Aug 24, 2018
Aug 3, 2018
2018 Madison Meeting Index Page
July 28 Saturday
Keynote Address 1 (Henle Lecture: Paul Lieberman)
July 29 Sunday
Keynote Address 1 (Henle Lecture: Paul Lieberman)
July 29 Sunday
- Global Changes in the Host B cell During the Pre-latent Phase of EBV Infection (Paulina Mrozek-Gorska)
- Single-Cell Analysis of Gammaherpesvirus Infection and Gene Expression (Lauren Oko)
- 3D architecture of host-pathogen genomic interactions during EBV reactivation (JJ Miranda)
- Modulation of Cellular CpG DNA Methylation by Kaposi’s Sarcoma Associated Herpesvirus (Guy Journo)
- Epstein-Barr Virus Nuclear Antigen EBNA-LP is Essential for Transforming Naive B Cells, and Supports the Activation of Viral Genes, but not EBNA2-Induced Cellular genes (Rob E. White)
- Polycomb Recruitment During Latency Establishment: A Comparative Study (Thomas Günther)
Aug 1, 2018
Joint Session #4
Session Chairs: Laurie Krug & Bettie Steinberg
See notes ^o^ (Pat Moore and Yuan Chang Lab)
Circular RNA
Lineage transcripts
Potentials of KSHV circular RNAs
High-risk E7 enable proteasome-mediated degradation of PTPN14
PTPN14-E7-UBR4
UBR4 required for transformation by E7
Allows E7 to inhibit apoptosis following detachment (?)
PTPN14
PTPN14/YAP/TEAD
Can’t reproduce the experiments, went to YAP expert, the YAP expert said: the situation got to be complicated XDD
Oral Talk #115
Virology. 2016 Aug;495:52-62. doi: 10.1016/j.virol.2016.05.005. Epub 2016 May 11.
Oral Talk #116
OPC, 90% OPC occur in tonsils
PLoS Pathog. 2015 Oct 2;11(10):e1005195. doi: 10.1371/journal.ppat.1005195. eCollection 2015 Oct.
Oral Talk #117
PARP Pol
CTCF-PARlyation
Oral Talk #112
Discovery and Characterization of Human γHerpesvirus-
Circular RNAs
Y Chang, N Lee3, PS Moore1,3, MA Nalesnik2, S Ranganathan2, KH Shair3, SH Swerdlow2, Tuna Toptan1,2, J Wendzicki1
1Hillman Cancer Center, 2University of Pittsburgh Medical Center, 3Department of Microbiology and Molecular Genetics
CircRNAs are formed by backsplicing of linear RNA and can be exonic and/or intronic. Due to their covalently joined circular nature, circRNAs are resistant to exonucleases and therefore highly stable. To date no circRNAs have been described from DNA tumor viruses. We performed RNA sequencing on exonuclease (RNaseR) treated EBV positive and negative post-transplant lymphoproliferative disease (PTLD) patient samples and identified EBV backspliced junction reads within the RPMS1 locus only in EBV positive PTLD. These junction reads were determined to be from circular RNAs by RT-PCR using divergent primers. They comprise 4 differentially spliced isoforms that vary in size based on specific combinations of intronic and exonic elements. In extended sampling, these viral circRNAs were found in all 8 EBV+ but none of 9 EBV- PTLD tested. We also verified the presence of circRPMS1 in all EBV+ cell lines tested, except in the B95-8 line, which harbors a deletion in this locus. CircRPMS1 forms are present during both latent and lytic replication, although we detected some variation in relative quantity from cell line to cell line. Furthermore, circRNAseq analysis of KSHV-infected PEL cell lines revealed several species of KSHV circRNAs, which can also be detected in KS lesions. Gammaherpesviruses generate circRNAs as a novel class of viral transcripts.
Circular RNAs
Y Chang, N Lee3, PS Moore1,3, MA Nalesnik2, S Ranganathan2, KH Shair3, SH Swerdlow2, Tuna Toptan1,2, J Wendzicki1
1Hillman Cancer Center, 2University of Pittsburgh Medical Center, 3Department of Microbiology and Molecular Genetics
CircRNAs are formed by backsplicing of linear RNA and can be exonic and/or intronic. Due to their covalently joined circular nature, circRNAs are resistant to exonucleases and therefore highly stable. To date no circRNAs have been described from DNA tumor viruses. We performed RNA sequencing on exonuclease (RNaseR) treated EBV positive and negative post-transplant lymphoproliferative disease (PTLD) patient samples and identified EBV backspliced junction reads within the RPMS1 locus only in EBV positive PTLD. These junction reads were determined to be from circular RNAs by RT-PCR using divergent primers. They comprise 4 differentially spliced isoforms that vary in size based on specific combinations of intronic and exonic elements. In extended sampling, these viral circRNAs were found in all 8 EBV+ but none of 9 EBV- PTLD tested. We also verified the presence of circRPMS1 in all EBV+ cell lines tested, except in the B95-8 line, which harbors a deletion in this locus. CircRPMS1 forms are present during both latent and lytic replication, although we detected some variation in relative quantity from cell line to cell line. Furthermore, circRNAseq analysis of KSHV-infected PEL cell lines revealed several species of KSHV circRNAs, which can also be detected in KS lesions. Gammaherpesviruses generate circRNAs as a novel class of viral transcripts.
See notes ^o^ (Pat Moore and Yuan Chang Lab)
Oral Talk #113
Human and Viral Circular RNAs during Infection
Takanobu Tagawa1, Joseph Ziegelbauer1
1HIV and AIDS Malignancy Branch, National Institute of Health
Non-coding RNAs have wide implications in host-virus interactions; Herpesvirus-encoded miRNAs reduce apoptosis, promote cell cycle and growth, and reduce immunogenicity; PAN RNA in KSHV and EBERs in EBV are involved in successful viral infection. Circular RNAs (circRNA) are novel single-stranded non-coding RNAs with long half-lives, sequence conservation, localization in cytoplasm, and abundant expression. CircRNAs are products of back-splicing and potential to antagonize RNA binding proteins, mRNAs, and miRNAs in sequence-dependent manner. Human circRNAs have been shown to inhibit human miRNAs.
We hypothesized that circRNAs influence viral infection by inhibiting host and/or viral factors. Viral miRNAs are particularly attractive targets of circRNAs since viral miRNAs are produced by same mechanism of human miRNAs and less likely to be distinguished through synthetic pathway. We found that many human circRNAs are enriched with predicted viral miRNA target sites. Screening with RNA-Seq analysis of KSHV-infected Human Umbilical Vein Endothelial Cells (HUVEC) identified hundreds of human circRNAs that are differentially regulated upon infection. We confirmed the expression changes with RT-qPCR and RNase R treatment. We are pursuing gain and loss of specific circRNAs to investigate their effects on infection.
Furthermore, we examined whether herpesviruses express their own circRNAs as in the case of viral miRNAs. We have identified multiple KSHV circRNAs expressed in KSHV- infected endothelial cells and primary effusion lymphoma cells. Upon the induction of lytic cycle, some viral circRNAs are up-regulated. To our knowledge, circRNAs have not yet been reported for sub-genomic viral RNAs.
We are continuing to investigate the functional roles of human and viral circRNAs in inhibiting or benefitting infection. Both miRNAs and circRNAs are by nature non- immunogenic and long-lived compared to conventional linear RNA molecules. This research has revealed new host-viral interactions with circRNAs that may play key roles for successful infection by herpesvirus.
Takanobu Tagawa1, Joseph Ziegelbauer1
1HIV and AIDS Malignancy Branch, National Institute of Health
Non-coding RNAs have wide implications in host-virus interactions; Herpesvirus-encoded miRNAs reduce apoptosis, promote cell cycle and growth, and reduce immunogenicity; PAN RNA in KSHV and EBERs in EBV are involved in successful viral infection. Circular RNAs (circRNA) are novel single-stranded non-coding RNAs with long half-lives, sequence conservation, localization in cytoplasm, and abundant expression. CircRNAs are products of back-splicing and potential to antagonize RNA binding proteins, mRNAs, and miRNAs in sequence-dependent manner. Human circRNAs have been shown to inhibit human miRNAs.
