore, a correlation between the elevation of caspase 3 activity and the accumulation of EBV DNA in the cells was observed. Activation of the mitochondrial or intrinsic pathway of apoptosis or expression of a dominant active form of caspase 3 showed stimulatory effect on EBV lytic replication as reflected by the ratio of GFP+ cells and level of EBV DNA. Finally, Z-DEVD-FMK, a highly specific inhibitor of caspase 3, ablated the stimulatory effect of 5FU or cisplatin. Similar findings were also obtained in NP460 nasopharyngeal epithelial cells and HK1 NPC cells infected with M81. Taken together, our work revealed that activation of caspase 3 facilitates EBV productive infection in epithelial cells. Our findings might also provide new strategies to improve the design of anti-EBV therapy.
Caspase 3 Activation Facilitates Lytic Replication of Epstein-
Barr Virus in Epithelial Cells
Dong-Yan Jin1, Tsz-Fung Cheng1, Ting-Hin Ho1, Kit-San Yuen1 1The University of Hong Kong
Epstein-Barr virus (EBV) undergoes lytic replication and also maintains latency states in B cells and epithelial cells. Both states are required for maintenance and oncogenesis of EBV. How lytic replication of EBV is regulated in epithelial cells is not well understood. Particularly, it is unclear how apoptosis and activation of caspase 3 might affect EBV lytic induction. (讚) In this study we found that activation of caspase 3 facilitates productive infection of EBV in epithelial cells.(這是不是和那令人不解的實驗結果有關?) Pro-caspase 3 was identified in a CRISPR activation screen for cellular dependency factors of EBV infection in HEK293 cells. The positive role of caspase 3 activation in de novo infection of EBV in HEK293 cells was verified. Pre-treatment with 5- flurouracil (5FU) and cisplatin was highly capable of sensitizing HEK293 cells to infection with M81 EBV, leading to much higher ratios of GFP+ cells. This phenotype was abrogated completely with the addition of QVD-OPh (QV), a potent and broad-spectrum caspase inhibitor. Furthermore, a correlation between the elevation of caspase 3 activity and the accumulation of EBV DNA in the cells was observed. Activation of the mitochondrial or intrinsic pathway of apoptosis or expression of a dominant active form of caspase 3 showed stimulatory effect on EBV lytic replication as reflected by the ratio of GFP+ cells and level of EBV DNA. Finally, Z-DEVD-FMK, a highly specific inhibitor of caspase 3, ablated the stimulatory effect of 5FU or cisplatin. Similar findings were also obtained in NP460 nasopharyngeal epithelial cells and HK1 NPC cells infected with M81. Taken together, our work revealed that activation of caspase 3 facilitates EBV productive infection in epithelial cells. Our findings might also provide new strategies to improve the design of anti-EBV therapy.
Oral Talk #27
EBNA3A Regulates Apoptosis at Early and Late Times after
Infection in Epstein Barr Virus-Infected B Cells
Joanne Dai1, Micah Luftig1
1Department of Molecular Genetics and Microbiology, Duke University
Epstein-Barr virus (EBV) is a highly prevalent pathogen that establishes latent infection in human B cells. In vivo, primary infection occurs in the oral cavity where viral latency induces naïve B cells to proliferate and differentiate to access the long-lived memory B-cell compartment. In contrast, during in vitro infection, EBV-infected B cells are immortalized and form lymphoblastoid cell lines (LCLs), which require LMP1 for proliferation and survival. Our lab has found that early-infected B cells and LCLs are transcriptionally distinct and differ most notably in that early-infected B cells express low levels of LMP1 and NFκB targets, despite undergoing rapid hyperproliferation with low levels of apoptosis. To understand how early-infected B cells survive in the absence of LMP1, we performed BH3 profiling and found that uninfected B cells depend on BCL-2, early-infected B cells depend on MCL-1 and BCL-2, and LCLs depend on BFL-1 for survival. Through GSEA, we found that early-infected B cells are transcriptionally similar to germinal center (GC) centroblasts and that LCLs are similar to centrocytes. To determine if apoptosis regulation in EBV- infected B cells was similar to that in maturing B cells, we then performed BH3 profiling (?) of human B-cell subsets from tonsillectomies. We found that human GC B cells rely on MCL-1 for survival, suggesting that early, rapidly proliferating EBV-infected B cells utilize the same survival program found in GC B cells. In addition, we have found that the viral protein EBNA3A is required for both early MCL-1 activation and late BFL-1 expression. EBNA3A promotes the accumulation of MCL-1 at the mitochondria and regulates BFL-1 transcription by coordinating chromatin looping. In sum, we have discovered a novel viral-specific mechanism of apoptosis regulation that mimics survival programs in normal B-cell maturation. These findings have important implications for both EBV latency establishment and EBV-positive lymphoma cell survival.
EBNA3A Regulates Apoptosis at Early and Late Times after
Infection in Epstein Barr Virus-Infected B Cells
Joanne Dai1, Micah Luftig1
1Department of Molecular Genetics and Microbiology, Duke University
Epstein-Barr virus (EBV) is a highly prevalent pathogen that establishes latent infection in human B cells. In vivo, primary infection occurs in the oral cavity where viral latency induces naïve B cells to proliferate and differentiate to access the long-lived memory B-cell compartment. In contrast, during in vitro infection, EBV-infected B cells are immortalized and form lymphoblastoid cell lines (LCLs), which require LMP1 for proliferation and survival. Our lab has found that early-infected B cells and LCLs are transcriptionally distinct and differ most notably in that early-infected B cells express low levels of LMP1 and NFκB targets, despite undergoing rapid hyperproliferation with low levels of apoptosis. To understand how early-infected B cells survive in the absence of LMP1, we performed BH3 profiling and found that uninfected B cells depend on BCL-2, early-infected B cells depend on MCL-1 and BCL-2, and LCLs depend on BFL-1 for survival. Through GSEA, we found that early-infected B cells are transcriptionally similar to germinal center (GC) centroblasts and that LCLs are similar to centrocytes. To determine if apoptosis regulation in EBV- infected B cells was similar to that in maturing B cells, we then performed BH3 profiling (?) of human B-cell subsets from tonsillectomies. We found that human GC B cells rely on MCL-1 for survival, suggesting that early, rapidly proliferating EBV-infected B cells utilize the same survival program found in GC B cells. In addition, we have found that the viral protein EBNA3A is required for both early MCL-1 activation and late BFL-1 expression. EBNA3A promotes the accumulation of MCL-1 at the mitochondria and regulates BFL-1 transcription by coordinating chromatin looping. In sum, we have discovered a novel viral-specific mechanism of apoptosis regulation that mimics survival programs in normal B-cell maturation. These findings have important implications for both EBV latency establishment and EBV-positive lymphoma cell survival.