Session Chairs: Scott Tibbetts & Erik Flemington
Oral Talk #54
Misprocessing of Cellular Noncoding RNAs as a Pathway for
Cell-Intrinsic Immune Activation
Yang Zhao1, Xiang Ye1, William Dunker1, Yu Song1, John Karijolich1
1Vanderbilt University School of Medicine
The RIG-I like receptors (RLRs) RIG-I and MDA5 are cytosolic RNA helicases best characterized as restriction factors for RNA viruses. However, evidence suggests RLRs participate in innate immune recognition of other pathogens, including DNA viruses.Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic human gammaherpesvirus and the etiological agent of Kaposi’s sarcoma and primary effusion lymphoma (PEL). Here, we demonstrate that RIG-I and MDA5 restrict KSHV lytic reactivation in PEL. Furthermore, we define the in vivo RLR substrates and demonstrate that RIG-I and MDA5-mediated restriction is facilitated exclusively by the recognition of host derived RNAs. Misprocessed noncoding RNAs are prominent RIG-I bound RNAs, and biochemical characterizations reveal that an infection-dependent reduction in a key RNA processing enzyme results in an accumulation of triphosphorylated noncoding RNAs, enabling their recognition by RIG-I. These finds reveal an intricate relationship between RNA processing and innate immunity, and demonstrate that an antiviral innate immune response can be elicited by the sensing of misprocessed cellular RNAs.
Cell-Intrinsic Immune Activation
Yang Zhao1, Xiang Ye1, William Dunker1, Yu Song1, John Karijolich1
1Vanderbilt University School of Medicine
The RIG-I like receptors (RLRs) RIG-I and MDA5 are cytosolic RNA helicases best characterized as restriction factors for RNA viruses. However, evidence suggests RLRs participate in innate immune recognition of other pathogens, including DNA viruses.Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic human gammaherpesvirus and the etiological agent of Kaposi’s sarcoma and primary effusion lymphoma (PEL). Here, we demonstrate that RIG-I and MDA5 restrict KSHV lytic reactivation in PEL. Furthermore, we define the in vivo RLR substrates and demonstrate that RIG-I and MDA5-mediated restriction is facilitated exclusively by the recognition of host derived RNAs. Misprocessed noncoding RNAs are prominent RIG-I bound RNAs, and biochemical characterizations reveal that an infection-dependent reduction in a key RNA processing enzyme results in an accumulation of triphosphorylated noncoding RNAs, enabling their recognition by RIG-I. These finds reveal an intricate relationship between RNA processing and innate immunity, and demonstrate that an antiviral innate immune response can be elicited by the sensing of misprocessed cellular RNAs.
Oral Talk #55
Identifying EBV microRNA Targets in B-cell Lymphoma and
Gastric Cancer Using CLASH
Nathan Ungerleider1, Whitney Bullard2, Mehmet Kara2, Scott Tibbetts2, Erik Flemington1
1Tulane University, 2University of Florida
MicroRNAs are 20-25 base noncoding RNAs that post-transcriptionally regulate expression of target mRNAs. The Epstein Barr Virus genome codes for up to 25 microRNAs from two loci (BART1-22 and BHRF1-3), each of which targets a distinct subset of host transcripts. To comprehensively map EBV microRNAs to their host targets with high specificity, we performed crosslinking, ligation, and sequencing of hybrids (CLASH) in Akata (B-cell lymphoma; latent and reactivated conditions) and SNU719 (gastric cancer) cells. With this approach, we found a total of 4,643 unique human mRNA targets of EBV microRNAs (87, 148, and 4,627 from latent Akata, reactivated Akata, and SNU719, respectively.) Of these, 52 target transcripts were found in all three samples, including those coding for pro- apoptotic proteins, nuclear import proteins, and proteins involved in translation. Interestingly, 9.7% of EBV microRNA interactions with human mRNA were non-canonical and occurred in the absence of a seed match. By utilizing CLASH to explore the EBV microRNA interactome, we have uncovered and quantified interactions that are difficult or impossible to detect using prediction based methods.