We hypothesized that circRNAs influence viral infection by inhibiting host and/or viral factors. Viral miRNAs are particularly attractive targets of circRNAs since viral miRNAs are produced by same mechanism of human miRNAs and less likely to be distinguished through synthetic pathway. We found that many human circRNAs are enriched with predicted viral miRNA target sites. Screening with RNA-Seq analysis of KSHV-infected Human Umbilical Vein Endothelial Cells (HUVEC) identified hundreds of human circRNAs that are differentially regulated upon infection. We confirmed the expression changes with RT-qPCR and RNase R treatment. We are pursuing gain and loss of specific circRNAs to investigate their effects on infection.
Furthermore, we examined whether herpesviruses express their own circRNAs as in the case of viral miRNAs. We have identified multiple KSHV circRNAs expressed in KSHV- infected endothelial cells and primary effusion lymphoma cells. Upon the induction of lytic cycle, some viral circRNAs are up-regulated. To our knowledge, circRNAs have not yet been reported for sub-genomic viral RNAs.
We are continuing to investigate the functional roles of human and viral circRNAs in inhibiting or benefitting infection. Both miRNAs and circRNAs are by nature non- immunogenic and long-lived compared to conventional linear RNA molecules. This research has revealed new host-viral interactions with circRNAs that may play key roles for successful infection by herpesvirus.
Circular RNA
- Product of back splicing
- Resistant to ending lease
- Identifying back-spploiced junctions
- Divergent primer PCR
- Resistant to RNase R
- KSHV infection up-regulation specific human circRNAs in endothelial cells
- SLK 3 days post infection
- Hsa_circ_0001400 suppresses latent and lytic gene expressions
- RTA
- But viral genome numbers are not affected
- Arise from lytic gene locus in unfected HUVECs
- PAN and ORF56!!
Lineage transcripts
- LANA
- RTA
- Kcirc3, 29, 57
Potentials of KSHV circular RNAs
- Decoy of antiviral RNAs and RBPs
- Long-lasting and cost effective
- Adaptive immune evasion
- Refjuction of lytic gene expression
- Spread via extracellular vehicles
- KSHV circRNAs May avoid recognition by RIG-1 (Chen et al, 2017)
Oral Talk #114
High-Risk HPV E7 Proteins Degrade the Tumor Suppressor
PTPN14 to Limit Keratinocyte Differentiation
Joshua Hatterschide1, Amelia E. Bohidar1, Elizabeth A. White1
1University of Pennsylvania Perelman School of Medicine
High-risk human papillomavirus (HPV) E7 oncoproteins have a transformation activity that is independent of their binding to the retinoblastoma tumor suppressor (RB1), but the molecular basis of this activity remains uncertain. We have previously determined that PTPN14, a non-receptor protein tyrosine phosphatase and putative tumor suppressor, interacts with many HPV E7 proteins. Oncogenic HPV E7 form a complex that includes the E3 ligase UBR4 and PTPN14 and use UBR4 to target PTPN14 for proteasome-mediated degradation. The ability of E7 to bind PTPN14 is correlated with its oncogenic activity in several cellular transformation assays.
We have continued to investigate the effects of PTPN14 degradation by E7 and the effects of PTPN14 knockdown or knockout in the absence of E7. We have characterized an HPV16 E7 variant that cannot bind to UBR4 but that retains the ability to inactivate RB1. Using this variant in primary keratinocyte immortalization assays has enabled us to separate the pro-proliferative effect of RB1 inactivation by HPV16 E7 from the effect of PTPN14 degradation. Gene expression analyses in cells producing HPV16 E7 or an E7 variant, or in cells depleted of PTPN14, suggest that PTPN14 promotes the expression of keratinocyte differentiation genes. We hypothesize that the degradation of PTPN14 by high-risk HPV E7 limits keratinocyte differentiation, and we are testing whether this is related to the RB1-independent transformation activity of high-risk HPV E7.
PTPN14 has been characterized as a negative regulator of YAP in the Hippo signaling pathway, but we find that a reduction in PTPN14 levels does not cause activation of pro- proliferative YAP target genes in human keratinocytes. We are testing whether the regulation of differentiation-related genes by PTPN14 is YAP-dependent or YAP- independent and whether this regulation requires the phosphatase activity of PTPN14. These experiments and other aspects of our studies will be discussed.
PTPN14 to Limit Keratinocyte Differentiation
Joshua Hatterschide1, Amelia E. Bohidar1, Elizabeth A. White1
1University of Pennsylvania Perelman School of Medicine
High-risk human papillomavirus (HPV) E7 oncoproteins have a transformation activity that is independent of their binding to the retinoblastoma tumor suppressor (RB1), but the molecular basis of this activity remains uncertain. We have previously determined that PTPN14, a non-receptor protein tyrosine phosphatase and putative tumor suppressor, interacts with many HPV E7 proteins. Oncogenic HPV E7 form a complex that includes the E3 ligase UBR4 and PTPN14 and use UBR4 to target PTPN14 for proteasome-mediated degradation. The ability of E7 to bind PTPN14 is correlated with its oncogenic activity in several cellular transformation assays.
We have continued to investigate the effects of PTPN14 degradation by E7 and the effects of PTPN14 knockdown or knockout in the absence of E7. We have characterized an HPV16 E7 variant that cannot bind to UBR4 but that retains the ability to inactivate RB1. Using this variant in primary keratinocyte immortalization assays has enabled us to separate the pro-proliferative effect of RB1 inactivation by HPV16 E7 from the effect of PTPN14 degradation. Gene expression analyses in cells producing HPV16 E7 or an E7 variant, or in cells depleted of PTPN14, suggest that PTPN14 promotes the expression of keratinocyte differentiation genes. We hypothesize that the degradation of PTPN14 by high-risk HPV E7 limits keratinocyte differentiation, and we are testing whether this is related to the RB1-independent transformation activity of high-risk HPV E7.
PTPN14 has been characterized as a negative regulator of YAP in the Hippo signaling pathway, but we find that a reduction in PTPN14 levels does not cause activation of pro- proliferative YAP target genes in human keratinocytes. We are testing whether the regulation of differentiation-related genes by PTPN14 is YAP-dependent or YAP- independent and whether this regulation requires the phosphatase activity of PTPN14. These experiments and other aspects of our studies will be discussed.
High-risk E7 enable proteasome-mediated degradation of PTPN14
PTPN14-E7-UBR4
UBR4 required for transformation by E7
Allows E7 to inhibit apoptosis following detachment (?)
PTPN14
- Non receptor tyrosine phosphatase
- Tumor suppressor in colorectal other cancers
- Relapse neuroblastoma
- Skin basal cell carcinoma
- PDAC
PTPN14/YAP/TEAD
Can’t reproduce the experiments, went to YAP expert, the YAP expert said: the situation got to be complicated XDD
Oral Talk #115
Human Papillomavirus E7 Oncoprotein Promotes the Epstein-
Barr Virus Lytic Life Cycle in Immortalized Oral Keratinocytes
Kathleen Makielski1, Denis Lee1, Mitchell Hayes1, Bill Sugden1, Paul F. Lambert1
1University of Wisconsin-Madison
Epstein-Barr virus (EBV) and human papillomaviruses (HPVs) are human tumor viruses that cause head and neck cancers. Both viruses infect and replicate in upper aerodigestive tract epithelia. Some studies have detected co-infection in both oropharyngeal and nasopharyngeal cancers raising the possibility that these viruses could potentially affect each other’s life cycles and/or oncogenic potential. Our lab has established an in vitro model system using organotypic raft cultures to test the effects of EBV and HPV on each other in stratified squamous oral epithelial cells. We found that the presence of HPV promotes EBV’s lytic life cycle in suprabasal layers of the oral epithelium. Specifically, HPV promotes expression of an EBV immediate-early protein, Z, EBV genome amplification, and EBV infectious virus production in raft cultures. HPV’s effects on EBV’s lytic life cycle correlate with the ability of the high-risk HPV oncoprotein, E7, to bind pocket proteins. However, E7’s ability to degrade pocket proteins as well as E7’s ability to specifically bind Rb are not necessary for its promotion of the EBV lytic life cycle. Ongoing studies designed to reveal which pocket protein(s) are critical targets in mediating HPV E7 oncoprotein’s effect on the EBV life cycle will shed further light on what controls the lytic phase of the EBV life cycle in human oral epithelial cells.