Gastric Cancer Using CLASH
Nathan Ungerleider1, Whitney Bullard2, Mehmet Kara2, Scott Tibbetts2, Erik Flemington1
1Tulane University, 2University of Florida
MicroRNAs are 20-25 base noncoding RNAs that post-transcriptionally regulate expression of target mRNAs. The Epstein Barr Virus genome codes for up to 25 microRNAs from two loci (BART1-22 and BHRF1-3), each of which targets a distinct subset of host transcripts. To comprehensively map EBV microRNAs to their host targets with high specificity, we performed crosslinking, ligation, and sequencing of hybrids (CLASH) in Akata (B-cell lymphoma; latent and reactivated conditions) and SNU719 (gastric cancer) cells. With this approach, we found a total of 4,643 unique human mRNA targets of EBV microRNAs (87, 148, and 4,627 from latent Akata, reactivated Akata, and SNU719, respectively.) Of these, 52 target transcripts were found in all three samples, including those coding for pro- apoptotic proteins, nuclear import proteins, and proteins involved in translation. Interestingly, 9.7% of EBV microRNA interactions with human mRNA were non-canonical and occurred in the absence of a seed match. By utilizing CLASH to explore the EBV microRNA interactome, we have uncovered and quantified interactions that are difficult or impossible to detect using prediction based methods.
Nearly 10% of gastric tumor are EBV +
In EBV+gastric gastric tumors EBV miRNAs are most abundant
CLASH： crosslinkingm， ligation， ans sequencing of hybrid
EBV-miR targets in SNU719 cells
EBV-miR targets in Akata cells -> mostly about the same as those in SNU719
- EBV miRs have similar targets in BL (Akata) and gastric cancer (SNU719)
Example seed matches
- BAT3::IPO7
- BART7::GFPT1
- BART2::DAZAP2
EBV miRs have higher affinity for human transcripts than human miRs
Supplementary (3‘ of seed) complementarity can increase ternary AGO-miR-mRNA complex formation
miRNa affinity for targets and GO analysis
- viral carcinogenesis
- cell cycle
- protein procesing in ER
- adherent junction
Oral Talk #56
Transcriptional Regulation of Viral ncRNAs
Ashley N. Knox1, Eva Medina1, Lauren Oko1, Linda F.van Dyk1
1Department of Immunology & Microbiology, University of Colorado Denver | Anschutz Medical Campus
While viral RNA is known to play vital roles in viral infection and replication, the regulation of viral RNA polymerase III (pol III)-transcribed non-coding RNAs (ncRNAs) remains unclear. The gammaherpesviruses (γHVs) contain multiple ncRNAs that are highly expressed during infection. To study the contribution of pol III ncRNAs to viral infection, we have used a murine model of γHV (MHV68) that expresses pol III ncRNAs. Transcription by pol III imparts specific characteristics such as a 5’-triphosphate and 3’-oligouridylate sequence, which can be recognized and bound by host RNA-binding proteins. These RBP- RNA interactions may trigger innate immune signaling pathways that drive pathogenesis. Therefore, detailing how these viral ncRNAs are expressed is integral to understanding their role in pathogenesis. To compare the transcriptional regulation of pol III ncRNAs, we cloned several types of pol III promoters into a newly generated luciferase reporter we have optimized to measure pol III promoter activity during infection. Promoters include those of host ncRNAs (5S rRNA, tRNA, U6 snRNA), and of the MHV68 TMERs, the EBV EBERs, and the adenovirus VA RNAs. Infection with MHV68 drives differential expression of pol III transcription machinery, resulting in simultaneous repression of U6 promoter activity and stimulation of the MHV68 TMER promoters. This TMER promoter induction was prevented by inhibitors of viral replication and protein synthesis, indicating that these aspects of infection are vital for driving transcriptional regulation. This analysis of pol III promoter activity indicates shared and unique regulation patterns and identifies the critical promoter and polymerase features for preferential expression during infection.