Barr Virus Lytic Life Cycle in Immortalized Oral Keratinocytes
Kathleen Makielski1, Denis Lee1, Mitchell Hayes1, Bill Sugden1, Paul F. Lambert1
1University of Wisconsin-Madison
Epstein-Barr virus (EBV) and human papillomaviruses (HPVs) are human tumor viruses that cause head and neck cancers. Both viruses infect and replicate in upper aerodigestive tract epithelia. Some studies have detected co-infection in both oropharyngeal and nasopharyngeal cancers raising the possibility that these viruses could potentially affect each other’s life cycles and/or oncogenic potential. Our lab has established an in vitro model system using organotypic raft cultures to test the effects of EBV and HPV on each other in stratified squamous oral epithelial cells. We found that the presence of HPV promotes EBV’s lytic life cycle in suprabasal layers of the oral epithelium. Specifically, HPV promotes expression of an EBV immediate-early protein, Z, EBV genome amplification, and EBV infectious virus production in raft cultures. HPV’s effects on EBV’s lytic life cycle correlate with the ability of the high-risk HPV oncoprotein, E7, to bind pocket proteins. However, E7’s ability to degrade pocket proteins as well as E7’s ability to specifically bind Rb are not necessary for its promotion of the EBV lytic life cycle. Ongoing studies designed to reveal which pocket protein(s) are critical targets in mediating HPV E7 oncoprotein’s effect on the EBV life cycle will shed further light on what controls the lytic phase of the EBV life cycle in human oral epithelial cells.
Virology. 2016 Aug;495:52-62. doi: 10.1016/j.virol.2016.05.005. Epub 2016 May 11.
Human papillomavirus promotes Epstein-Barr virus maintenance and lytic reactivation in immortalized oral keratinocytes.
Abstract
Epstein-Barr virus and human papillomaviruses are human tumor viruses that infect and replicate in upper aerodigestive tract epithelia and cause head and neck cancers. The productive phases of both viruses are tied to stratified epithelia highlighting the possibility that these viruses may affect each other's life cycles. Our lab has established an in vitro model system to test the effects of EBV and HPV co-infection in stratified squamous oral epithelial cells. Our results indicate that HPV increases maintenance of the EBV genome in the co-infected cells and promotes lytic reactivation of EBV in upper layers of stratified epithelium. Expression of the HPV oncogenes E6 and E7 were found to be necessary and sufficient to account for HPV-mediated lytic reactivation of EBV. Our findings indicate that HPV increases the capacity of epithelial cells to support the EBV life cycle, which could in turn increase EBV-mediated pathogenesis in the oral cavity. (PMID 27179345)
Oral Talk #116
Inhibition of Epstein-Barr Virus Replication in Human
Papillomavirus-Immortalized Keratinocytes
Joseph T Guidry1,2,3, Julia E Myers1,2, William K Songock1,2, Malgorzata Bienkowska-Haba1,2, Xiaohui Ma3, Cherie-Ann O Nathan3,4, Martin Sapp1,2,3, Jason M Bodily1,2,3, Rona S Scott1,2,3 1Department of Microbiology and Immunology, LSU Health Sciences Center, 2Center for Molecular and Tumor Virology, LSU Health Sciences Center, 3Feist-Weiller Cancer Center, LSU Health Sciences Center, 4Department of Otolaryngology, LSU Health Sciences Center
The detection of the oncogenic herpesvirus Epstein-Barr virus (EBV) in human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OSCC) implies a possible cofactorial role for EBV alongside HPV in OSCC pathogenesis. As EBV- associated carcinomas exhibit latent EBV infection, we hypothesized that HPV may delay epithelial differentiation required for EBV replication and promote EBV latency. To test our hypothesis, HPV16-transfected tonsillar keratinocytes grown in organotypic raft culture were infected via co-culture with EBV-positive Akata Burkitt’s lymphoma cells. Six days post-EBV infection, robust EBV replication was observed in HPV-negative tonsillar rafts, with little to no replication in HPV16-immortalized rafts. The reduction in EBV replication was independent of immortalization as human telomerase-immortalized normal oral keratinocytes supported robust EBV replication in raft culture. To identify the HPV factors responsible, human foreskin keratinocytes (HFK) expressing HPV16 oncogenes E6 and/or E7 showed that HPV16 E7 was sufficient to reduce EBV replication. EBV is dependent upon epithelial differentiation and the differentiation-dependent expression of the cellular transcription factor KLF4 for replication. While KLF4 RNA and protein levels were unaltered in HPV16-transfected or E6/E7-expressing rafts, several KLF4 cellular targets, including differentiation markers filaggrin and loricrin, were reduced. However, expression of the EBV immediate early genes, viral targets transcriptionally activated by KLF4, was not affected by HPV16. Instead, a block in EBV early gene expression was observed. These results suggest that HPV interference with EBV replication was independent of KLF4 activity on viral gene expression. Instead, HPV16 E7 interactions with retinoblastoma pocket proteins may result in a cellular reprogramming that is no longer conducive for EBV replication. HPV16 E7-mediated interruption of EBV replication may facilitate a latent EBV infection which would provide additional viral oncogene expression and contribute to the rapid progression of HPV+ OSCC.
Papillomavirus-Immortalized Keratinocytes
Joseph T Guidry1,2,3, Julia E Myers1,2, William K Songock1,2, Malgorzata Bienkowska-Haba1,2, Xiaohui Ma3, Cherie-Ann O Nathan3,4, Martin Sapp1,2,3, Jason M Bodily1,2,3, Rona S Scott1,2,3 1Department of Microbiology and Immunology, LSU Health Sciences Center, 2Center for Molecular and Tumor Virology, LSU Health Sciences Center, 3Feist-Weiller Cancer Center, LSU Health Sciences Center, 4Department of Otolaryngology, LSU Health Sciences Center
The detection of the oncogenic herpesvirus Epstein-Barr virus (EBV) in human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OSCC) implies a possible cofactorial role for EBV alongside HPV in OSCC pathogenesis. As EBV- associated carcinomas exhibit latent EBV infection, we hypothesized that HPV may delay epithelial differentiation required for EBV replication and promote EBV latency. To test our hypothesis, HPV16-transfected tonsillar keratinocytes grown in organotypic raft culture were infected via co-culture with EBV-positive Akata Burkitt’s lymphoma cells. Six days post-EBV infection, robust EBV replication was observed in HPV-negative tonsillar rafts, with little to no replication in HPV16-immortalized rafts. The reduction in EBV replication was independent of immortalization as human telomerase-immortalized normal oral keratinocytes supported robust EBV replication in raft culture. To identify the HPV factors responsible, human foreskin keratinocytes (HFK) expressing HPV16 oncogenes E6 and/or E7 showed that HPV16 E7 was sufficient to reduce EBV replication. EBV is dependent upon epithelial differentiation and the differentiation-dependent expression of the cellular transcription factor KLF4 for replication. While KLF4 RNA and protein levels were unaltered in HPV16-transfected or E6/E7-expressing rafts, several KLF4 cellular targets, including differentiation markers filaggrin and loricrin, were reduced. However, expression of the EBV immediate early genes, viral targets transcriptionally activated by KLF4, was not affected by HPV16. Instead, a block in EBV early gene expression was observed. These results suggest that HPV interference with EBV replication was independent of KLF4 activity on viral gene expression. Instead, HPV16 E7 interactions with retinoblastoma pocket proteins may result in a cellular reprogramming that is no longer conducive for EBV replication. HPV16 E7-mediated interruption of EBV replication may facilitate a latent EBV infection which would provide additional viral oncogene expression and contribute to the rapid progression of HPV+ OSCC.
OPC, 90% OPC occur in tonsils
PLoS Pathog. 2015 Oct 2;11(10):e1005195. doi: 10.1371/journal.ppat.1005195. eCollection 2015 Oct.
Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.
Nawandar DM1, Wang A1, Makielski K2, Lee D2, Ma S2, Barlow E2, Reusch J1, Jiang R3, Wille CK4, Greenspan D5, Greenspan JS5, Mertz JE2, Hutt-Fletcher L3, Johannsen EC6, Lambert PF2, Kenney SC6.