Ashley N. Knox1, Eva Medina1, Lauren Oko1, Linda F.van Dyk1
1Department of Immunology & Microbiology, University of Colorado Denver | Anschutz Medical Campus
While viral RNA is known to play vital roles in viral infection and replication, the regulation of viral RNA polymerase III (pol III)-transcribed non-coding RNAs (ncRNAs) remains unclear. The gammaherpesviruses (γHVs) contain multiple ncRNAs that are highly expressed during infection. To study the contribution of pol III ncRNAs to viral infection, we have used a murine model of γHV (MHV68) that expresses pol III ncRNAs. Transcription by pol III imparts specific characteristics such as a 5’-triphosphate and 3’-oligouridylate sequence, which can be recognized and bound by host RNA-binding proteins. These RBP- RNA interactions may trigger innate immune signaling pathways that drive pathogenesis. Therefore, detailing how these viral ncRNAs are expressed is integral to understanding their role in pathogenesis. To compare the transcriptional regulation of pol III ncRNAs, we cloned several types of pol III promoters into a newly generated luciferase reporter we have optimized to measure pol III promoter activity during infection. Promoters include those of host ncRNAs (5S rRNA, tRNA, U6 snRNA), and of the MHV68 TMERs, the EBV EBERs, and the adenovirus VA RNAs. Infection with MHV68 drives differential expression of pol III transcription machinery, resulting in simultaneous repression of U6 promoter activity and stimulation of the MHV68 TMER promoters. This TMER promoter induction was prevented by inhibitors of viral replication and protein synthesis, indicating that these aspects of infection are vital for driving transcriptional regulation. This analysis of pol III promoter activity indicates shared and unique regulation patterns and identifies the critical promoter and polymerase features for preferential expression during infection.
viral ncRNAs=highly abundant transcripts in infected cells
rHV infection alters pol III activity
EBV infection upregulates TFIIIC, increased expression of EBERs and the transcripts from type 1a and 2 pol III promoters
EBV and KSHV upregulate host vault RNA expression increased apoptotic resistance and cell proliferation enhanced EBV establishment
Structure of pol III promoters
type 2 of Pol III promoters are used for viral ncRNA
Activity of pol III promoters can be measured using a luciferase reporter
- luciferase assay to measure promoter activity of both gene internal and external pol III promoters
- allow direct comparison of pol III promoters outside
- pol III transcription is stopped by TTTTTT stretch
Experimental design: measuring pol III promoter activiyt during rHV68 infection
infection with rHV68 differentially alters pol III promoter activity
- TMER 1 is the only one cellular promoters upregulated during rHV68 infection
- EBV is upregulated by rHV68 infection
- pol III opromoters respond differently to infection （using PAA or CHX
- inhibition of viral early gene expression attenuates changes in pol III promoter activity
What is the mechanism behind changes in pol III promtoer ctivity during infection？
- CHX
- inhibition of viral life cycle attenuates changes in pol III promoter activity
Next questions:
are differences among pol III type 2 promoters due to minimal promoters or require upstream regions？
Do diferences … to specific pol III subunits, transcription
Oral Talk #57
EBV Encoded miR-BART10-3p and miR-BART22 Promote
Invasion and Metastasis by Activating Canonical Wnt/β-catenin
Signaling Pathway in EBV-Associated Gastric Carcinoma
Min Dong1, Li-ping Gong2, Jian-ning Chen2, Xiao-fang Zhang2, Yi-wang Zhang2, Chun-kui Shao2
1Department of Medical Oncology, The Third Affiliated Hospital, Sun Yat-sen University, 2Department of Pathology, The Third Affiliated Hospital, Sun Yat-sen University
Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) accounts for approximately 10% of gastric carcinoma worldwide. Compared with other subtypes of gastric carcinoma, EBVaGC has distinct clinicopathological features and molecular abnormality, but the mechanisms of tumorigenesis and progression of EBVaGC are largely unknown. In recent years, an increasing number of studies have demonstrated that EBV- encoded microRNAs (EBV miRNAs) play important roles in the development and progression of EBV-associated tumors, including nasopharyngeal carcinoma and EBV- associated lymphoma. However, little has been reported about the expression and roles of EBV miRNAs in EBVaGC. In this study, we constructed a comprehensive profiling of EBV miRNAs expressed in EBVaGC by deep sequencing, and found ebv-miR-BART10-3p (BART10-3p) and ebv-miR-BART22 (BART22) were abundantly expressed. qRT-PCR analysis in 71 EBVaGC samples suggested that high expression of BART10-3p or BART22 was significantly associated with lymph node metastasis and worse 5-year overall survival. Furthermore, transwell assays demonstrated that both BART10-3p and BART22 enhanced cell migration and invasion. Bioinformatics analysis and luciferase reporter assay demonstrated that BART10-3p and BART22 directly target adenomatous polyposis coli (APC) and Dickkopf1 (DKK1). Forced expression of BART10-3p or BART22 in EBV- negative AGS and BGC823 cells upregulated the protein level of β-catenin and facilitated its transition from cytoplasm to nucleus thus activating the Wnt/β-catenin signaling pathway, and consequently upregulated the expression of Twist and downregulated the expression of E-cadherin. Reversely, reduced expression of endogenous BART10-3p or BART22 in EBV-positive SNU719 cells led to downregulation of β-catenin and Twist and upregulation of E-cadherin. These findings suggest that BART10-3p and BART22 play a vital role in promoting invasion and metastasis of EBVaGC by targeting APC and DKK1 and activating Wnt/β-catenin signaling pathway, providing novel prognostic biomarkers and therapeutic targets.
Invasion and Metastasis by Activating Canonical Wnt/β-catenin
Signaling Pathway in EBV-Associated Gastric Carcinoma
Min Dong1, Li-ping Gong2, Jian-ning Chen2, Xiao-fang Zhang2, Yi-wang Zhang2, Chun-kui Shao2
1Department of Medical Oncology, The Third Affiliated Hospital, Sun Yat-sen University, 2Department of Pathology, The Third Affiliated Hospital, Sun Yat-sen University
Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) accounts for approximately 10% of gastric carcinoma worldwide. Compared with other subtypes of gastric carcinoma, EBVaGC has distinct clinicopathological features and molecular abnormality, but the mechanisms of tumorigenesis and progression of EBVaGC are largely unknown. In recent years, an increasing number of studies have demonstrated that EBV- encoded microRNAs (EBV miRNAs) play important roles in the development and progression of EBV-associated tumors, including nasopharyngeal carcinoma and EBV- associated lymphoma. However, little has been reported about the expression and roles of EBV miRNAs in EBVaGC. In this study, we constructed a comprehensive profiling of EBV miRNAs expressed in EBVaGC by deep sequencing, and found ebv-miR-BART10-3p (BART10-3p) and ebv-miR-BART22 (BART22) were abundantly expressed. qRT-PCR analysis in 71 EBVaGC samples suggested that high expression of BART10-3p or BART22 was significantly associated with lymph node metastasis and worse 5-year overall survival. Furthermore, transwell assays demonstrated that both BART10-3p and BART22 enhanced cell migration and invasion. Bioinformatics analysis and luciferase reporter assay demonstrated that BART10-3p and BART22 directly target adenomatous polyposis coli (APC) and Dickkopf1 (DKK1). Forced expression of BART10-3p or BART22 in EBV- negative AGS and BGC823 cells upregulated the protein level of β-catenin and facilitated its transition from cytoplasm to nucleus thus activating the Wnt/β-catenin signaling pathway, and consequently upregulated the expression of Twist and downregulated the expression of E-cadherin. Reversely, reduced expression of endogenous BART10-3p or BART22 in EBV-positive SNU719 cells led to downregulation of β-catenin and Twist and upregulation of E-cadherin. These findings suggest that BART10-3p and BART22 play a vital role in promoting invasion and metastasis of EBVaGC by targeting APC and DKK1 and activating Wnt/β-catenin signaling pathway, providing novel prognostic biomarkers and therapeutic targets.
EBV associated gastric carcinoma, EBaGC has..
EBV miRNAs: 44 EBV-encoded miRNAs (ebv-miR-BARTs)
relateive expression levels of EBV miRNAs in SNU-719 and YCCEL1 cell lines
- BART10-3p and BART22 are highly expressed in EBVaGC
- Expressionof BART10-3p and BART22 in 71 EBVaGC cases
- forced expression of BART10 and BART22 increases EBaGC cell lines proliferation, invasion,migration
Enrichment analysis of predicated BART10-3p and BART22 targets in KEGG signaling pathway database
- APC and DKK1 inhibitors of WNT signaling are potential targets of BAR10-3p and BART22
Oral Talk #58
A Herpesvirus Utilizes a Sm-class Non-Coding RNAs as a
miRNA Adaptor to Modulate Host Gene Expression
Carlos Gorbea1, Tim Mosbruger2, Demian Cazalla1
1University of Utah, 2Children’s Hospital of Philadelphia
Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that infects T cells and causes aggressive lymphomas and leukemias in New World primates. HVS expresses seven Sm- class RNAs called HSURs (Herpesvirus saimiri U-rich RNAs). We have recently shown that one of these ncRNAs, HSUR 2, functions as a miRNA adaptor that base-pairs with host mRNAs and recruits host miR-142-3p and miR-16 to repress target mRNA expression1. Using this mechanism, HSUR 2 modulates the expression of several transcripts that encode proteins with roles in different cellular processes including apoptosis, regulation of cell cycle, and innate immune response1. It is currently not clear how HSUR 2 specifically targets this diverse group of mRNAs. Psoralen crosslinking experiments showed that interactions between HSUR 2 and target mRNAs involve direct base-pairing, strongly suggesting that complementarity between sequences present in HSUR 2 and target mRNAs may dictate target mRNA recognition and specificity.