Abstract
Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells. (PMID:26431332)
Oral Talk #117
EBV Hijacks PARP1 Activity to Control Both the Viral and
Cellular Epigenome
Lena Lupey - Green1, Lisa Caruso1, Kayla Martin1, Michael Hulse1, Jozef Madzo1, Sarah Johnson1, Italo Tempera1,
1Fels Institute for Cancer Research - School of Medicine Temple University
EBV establishes a latent infection with complex and dynamic gene expression patterns. These gene expression patterns can adapt to diverse host cell types and immunological responses. The changes in viral gene expression are regulated by both viral and cellular factors, including epigenetic modulators. We previously discovered that an important epigenetic feature of EBV is the three-dimensional (3D) structure of the viral chromosome. Changes in EBV 3D chromatin structure correlates with different patterns of gene expression. The primary factor that mediates the 3D genome structure in EBV is CTCF. Our recent work reveals that EBV activates PARP1, which has been shown to be important in regulating CTCF chromatin loop formation. We performed ChIPseq in EBV+ cells to assess PARP1 binding across the EBV genome. We identified numerous PARP1 binding sites, including OriP and the active Cp promoter. We also identified numerous sites at which PARP1 and CTCF colocalize, including the Cp promoter. ChIPseq in EBV+ cells treated with and without the PARP inhibitor olaparib revealed some changes in CTCF binding across the genome, including at Cp, where CTCF was evicted from the region. By FAIRE-seq and ChIP-seq we determined that changes in CTCF binding correlate with changes in chromatin accessibility and accumulation of repressive epigenetic marks. RNA- seq analysis confirmed that these changes in the chromatin structure correlate with changes in viral gene expression. PARP1 and CTCF also regulate cellular chromatin, therefore we determined whether EBV can alter host gene expression through PARP1 in a similar way. We found that EBV activation of PARP1 changes PARP1 and CTCF binding across the human genome, altering the epigenetic landscape of several cellular genes. Overall, our data indicate that EBV utilizes PARP1 to induce both viral and human epigenetic programs that can sustain EBV persistent infection.
Cellular Epigenome
Lena Lupey - Green1, Lisa Caruso1, Kayla Martin1, Michael Hulse1, Jozef Madzo1, Sarah Johnson1, Italo Tempera1,
1Fels Institute for Cancer Research - School of Medicine Temple University
EBV establishes a latent infection with complex and dynamic gene expression patterns. These gene expression patterns can adapt to diverse host cell types and immunological responses. The changes in viral gene expression are regulated by both viral and cellular factors, including epigenetic modulators. We previously discovered that an important epigenetic feature of EBV is the three-dimensional (3D) structure of the viral chromosome. Changes in EBV 3D chromatin structure correlates with different patterns of gene expression. The primary factor that mediates the 3D genome structure in EBV is CTCF. Our recent work reveals that EBV activates PARP1, which has been shown to be important in regulating CTCF chromatin loop formation. We performed ChIPseq in EBV+ cells to assess PARP1 binding across the EBV genome. We identified numerous PARP1 binding sites, including OriP and the active Cp promoter. We also identified numerous sites at which PARP1 and CTCF colocalize, including the Cp promoter. ChIPseq in EBV+ cells treated with and without the PARP inhibitor olaparib revealed some changes in CTCF binding across the genome, including at Cp, where CTCF was evicted from the region. By FAIRE-seq and ChIP-seq we determined that changes in CTCF binding correlate with changes in chromatin accessibility and accumulation of repressive epigenetic marks. RNA- seq analysis confirmed that these changes in the chromatin structure correlate with changes in viral gene expression. PARP1 and CTCF also regulate cellular chromatin, therefore we determined whether EBV can alter host gene expression through PARP1 in a similar way. We found that EBV activation of PARP1 changes PARP1 and CTCF binding across the human genome, altering the epigenetic landscape of several cellular genes. Overall, our data indicate that EBV utilizes PARP1 to induce both viral and human epigenetic programs that can sustain EBV persistent infection.
PARP Pol
CTCF-PARlyation
The viral LMP1 activates PARP1
PARP1 binds to EBV genome during latency (Lupercalia-Green et al JV 2018)
(Cool should check this up, there are peaks!)
PARP1 inhibitionalters CTCF binding. (+ Olaparib)
LMP2 regulates EBV genome chromatin structure through PARP1
PRPPi (olaparib) offsets LMP10mediatd gene activation
6:00 PM – 11:00 PM Closing Banquet Great Hall
PARP1 binds to EBV genome during latency (Lupercalia-Green et al JV 2018)
(Cool should check this up, there are peaks!)
PARP1 inhibitionalters CTCF binding. (+ Olaparib)
- PARP1 inhibition alters CTCF binding
- Represses Cp promoter activity (Cp-Type III, QP -Type I)
LMP2 regulates EBV genome chromatin structure through PARP1
PRPPi (olaparib) offsets LMP10mediatd gene activation
- Targets are involved in cell metabolism
- By 3C-Hi contact
- AB compartment switch ~ 200kb resolution
- LMP1 regulates both host and viral genomes chromatin structure through PARP1
6:00 PM – 11:00 PM Closing Banquet Great Hall
Joint Session #3
Session Chairs: Michelle West & Doug Lowy
Oral Talk #107
Label with tandem-mass-tag (TMT) based MS3 mass spectrometry
CD23+ of time points 0, 2, 4, 7, 10, 14, 18,21, 28, 35
Day 7, LMP1 and LMP2 —> Burkitt like
Day 14 lymphoblastoid
Latency III antigen expression profiles
See notes for the rest of the talk (extremely fast!)
Six proteins
* Integrative approach to identify gene targets of KSHV transcription
* ORF24 interacts with RNA Pol II through leucine
* ORF24-Pol II interaction mutant does not produce infectious visions and is detective in alert gene transcription
* Distinct gene clusters that show differential dependence on late gene transcription complex
* ORF24 - dependent
* ChIP-seq of ORF34-HA shows binding of ORF34 across the viral genome
* These represent ORF24 dependent
* Overlap of RNA-seq and ChIP-seq data distinguishes the direct and index
* TATTTAA (direct target short motif)
* Tang et al J Viral 2004 p2609
* K8.1 RSNYS motif
* Expand mitigation us aknost exclusively in direct targets of ORF24 complex
* ORF59 is the only outlier of the
* TATT motif may be important for recognition but downstream sequence sequ is potentially important for transcription
Oral Talk #109
955 MCP and SCP complex in each capsid
High pressure during DNA packaging
Oral Talk #107
EBV-Induced Metabolic Dependency Factors in B-Cell Transformation
Liang-Wei Wang1,2, Ina Ersing1, Luis Nobre3, Hongying Shen4, Stephen Trudeau1, Camille Ashbaugh1, Steve Gygi5, Vamsi Mootha4, Michael Weekes3, Benjamin Gewurz1,2
1Division of Infectious Disease, Brigham & Women's Hospital, 2Program in Virology, Harvard Medical School, 3Cambridge Instiute for Medical Research, University of Cambridge, 4Department of Molecular Biology, Massachusetts General Hospital, 5Department of Cell Biology, Harvard Medical School
Epstein-Barr virus (EBV) transforms B-cells into continuously proliferating lymphoblastoid B-cell lines (LCL). Knowledge of how EBV remodels host metabolic pathways to support rapid B-cell outgrowth and to overcome metabolic redox stress remains incomplete. To gain insights into EBV-induced metabolic dependency factors, we constructed a proteomic map of B-cell transformation. Primary B-cells from 12 human donors were profiled by tandem-mass-tag mass spectrometry at rest and at 9 timepoints after EBV infection. This approach generated expression profiles of >6500 host and 17 viral proteins and highlighted EBV-induced pathways. Glycolysis, cholesterol and fatty acid biosynthesis pathways were highly upregulated cytosolic metabolic pathways, each if of which were critical for EBV- driven B-cell outgrowth. Likewise, EBV remodeled B-cell mitochondria, with the one-carbon (1C) metabolism pathway among the most highly EBV-induced. Mitochondrial 1C uses folate carriers to interconvert serine into glycine, formate and NADPH building blocks. EBNA2 and cMyc were critical for upregulation of key mitochondrial 1C enzymes including MTHFD2, which is highly expressed during embryogenesis but not in most adult tissues. Chemical and CRISPR genetic analysis underscored MTHFD2 and 1C pathway roles in EBV-driven B-cell growth and survival. MTHFD2 was important for intramitochondrial NADPH generation, and compartment-specific perturbation of mitochondrial NADPH levels diminished LCL growth and survival. Isotope tracing studies further supported EBV-induced 1C pathway roles in purine nucleotide synthesis and NADPH production. To fuel 1C metabolism, EBV upregulated import and de novo synthesis of serine, each of which were found to be important dependency factors. 1C-derived glycine also supported glutathione synthesis, which together with NADPH had redox defense roles. Our results highlight mitochondrial 1C as a key EBV-induced metabolic dependency factor and potential therapeutic target.