We had previously developed a method termed RNA-RNA interaction identification by crosslinking and capture (RICC), which combines in vivo psoralen crosslinking and specific HSUR 2 capture using biotinylated probe hybridization, with high-throughput sequencing (seq) to identify crosslinked RNAs1. We have now successfully modified RICC-seq (iRICC- seq) to determine sequences involved in the interactions between HSUR 2 and its target mRNAs with individual nucleotide resolution. HSUR 2 binds primarily to the 3′ UTRs of target mRNAs at unique positions in some targets, but it can also bind some other targets at more than one position in their 3′ UTR. The data obtained with iRICC-seq also showed that HSUR 2 binds some of its targets using the same sequence that is used for binding miR-16, partially explaining why HSUR 2 does not require miR-16 activity to repress some of its targets1.
miRNA Adaptor to Modulate Host Gene Expression
Carlos Gorbea1, Tim Mosbruger2, Demian Cazalla1
1University of Utah, 2Children’s Hospital of Philadelphia
Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that infects T cells and causes aggressive lymphomas and leukemias in New World primates. HVS expresses seven Sm- class RNAs called HSURs (Herpesvirus saimiri U-rich RNAs). We have recently shown that one of these ncRNAs, HSUR 2, functions as a miRNA adaptor that base-pairs with host mRNAs and recruits host miR-142-3p and miR-16 to repress target mRNA expression1. Using this mechanism, HSUR 2 modulates the expression of several transcripts that encode proteins with roles in different cellular processes including apoptosis, regulation of cell cycle, and innate immune response1. It is currently not clear how HSUR 2 specifically targets this diverse group of mRNAs. Psoralen crosslinking experiments showed that interactions between HSUR 2 and target mRNAs involve direct base-pairing, strongly suggesting that complementarity between sequences present in HSUR 2 and target mRNAs may dictate target mRNA recognition and specificity.
We had previously developed a method termed RNA-RNA interaction identification by crosslinking and capture (RICC), which combines in vivo psoralen crosslinking and specific HSUR 2 capture using biotinylated probe hybridization, with high-throughput sequencing (seq) to identify crosslinked RNAs1. We have now successfully modified RICC-seq (iRICC- seq) to determine sequences involved in the interactions between HSUR 2 and its target mRNAs with individual nucleotide resolution. HSUR 2 binds primarily to the 3′ UTRs of target mRNAs at unique positions in some targets, but it can also bind some other targets at more than one position in their 3′ UTR. The data obtained with iRICC-seq also showed that HSUR 2 binds some of its targets using the same sequence that is used for binding miR-16, partially explaining why HSUR 2 does not require miR-16 activity to repress some of its targets1.
2018 International Conference on EBV & KSHV
1Gorbea, c. et al. Nature 550(7675):275-279 (2017).
HVS U-rich, Sm-class RBAs (HSURs) 超時太久!