Liang-Wei Wang1,2, Ina Ersing1, Luis Nobre3, Hongying Shen4, Stephen Trudeau1, Camille Ashbaugh1, Steve Gygi5, Vamsi Mootha4, Michael Weekes3, Benjamin Gewurz1,2
1Division of Infectious Disease, Brigham & Women's Hospital, 2Program in Virology, Harvard Medical School, 3Cambridge Instiute for Medical Research, University of Cambridge, 4Department of Molecular Biology, Massachusetts General Hospital, 5Department of Cell Biology, Harvard Medical School
Epstein-Barr virus (EBV) transforms B-cells into continuously proliferating lymphoblastoid B-cell lines (LCL). Knowledge of how EBV remodels host metabolic pathways to support rapid B-cell outgrowth and to overcome metabolic redox stress remains incomplete. To gain insights into EBV-induced metabolic dependency factors, we constructed a proteomic map of B-cell transformation. Primary B-cells from 12 human donors were profiled by tandem-mass-tag mass spectrometry at rest and at 9 timepoints after EBV infection. This approach generated expression profiles of >6500 host and 17 viral proteins and highlighted EBV-induced pathways. Glycolysis, cholesterol and fatty acid biosynthesis pathways were highly upregulated cytosolic metabolic pathways, each if of which were critical for EBV- driven B-cell outgrowth. Likewise, EBV remodeled B-cell mitochondria, with the one-carbon (1C) metabolism pathway among the most highly EBV-induced. Mitochondrial 1C uses folate carriers to interconvert serine into glycine, formate and NADPH building blocks. EBNA2 and cMyc were critical for upregulation of key mitochondrial 1C enzymes including MTHFD2, which is highly expressed during embryogenesis but not in most adult tissues. Chemical and CRISPR genetic analysis underscored MTHFD2 and 1C pathway roles in EBV-driven B-cell growth and survival. MTHFD2 was important for intramitochondrial NADPH generation, and compartment-specific perturbation of mitochondrial NADPH levels diminished LCL growth and survival. Isotope tracing studies further supported EBV-induced 1C pathway roles in purine nucleotide synthesis and NADPH production. To fuel 1C metabolism, EBV upregulated import and de novo synthesis of serine, each of which were found to be important dependency factors. 1C-derived glycine also supported glutathione synthesis, which together with NADPH had redox defense roles. Our results highlight mitochondrial 1C as a key EBV-induced metabolic dependency factor and potential therapeutic target.
Label with tandem-mass-tag (TMT) based MS3 mass spectrometry
CD23+ of time points 0, 2, 4, 7, 10, 14, 18,21, 28, 35
Day 7, LMP1 and LMP2 —> Burkitt like
Day 14 lymphoblastoid
Latency III antigen expression profiles
See notes for the rest of the talk (extremely fast!)
A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells.
Cell Reports 2017/05 pdf 4986
Oral Talk #108
Identifying Genome-Wide Targets of the Late Gene Transcription Complex in KSHV
Divya Nandakumar1, Britt Glaunsinger1,2
1University of California Berkeley, 2Howard Hughes Medical Institute
Herpesviruses compete with the tightly regulated cellular transcription by encoding transcription factors that divert the transcriptional machinery from the host to the viral genome. In β and γ-herpesviruses, a viral pre-initiation complex (vPIC) orchestrates the transcription of genes expressed late in infection by hijacking the host RNA polymerase. Identifying candidate genes regulated by the vPIC will be key to detecting promoter elements that are important for vPIC recognition and potentially offer insights on the mechanism of switch from early to late gene expression. In this study, we have used an integrative approach to identify targets of the vPIC using a combination of ChIP-Seq, RNA- Seq and reporter assays. Given that most viral proteins have multiple functions, a targeted approach using site specific mutants in the vPIC that exclusively inhibit transcription would provide more accurate information on vPIC targets. The core component of the vPIC is ORF24, a viral TATA Binding Protein (TBP) that displays putative structural mimicry to cellular TBP but unlike TBP binds directly to both RNA polymerase II and promoter DNA. We have used two specific mutants of ORF24 (a RNA polymerase II interaction mutant and a mutant of ORF24 that disrupts vPIC formation) to identify the functional gene targets of the vPIC. Additionally, we have integrated the differential expression information with genome-wide binding data of the vPIC, generated using an endogenously tagged component of the vPIC, to differentiate the direct and indirect targets of the complex during infection. Our results indicate that the spectrum of genes regulated by the vPIC extends beyond late genes and includes early genes that continue to be expressed at later stages of infection. The results also hint at a link between DNA replication and transcription and current studies are underway to validate the functional significance of the genome-wide analysis.
Divya Nandakumar1, Britt Glaunsinger1,2
1University of California Berkeley, 2Howard Hughes Medical Institute
Herpesviruses compete with the tightly regulated cellular transcription by encoding transcription factors that divert the transcriptional machinery from the host to the viral genome. In β and γ-herpesviruses, a viral pre-initiation complex (vPIC) orchestrates the transcription of genes expressed late in infection by hijacking the host RNA polymerase. Identifying candidate genes regulated by the vPIC will be key to detecting promoter elements that are important for vPIC recognition and potentially offer insights on the mechanism of switch from early to late gene expression. In this study, we have used an integrative approach to identify targets of the vPIC using a combination of ChIP-Seq, RNA- Seq and reporter assays. Given that most viral proteins have multiple functions, a targeted approach using site specific mutants in the vPIC that exclusively inhibit transcription would provide more accurate information on vPIC targets. The core component of the vPIC is ORF24, a viral TATA Binding Protein (TBP) that displays putative structural mimicry to cellular TBP but unlike TBP binds directly to both RNA polymerase II and promoter DNA. We have used two specific mutants of ORF24 (a RNA polymerase II interaction mutant and a mutant of ORF24 that disrupts vPIC formation) to identify the functional gene targets of the vPIC. Additionally, we have integrated the differential expression information with genome-wide binding data of the vPIC, generated using an endogenously tagged component of the vPIC, to differentiate the direct and indirect targets of the complex during infection. Our results indicate that the spectrum of genes regulated by the vPIC extends beyond late genes and includes early genes that continue to be expressed at later stages of infection. The results also hint at a link between DNA replication and transcription and current studies are underway to validate the functional significance of the genome-wide analysis.
Six proteins
- ORF24
- ORF31
- ORF34
- ORF18
- ORF30
- ORF66
* Integrative approach to identify gene targets of KSHV transcription
* ORF24 interacts with RNA Pol II through leucine
* ORF24-Pol II interaction mutant does not produce infectious visions and is detective in alert gene transcription
* Distinct gene clusters that show differential dependence on late gene transcription complex
* ORF24 - dependent
* ChIP-seq of ORF34-HA shows binding of ORF34 across the viral genome
* These represent ORF24 dependent
* Overlap of RNA-seq and ChIP-seq data distinguishes the direct and index
* TATTTAA (direct target short motif)
* Tang et al J Viral 2004 p2609
* K8.1 RSNYS motif
* Expand mitigation us aknost exclusively in direct targets of ORF24 complex
* ORF59 is the only outlier of the
* TATT motif may be important for recognition but downstream sequence sequ is potentially important for transcription
Oral Talk #109
High Risk α-HPV Oncogenes Induce and Disrupt Translesion
Synthesis
Sebastian O. Wendel1, Laura Brown2, Jazmine Snow1, Tyler Bastian3, Vaibhav Murthy1, Daniel Neill2, Monica Gamez1, Andrew Kahn1, Dalton Dacus1, Kevin Ault4, Ossama Tawfik5, Cen Wu3, Nicholas A. Wallace1
1Division of Biology, Kansas State University, 2Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, 3Department of Statistics, Kansas State University, 4Department of Obstetrics and Gynecology, University of Kansas Medical Center, 5MAWD Pathology Group
Translesion synthesis (TLS) is an essential DNA-damage tolerance pathway needed during replication. It prevents minor DNA lesions from causing replication fork collapse and descending into more deleterious double strand DNA breaks. Increased proliferation induces TLS to cope with the associated elevation in replication stress. Our bioinformatics analysis shows that increased TLS expression correlates with unfavorable outcomes of patient survival data for several cancer types. Cervical cancers, almost exclusively caused by high risk α-HPV, were the only exception that we found. Here, increased TLS expression is predictive of favorable outcomes. An in-depth bioinformatics analysis of RNAseq data from cervical cancers revealed a general upregulation of the TLS pathway, with the notable exception of POL-η, the lesion bypassing TLS polymerase. HPV E7 driven proliferation causes replication stress. In addition to increased mRNA, a subset of TLS- pathway proteins is stabilized in α-HPV oncogene expressing cervical cancer cell lines. Increases in TLS expression (protein abundance and RNAseq) correlate with disease progression. However, we show that TLS-functionality falls apart in the presence of E6. HPV E6 interferes with the induction of the TLS polymerase POL-η by degrading p53 and blocking its role as transcription factor for POL-η induction. HPV-positive cervical cancer cell lines fail to increase POL-η abundance or concentrate the polymerase in response to extrinsic DNA damage. This increases replication fork stall and collapse, indications of failed TLS. Consequently, we show that cervical cancer cell lines exhibit a significant sensitivity to chemical DNA-crosslinkers as well as UV radiation. We also provide evidence of a subpopulation (~20%) of cervical cancer patients that have increased expression of y-family polymerases. These women have a dramatically decreased survival. Our data makes the case that α-HPV oncogenes E6 and E7 both disrupt and induce the TLS pathway. Further, because TLS mitigates the damage from crosslinking agents, y-family polymerase expression could potentially be used as a diagnostic to identify cervical cancer patients that are unlikely to respond to the standard of care, Cisplatin. Resolving cervical cancer patient data by stage suggests a progression and correlation of RNAseq and immunohistochemistry data with increased cancer stage.
Synthesis
Sebastian O. Wendel1, Laura Brown2, Jazmine Snow1, Tyler Bastian3, Vaibhav Murthy1, Daniel Neill2, Monica Gamez1, Andrew Kahn1, Dalton Dacus1, Kevin Ault4, Ossama Tawfik5, Cen Wu3, Nicholas A. Wallace1
1Division of Biology, Kansas State University, 2Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, 3Department of Statistics, Kansas State University, 4Department of Obstetrics and Gynecology, University of Kansas Medical Center, 5MAWD Pathology Group
Translesion synthesis (TLS) is an essential DNA-damage tolerance pathway needed during replication. It prevents minor DNA lesions from causing replication fork collapse and descending into more deleterious double strand DNA breaks. Increased proliferation induces TLS to cope with the associated elevation in replication stress. Our bioinformatics analysis shows that increased TLS expression correlates with unfavorable outcomes of patient survival data for several cancer types. Cervical cancers, almost exclusively caused by high risk α-HPV, were the only exception that we found. Here, increased TLS expression is predictive of favorable outcomes. An in-depth bioinformatics analysis of RNAseq data from cervical cancers revealed a general upregulation of the TLS pathway, with the notable exception of POL-η, the lesion bypassing TLS polymerase. HPV E7 driven proliferation causes replication stress. In addition to increased mRNA, a subset of TLS- pathway proteins is stabilized in α-HPV oncogene expressing cervical cancer cell lines. Increases in TLS expression (protein abundance and RNAseq) correlate with disease progression. However, we show that TLS-functionality falls apart in the presence of E6. HPV E6 interferes with the induction of the TLS polymerase POL-η by degrading p53 and blocking its role as transcription factor for POL-η induction. HPV-positive cervical cancer cell lines fail to increase POL-η abundance or concentrate the polymerase in response to extrinsic DNA damage. This increases replication fork stall and collapse, indications of failed TLS. Consequently, we show that cervical cancer cell lines exhibit a significant sensitivity to chemical DNA-crosslinkers as well as UV radiation. We also provide evidence of a subpopulation (~20%) of cervical cancer patients that have increased expression of y-family polymerases. These women have a dramatically decreased survival. Our data makes the case that α-HPV oncogenes E6 and E7 both disrupt and induce the TLS pathway. Further, because TLS mitigates the damage from crosslinking agents, y-family polymerase expression could potentially be used as a diagnostic to identify cervical cancer patients that are unlikely to respond to the standard of care, Cisplatin. Resolving cervical cancer patient data by stage suggests a progression and correlation of RNAseq and immunohistochemistry data with increased cancer stage.
- Hyperproliferation of
- TLS abundance correlated with proliferation
- TLS pathway
- upregulation correlates with survival in cervial cancer
- 5 datasets merged
- 188 subjects
- 5 major TLS related enzymes (POLH, …) are either located at non-changed groups
- POLH abundance in response to UV radiation
- POLH expression correlates with p53 expression
- Nonetheless, About 20% patients with TLS proteins go down associated with poor prognosis
Oral Talk #110
High Risk HPV Oncogenes Impair the Fanconi Anemia DNA
Repair Pathway
Sujita Khanal1, Denise Galloway1
1Division of Human Biology, Fred Hutchinson Cancer Research Center
Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and oropharyngeal cancers. Our study showed that E6/E7 expressing cells are hypersensitive to the DNA crosslinking agent cisplatin and have defects in repairing DNA inter strand cross-links (ICL), suggesting that HPV oncogenes impair the Fanconi anemia (FA) pathway. Monoubiquitination and foci formation of FancD2 are functional activators of the FA pathway. Consistent with previous studies, we found that E6/E7 increased FancD2 monoubiquitination and foci formation. However, using an I-SceI co-localization assay revealed that HPV E6 and E7 either expressing individually or together impair the co- localization of FancD2 with double-strand breaks (pH2AX). Recently, de-ubiquitination of FancD2 was recognized to be equally important as ubiquitination for effective ICL repair. We demonstrated that E6 caused delayed FancD2 de-ubiquitination, thereby impairing ICL repair. E6 cells also showed defects in repair proteins, Rad51 and Palb2, which are downstream of FancD2 in the FA pathway. Using genetic epistasis analyses, we showed that mislocalization defect of Rad51 in E6 cells is dependent on FancD2. Further, E6 reduced the expression and foci formation of Palb2. These findings uncover unique mechanisms by which HPV oncogenes (notably E6) impair the completion of the FA repair pathway, though they activate the initiation of the pathway.
Repair Pathway
Sujita Khanal1, Denise Galloway1
1Division of Human Biology, Fred Hutchinson Cancer Research Center
Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and oropharyngeal cancers. Our study showed that E6/E7 expressing cells are hypersensitive to the DNA crosslinking agent cisplatin and have defects in repairing DNA inter strand cross-links (ICL), suggesting that HPV oncogenes impair the Fanconi anemia (FA) pathway. Monoubiquitination and foci formation of FancD2 are functional activators of the FA pathway. Consistent with previous studies, we found that E6/E7 increased FancD2 monoubiquitination and foci formation. However, using an I-SceI co-localization assay revealed that HPV E6 and E7 either expressing individually or together impair the co- localization of FancD2 with double-strand breaks (pH2AX). Recently, de-ubiquitination of FancD2 was recognized to be equally important as ubiquitination for effective ICL repair. We demonstrated that E6 caused delayed FancD2 de-ubiquitination, thereby impairing ICL repair. E6 cells also showed defects in repair proteins, Rad51 and Palb2, which are downstream of FancD2 in the FA pathway. Using genetic epistasis analyses, we showed that mislocalization defect of Rad51 in E6 cells is dependent on FancD2. Further, E6 reduced the expression and foci formation of Palb2. These findings uncover unique mechanisms by which HPV oncogenes (notably E6) impair the completion of the FA repair pathway, though they activate the initiation of the pathway.