Oral Talk #59
Impact of Demethylating Agents on Nasopharyngeal
Carcinoma Xenografts: De Novo Expression of Cp/Wp
Transcripts and BHRF1 microRNAs
Pierre Busson1, Nikifroros Kapetanakis1, Aaron Nguyen2
1UMR 8126 CNRS – Gustave Roussy, 2Celgene
Demethylating agents are expected to increase the immunogenicity of malignant cells by changing their protein profile; for example by increasing the expression of endogenous retroviral sequences. Inspired by this approach, we undertook to use 5 azacitidine (5 aza) and 5-aza-2’-deoxycytidine (Decitabine) for increasing the range and the abundance of EBV products in nasopharyngeal carcinoma (NPC) cells. We used the C666-1 NPC cell line which was initially derived from a primary tumor and which is propagated either in vitro or into nude mice as a cell-derived xenograft. We also used 3 patient-derived xenografts propagated into nude mice: C15, C17 and C18 which were derived from a primary tumor and two metastatic lesions, respectively. A tumor growth delay was recorded for all tumor lines with a much greater response for the C15 and C666-1, indicating that both agents have a cell-intrinsic inhibitory effect, especially in cells derived from primary tumors. Regarding viral protein expression, the most remarkable observation was a dose- dependent increase in EBNA1 and BZLF1 expression in the C666-1 and C18 tumors. Regarding viral microRNAs, we noted a de novo dose-dependent expression of the BHRF1 microRNAs with maximal levels in the C15 and C666-1 tumors, the most responsive in terms of tumor growth reduction. Plasma abundance of these microRNAs was correlated to their levels in the tumor. Simultaneously large transcripts originating from the Cp/Wp promoter were detected in the tumors. We also found some evidence of concomitant expression of the EBNA-LP protein in treated C666-1 cells (a point currently under verification). In conclusion, 5 Aza and decitabine have multiple effects on viral expression in malignant NPC cells including enhanced expression of immunogenic proteins and de novo expression of the BHRF1 microRNAs. Additional work will be required to investigate similar possible responses in malignant NPC cells in clinical specimens.
Carcinoma Xenografts: De Novo Expression of Cp/Wp
Transcripts and BHRF1 microRNAs
Pierre Busson1, Nikifroros Kapetanakis1, Aaron Nguyen2
1UMR 8126 CNRS – Gustave Roussy, 2Celgene
Demethylating agents are expected to increase the immunogenicity of malignant cells by changing their protein profile; for example by increasing the expression of endogenous retroviral sequences. Inspired by this approach, we undertook to use 5 azacitidine (5 aza) and 5-aza-2’-deoxycytidine (Decitabine) for increasing the range and the abundance of EBV products in nasopharyngeal carcinoma (NPC) cells. We used the C666-1 NPC cell line which was initially derived from a primary tumor and which is propagated either in vitro or into nude mice as a cell-derived xenograft. We also used 3 patient-derived xenografts propagated into nude mice: C15, C17 and C18 which were derived from a primary tumor and two metastatic lesions, respectively. A tumor growth delay was recorded for all tumor lines with a much greater response for the C15 and C666-1, indicating that both agents have a cell-intrinsic inhibitory effect, especially in cells derived from primary tumors. Regarding viral protein expression, the most remarkable observation was a dose- dependent increase in EBNA1 and BZLF1 expression in the C666-1 and C18 tumors. Regarding viral microRNAs, we noted a de novo dose-dependent expression of the BHRF1 microRNAs with maximal levels in the C15 and C666-1 tumors, the most responsive in terms of tumor growth reduction. Plasma abundance of these microRNAs was correlated to their levels in the tumor. Simultaneously large transcripts originating from the Cp/Wp promoter were detected in the tumors. We also found some evidence of concomitant expression of the EBNA-LP protein in treated C666-1 cells (a point currently under verification). In conclusion, 5 Aza and decitabine have multiple effects on viral expression in malignant NPC cells including enhanced expression of immunogenic proteins and de novo expression of the BHRF1 microRNAs. Additional work will be required to investigate similar possible responses in malignant NPC cells in clinical specimens.