Oral Talk #111
Structure-Guided Identification of Interaction Hotspot for
Developing Inhibitors Interrupting KSHV Capsid Assembly
Danyang Gong1, Xinghong Dai1, Ting-Ting Wu1, Hong Z. Zhou1, Ren Sun1
1UCLA
Kaposi’s sarcoma-associated herpesvirus (KSHV) capsid, like those of the other eight human herpesviruses, is highly pressurized by its dsDNA genome. Indeed, capsid assembly and genome packaging of herpesviruses are prone to interruption and thus can be targeted for structure-guided antiviral development. However, with nearly 3,000 proteins and over 1,300 Å in diameter, herpesvirus capsids present a formidable challenge to atomic structure determination and functional mapping of molecular interactions. By employing cryo electron microscopy (cryoEM) with highly purified KSHV virions, we resolved a 4.2 Å resolution structure of the KSHV capsid, and its atomic model containing unique conformers of the major capsid protein (MCP), the smallest capsid protein (SCP), the triplex proteins Tri1 and Tri2. Guided by the atomic resolution structure of KSHV in addition to functional analysis results, we have identified interaction hotspots essential for KSHV capsid assembly and replication, especially a groove on the upper domain of MCP enabling its interaction with a long helix on the SCP. More importantly, this groove could be targeted by SCP helix-mimicking polypeptides for inhibiting KSHV replication, making it a promising druggable site for developing inhibitors. Furthermore, the in vitro and in vivo MCP-SCP interaction systems are established and applied to identify chemical inhibitors using high throughput chemical screening.
Developing Inhibitors Interrupting KSHV Capsid Assembly
Danyang Gong1, Xinghong Dai1, Ting-Ting Wu1, Hong Z. Zhou1, Ren Sun1
1UCLA
Kaposi’s sarcoma-associated herpesvirus (KSHV) capsid, like those of the other eight human herpesviruses, is highly pressurized by its dsDNA genome. Indeed, capsid assembly and genome packaging of herpesviruses are prone to interruption and thus can be targeted for structure-guided antiviral development. However, with nearly 3,000 proteins and over 1,300 Å in diameter, herpesvirus capsids present a formidable challenge to atomic structure determination and functional mapping of molecular interactions. By employing cryo electron microscopy (cryoEM) with highly purified KSHV virions, we resolved a 4.2 Å resolution structure of the KSHV capsid, and its atomic model containing unique conformers of the major capsid protein (MCP), the smallest capsid protein (SCP), the triplex proteins Tri1 and Tri2. Guided by the atomic resolution structure of KSHV in addition to functional analysis results, we have identified interaction hotspots essential for KSHV capsid assembly and replication, especially a groove on the upper domain of MCP enabling its interaction with a long helix on the SCP. More importantly, this groove could be targeted by SCP helix-mimicking polypeptides for inhibiting KSHV replication, making it a promising druggable site for developing inhibitors. Furthermore, the in vitro and in vivo MCP-SCP interaction systems are established and applied to identify chemical inhibitors using high throughput chemical screening.
955 MCP and SCP complex in each capsid
High pressure during DNA packaging
Joint Session #2
Session Chairs: Jeffrey Cohen & Miranda Thomas
J Immunol. 2010 Feb 1;184(3):1242-50. doi: 10.4049/jimmunol.0902191. Epub 2009 Dec 18.
Front Immunol. 2017 May 4;8:524. doi: 10.3389/fimmu.2017.00524. eCollection 2017.
Combination immunotherapy for TC-1 tumor HPV E6/E7 DNA immunization and anti-PDL1 J Immunotherapeutics.
Chandra J (unpublished)Turing K (unpublished)
Another reason for logical immunosuppression loss of UIL33 in E7 KC demonstrated by subtle cell RNAseq Kelvin Tuong : unpublished)
A further reason for failed immunotherapy
Endogenous antigen presentation by hyperproliferative keratinocytes is impaired (Paula Kuo, unpublished)
Conclusion: Hyperproliferative epithelial cells (induced by HPV16 E7), rather than E7 induced signaling patwhays Induced IFNG dependent local immune regulation, inhibiting effector T cell function
APOBEC3 restricts a number of viruses including …. but not DNA viruses
A3B is an essential DDR activator
EBV BORF2 specifically pulls down A3B loop 7
BORF2 aborages A3B demitasse activity
BORF2 relicalizes endogenous A3B
Transferred BORF2 is sufficient for A3B relicalization
A3B in EBVdeltaBORF2 goes to DNA replication sites
3D-PCR differentiated mutated vs WT sequences
EBV delta BORF2 is sensitive to A3B deamination
A3B-mediated mutation burden in lytic EBVdletaBORF2
HAdenovirs (HAdV) efficiently suppresses host DDR
A3A is not targeted by the HAdV E3 unique tin ligase
Consequences of uracil in DNA
ORF54 (dUTPas) KO mice
ORF46 and ORF54 KO showed decreased ??
Impaired replication and destabilized viral genome
PLoS Pathogens 2018
BPLF1, the largest tegument protein of EBV
72 BPLF1 interacting proteins identified, highest hit
YWHA contains 14-3-3 proteins
Highly conserved ubiquitous proteins
Seven members: b, epsilon, gamma..
E3 ligase: TRIM25
BPLF1 promotes auto-ubiquitination of TRIM25
BPLF2 inhibits RIG-1 ubiquitination
BPLF1 BM does not promote autoubiquitination of TRIM25
Oral Talk #102
Local Dysregulation of the Immune Response Impairs
Immunotherapy for HPV Associated Premalignancy
Ian H. Frazer1, Zewen K. Tuong1, Siok Min Teoh1, Paula Kuo1, Abate Assefa Bashaw1, Stephen R. Mattarollo1, Graham R. Leggatt1, Joseph Powell1
1University of Queensland UQDI
Background: Persistence of infection is a rare consequence of exposure to high risk HPV, leading to hyperplasia and eventual neoplasia of anogenital epithelia. Transplantable tumour models expressing HPV E7 protein are susceptible to immunotherapies that have not proven successful in eradicating HPV associated human disease.
Aim: To determine the consequences of expression of E7 protein on local cellular immunity and on expression of immunoregulatory genes.
Approach: Examine, using RNAseq and measures of cellular immunity, the consequences of expression of E7 as a transgene in hyperplastic murine epithelium.
Results: RNAseq demonstrates comparable HPV associated changes in gene expression in hyperplastic E7 transgenic human CIN3 tissue. HPV E7 associated overexpression of immunoregulatory genes, together with altered local cytokine and chemokine expression, and local antigen presenting cell function is seen in murine E7 transgenic skin. These changes are not seen in normoplastic E7 transgenic murine epithelium also expressing a mutated Rb gene, demonstrating that local immunoregulation is associated with hyperplasia rather than E7 expression. Single cell RNAseq reveals further dysregulation of expression of host protective immunoregulatory proteins in basal keratinocytes.
Conclusion: Effective immunotherapy for HPV E7 associated disease will likely require correction of the local immunoregulatory environment, in addition to E7 specific immunotherapy.
HPV VLP vaccines are not therapeutic! need something else for people who are infected
Immunotherapy for HPV infection and associated cancer
Immunotherapy for HPV Associated Premalignancy
Ian H. Frazer1, Zewen K. Tuong1, Siok Min Teoh1, Paula Kuo1, Abate Assefa Bashaw1, Stephen R. Mattarollo1, Graham R. Leggatt1, Joseph Powell1
1University of Queensland UQDI
Background: Persistence of infection is a rare consequence of exposure to high risk HPV, leading to hyperplasia and eventual neoplasia of anogenital epithelia. Transplantable tumour models expressing HPV E7 protein are susceptible to immunotherapies that have not proven successful in eradicating HPV associated human disease.
Aim: To determine the consequences of expression of E7 protein on local cellular immunity and on expression of immunoregulatory genes.