NPC: Epigenetic contribution to the oncogenesis
EBV+ epithelial (type II latency)
Robust expression of BARTs, absence of BHRF1
EBV+ LCL (type III latency)
Abundant BHRFI, moderate BARTs
Demethylating agents
- 5-azacytidine
- 5-aza deoxy cytidine
Hypothesis:
variation in the profile of plasma viral muRNAs = surrogate markers of anti=tumor activity
Biological resources : EBV positive NPC models
C666-1 NPC cell line (propagated in vitro, also used a a xenografts (CDX)
NPC PDX C15, C17, C18
Impact of demethylating agents on the growth of NPC xenografts
5-Aza DAC: significant regression of tumor
Changes in EBV protein expression in xenogrageted NPC cells treated by 5-Aza
- C666-1 and C18, Zta and ___ are affected
MiRNAs
- Profiling of viral mIRNAs in the 4 NPC tumor models at baseline
- Changes in viral miRNA abundance in xenografts tumors and mouse plasma under treatment with 5-aka
- Consistent de nova detection of BHRF1 in tumors and mouse plasma samples
- De novo expression of transcripts reminiscent of latency III
- No concomitant detection f the BHRF1 protein
- Same sickle gin mechanism as in LCL? (Xiang and Keiff,m 20011)
- Possible detection of EBNA-LP
- Conclusions
- 5-aza induces de novo expression of miR BHRF1 in NPC cell line and PDX
- Concomitant BHRF1 expression
- Suggesting that epigenetic agents in NPC cells can modify that paternity of latent gene expression (not only the expression of lytic genes
- Detection of miR BHRF1 in cinical specimens requires further investigation
Oral Talk #60
The Secondary Structure of the MHV68 Noncoding RNA TMER4 is Essential for Function at a Critical in vivo Dissemination
Bottleneck
Brett A. Hoffman1, Emily R. Feldman1, Yiping Wang1, Scott A.Tibbetts1
1University of Florida
The ability of EBV and KSHV to establish lifelong latency is a critical factor in their ability to cause pathogenesis; however, the precise determinants that mediate in vivo latency remain unclear due to the strict species specificity of these viruses. Murine gammaherpesvirus 68 (MHV68) infection of laboratory mice is a robust system to model natural in vivo gammaherpesvirus infection. Like EBV and KSHV, MHV68 establishes lifelong latency in B cells and is associated with the development of lymphoproliferative disorders and lymphomas. Using this system we have previously demonstrated a crucial in vivo role for the viral noncoding RNA TMER4 in MHV68 dissemination and establishment of peripheral latency. TMER4 is a 203 nt RNA that is comprised of a tRNA-like element plus two pre-miRNA stem-loops, encoding up to four mature miRNAs. We sought to define the component of TMER4 required for this critical in vivo function. As expected, in infected cells both full-length TMER4 and TMER4-derived mature miRNAs were both readily detectable. However, an approximately 145 nt species corresponding to the size of the vtRNA plus a single stem-loop was the most stable form of TMER4 present in infected cells. Interestingly, mutation of either of the miRNA seed sequences had no effect on TMER4 activity in vivo, demonstrating that it functions independent of miRNA targeting. Additional mutagenesis of the TMER4 structure in the context of the virus revealed that stem-loop secondary structure is essential for activity, but that full-length stem-loop is not required. These findings are now aiding in additional mechanistic studies to determine the specific molecular function of TMER4 at this critical in vivo bottleneck.
Bottleneck
Brett A. Hoffman1, Emily R. Feldman1, Yiping Wang1, Scott A.Tibbetts1
1University of Florida
The ability of EBV and KSHV to establish lifelong latency is a critical factor in their ability to cause pathogenesis; however, the precise determinants that mediate in vivo latency remain unclear due to the strict species specificity of these viruses. Murine gammaherpesvirus 68 (MHV68) infection of laboratory mice is a robust system to model natural in vivo gammaherpesvirus infection. Like EBV and KSHV, MHV68 establishes lifelong latency in B cells and is associated with the development of lymphoproliferative disorders and lymphomas. Using this system we have previously demonstrated a crucial in vivo role for the viral noncoding RNA TMER4 in MHV68 dissemination and establishment of peripheral latency. TMER4 is a 203 nt RNA that is comprised of a tRNA-like element plus two pre-miRNA stem-loops, encoding up to four mature miRNAs. We sought to define the component of TMER4 required for this critical in vivo function. As expected, in infected cells both full-length TMER4 and TMER4-derived mature miRNAs were both readily detectable. However, an approximately 145 nt species corresponding to the size of the vtRNA plus a single stem-loop was the most stable form of TMER4 present in infected cells. Interestingly, mutation of either of the miRNA seed sequences had no effect on TMER4 activity in vivo, demonstrating that it functions independent of miRNA targeting. Additional mutagenesis of the TMER4 structure in the context of the virus revealed that stem-loop secondary structure is essential for activity, but that full-length stem-loop is not required. These findings are now aiding in additional mechanistic studies to determine the specific molecular function of TMER4 at this critical in vivo bottleneck.
MHV68: TMERs tRNA -miRNA-encoding RNAs)
8 TMERAS in MHV68 N terminus
TMER4 is required for heamato- dissemination
- Dramatic attenuation in the blood samples, suggesting TMER4 is involved in virus egress from eh lymph node
- TMER4 function is independent of miRNAs (change stem loop TMER4 still work fine)
- Stem-loop II is required for function
- Stem-loop size not critical
Is TMER4 function conserved among gammaherpesivruese?
- ~150 Nat TMER4 species is predominantly expressed and functional
- Function is
- EBERs!
- Both TMER4 and EBERs are highly abundant
- Can EBER1 expression rescue TMER4 in vivo function, yes! Strongly suggests a conserved in vivo function for MHV68 TMER4 and EBV EBER1
Questions:
In vivo B memory cells dropped, T cells not analyzed yet
Oral Talk #61
Epstein Barr Virus CircRNAome
Epstein Barr Virus CircRNAome
Nathan Ungerleider1, Monica Concha1, Zhen Lin1, Claire Roberts1, Xia Wang1, Subing Cao1, Walter Moss3, Yi Yu1, Scott Tibbetts2, Rolf Renne2, Yan Dong1, Erik Flemington1
1Tulane University, 2University of Florida, 3Iowa State University
Our appreciation for the extent of Epstein Barr virus (EBV) transcriptome complexity continues to grow through findings of EBV encoded microRNAs, new long non-coding RNAs as well as more recent findings of over a hundred newly discovered polyadenylated lytic transcripts. Here, we used RNase R-sequencing to elucidate the EBV circular RNAome (circRNAome). By performing RNase R-sequencing in cell models representing type I, II, and III latency and during reactivation, we identified EBV encoded circular RNAs expressed from latency Cp driven transcription, from latency expression of the long non- coding RPMS1 locus, from within the LMP2 gene, and we identified a highly expressed circular RNA derived from intragenic back-splicing of the BHLF1 gene. While expression of most of these circular RNAs correlate with the expression of the respective linear transcripts, circLMP2 is uniquely expressed during reactivation. Here we present the exonic structures and the nuclear/cytoplasmic localization of EBV encoded circular RNAs and show initial insights into phenotypic alterations mediated by one of these circular RNAs.
1Tulane University, 2University of Florida, 3Iowa State University
Our appreciation for the extent of Epstein Barr virus (EBV) transcriptome complexity continues to grow through findings of EBV encoded microRNAs, new long non-coding RNAs as well as more recent findings of over a hundred newly discovered polyadenylated lytic transcripts. Here, we used RNase R-sequencing to elucidate the EBV circular RNAome (circRNAome). By performing RNase R-sequencing in cell models representing type I, II, and III latency and during reactivation, we identified EBV encoded circular RNAs expressed from latency Cp driven transcription, from latency expression of the long non- coding RPMS1 locus, from within the LMP2 gene, and we identified a highly expressed circular RNA derived from intragenic back-splicing of the BHLF1 gene. While expression of most of these circular RNAs correlate with the expression of the respective linear transcripts, circLMP2 is uniquely expressed during reactivation. Here we present the exonic structures and the nuclear/cytoplasmic localization of EBV encoded circular RNAs and show initial insights into phenotypic alterations mediated by one of these circular RNAs.
Transcriptome resukltion throughinterartion f multi platform data TRIMD – O’Grady et al NAR 2016
Global EBV polyA lytic Transcriptome analysis (found new transcripts!)
Virus non-coding RNAs effector of hose cell remodeling with minimal immunological impact
- EBERs I and 2
- RPMS2 (lncRNA)
- MiRNAs
- circRNA?
RNAase R-seq EBV middles
Cell type I )Akata, Mutu I, Sav I)
B cell (type III) Mutu III IB4, Jijoye, JY
Type II (SNU719 and. YCCEL1)
Importance of RNase R seq versus Ribo-depletion R-seq
Backsplice read enrichment distribution by using RNase R-seq
EBV circRNA candidates
Type III latency
- W1_C1
- LMP2 E8_E2 identified only in reactivation
- BHRF1 also abundant
Viral CircRNA similar to cellular circRNAs
Latency