Approach: Examine, using RNAseq and measures of cellular immunity, the consequences of expression of E7 as a transgene in hyperplastic murine epithelium.
Results: RNAseq demonstrates comparable HPV associated changes in gene expression in hyperplastic E7 transgenic human CIN3 tissue. HPV E7 associated overexpression of immunoregulatory genes, together with altered local cytokine and chemokine expression, and local antigen presenting cell function is seen in murine E7 transgenic skin. These changes are not seen in normoplastic E7 transgenic murine epithelium also expressing a mutated Rb gene, demonstrating that local immunoregulation is associated with hyperplasia rather than E7 expression. Single cell RNAseq reveals further dysregulation of expression of host protective immunoregulatory proteins in basal keratinocytes.
Conclusion: Effective immunotherapy for HPV E7 associated disease will likely require correction of the local immunoregulatory environment, in addition to E7 specific immunotherapy.
HPV VLP vaccines are not therapeutic! need something else for people who are infected
Immunotherapy for HPV infection and associated cancer
E6/E7 recombinant
E6 and E7 protein and adjuvant immunotherapy: can we eliminate existing HPV associated CIN?
Modeling epithelial cancer immunotherapy keratinocytes-immune Cell interaction
Donors K14 transgenic
Recipient (allo matched nontransgenic)
Antigen expressed billy in basal keratinoicuytes
Grafters expression som antigens eg OVA
HPV16E7 mediated skin hyperplasia looks like CIN
K14R7 sumo shares …
E6 and E7 protein and adjuvant immunotherapy: can we eliminate existing HPV associated CIN?
Modeling epithelial cancer immunotherapy keratinocytes-immune Cell interaction
Donors K14 transgenic
Recipient (allo matched nontransgenic)
Antigen expressed billy in basal keratinoicuytes
Grafters expression som antigens eg OVA
HPV16E7 mediated skin hyperplasia looks like CIN
K14R7 sumo shares …
- A donor skin resident immunocyte inhibits rejection of E7 transgenic mice
The cell inhibiting E7 graft rejection is an NKT cell!
- Local IFNG production by iNKT cells inhibits graft rejection
Reconstitution with NKT cells but not IFNG-/0 NKT cells restores graft survival
Upregulation of multiple immune checkpoint molecules in K14E7 skin by IFNG
IFNG increases PD1, CTLA4, TIM3, LAG3, PDL1, PDL2. (J Immunol 184(3):1242-
IFNG increases PD1, CTLA4, TIM3, LAG3, PDL1, PDL2. (J Immunol 184(3):1242-
J Immunol. 2010 Feb 1;184(3):1242-50. doi: 10.4049/jimmunol.0902191. Epub 2009 Dec 18.
Invariant NKT cells in hyperplastic skin induce a local immune suppressive environment by IFN-gamma production.
Abstract
NKT cells can promote or inhibit adaptive immune responses. Cutaneous immunity is tightly regulated by cooperation between innate and adaptive immune processes, but the role of NKT cells in regulating cutaneous immunity is largely unknown. In this study, we show, in a mouse model, that skin-infiltrating CD1d-restricted NKT cells in HPV16-E7 transgenic hyperplastic skin produce IFN-gamma, which can prevent rejection of HPV16-E7-expressing skin grafts. Suppression of graft rejection is associated with the accumulation of CD1d(hi)-expressing CD11c(+)F4/80(hi) myeloid cells in hyperplastic skin. Blockade of CD1d, removal of NKT cells, or local inhibition of IFN-gamma signaling is sufficient to restore immune-mediated graft rejection. Thus, inhibition of NKT cell recruitment or function may enable effective immunity against tumor and viral Ags expressed in epithelial cells.
EBioMedicine. 2016 Jul;9:314-323. doi: 10.1016/j.ebiom.2016.06.011. Epub 2016 Jun 7.
A Mouse Model of Hyperproliferative Human Epithelium Validated by Keratin Profiling Shows an Aberrant Cytoskeletal Response to Injury.
Author information
- 1
- The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, QLD, Australia.
- 2
- McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, USA.
- 3
- The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, QLD, Australia. Electronic address: i.frazer@uq.edu.au.
Abstract
A validated animal model would assist with research on the immunological consequences of the chronic expression of stress keratins KRT6, KRT16, and KRT17, as observed in human pre-malignant hyperproliferative epithelium. Here we examine keratin gene expression profile in skin from mice expressing the E7 oncoprotein of HPV16 (K14E7) demonstrating persistently hyperproliferative epithelium, in nontransgenic mouse skin, and in hyperproliferative actinic keratosis lesions from human skin. We demonstrate that K14E7 mouse skin overexpresses stress keratins in a similar manner to human actinic keratoses, that overexpression is a consequence of epithelial hyperproliferation induced by E7, and that overexpression further increases in response to injury. As stress keratins modify local immunity and epithelial cell function and differentiation, the K14E7 mouse model should permit study of how continued overexpression of stress keratins impacts on epithelial tumor development and on local innate and adaptive immunity. (PMID:27333029)
Epithelial hyperplasia rather than E7 is the driver of the lymphocytic infiltrate in E7 tg skin. (Front Immunol 2017 Paula Kuo)
Front Immunol. 2017 May 4;8:524. doi: 10.3389/fimmu.2017.00524. eCollection 2017.
HPV16-E7-Specific Activated CD8 T Cells in E7 Transgenic Skin and Skin Grafts.
Abstract
Human papillomavirus (HPV) 16 E7 (E7) protein expression in skin promotes epithelial hyperproliferation and transformation to malignancy. Grafts of murine skin expressing E7 protein as a transgene in keratinocytes are not rejected from immunocompetent recipients, whereas grafts expressing ovalbumin (OVA), with or without coexpression of E7 protein, are promptly rejected, demonstrating that E7-associated non-antigen-specific local immunosuppression is not a major determinant of lack of rejection of E7 transgenic skin. To determine whether failure of rejection of E7 skin grafts is due to failure to attract E7-specific effector T cells, E7- and OVA-specific effector CD8+ T cells, activated in vitro, were transferred to animals bearing E7 transgenic skin grafts. Three days after T cell transfer, E7-specific T cells were present in significantly greater numbers than OVA-specific T cells in the grafted skin on animals bearing recently placed or healed E7 grafts, without graft rejection, and also in the ear skin of E7 transgenic animals, without obvious pathology. E7 and OVA-specific T cells were present in lesser numbers in healed E7 grafts than in recently placed grafts and in lesser numbers in recently placed E7 transgenic epidermal grafts without E7-associated hyperproliferation, derived from E7 transgenic mice with a mutated retinoblastoma gene. These data demonstrate that effector T cells are to some extent attracted to E7 transgenic skin specifically by E7 expression, but in large measure non-specifically by the epithelial proliferation associated with E7 expression, and by the local inflammation produced by grafting. Failure of E7 graft rejection was observed despite trafficking of E7-specific effector T cells to E7-expressing epithelium, a finding of consequence for immunotherapy of HPV 16 E7-associated human cancers. (PubMed: 28523003)
Murine HPV16 E7-expressing transgenic skin effectively emulates the cellular and molecular features of human high-grade squamous intraepithelial lesions.
Abstract
Currently available vaccines prevent HPV infection and development of HPV-associated malignancies, but do not cure existing HPV infections and dysplastic lesions. Persistence of infection(s) in immunocompetent patients may reflect induction of local immunosuppressive mechanisms by HPV, providing a target for therapeutic intervention. We have proposed that a mouse, expressing HPV16 E7 oncoprotein under a Keratin 14 promoter (K14E7 mice), and which develops epithelial hyperplasia, may assist with understanding local immune suppression mechanisms that support persistence of HPV oncogene-induced epithelial hyperplasia. K14E7 skin grafts recruit immune cells from immunocompetent hosts, but consistently fail to be rejected. Here, we review the literature on HPV-associated local immunoregulation, and compare the findings with published observations on the K14E7 transgenic murine model, including comparison of the transcriptome of human HPV-infected pre-malignancies with that of murine K14E7 transgenic skin. We argue from the similarity of i) the literature findings and ii) the transcriptome profiles that murine K14E7 transgenic skin recapitulates the cellular and secreted protein profiles of high-grade HPV-associated lesions in human subjects. We propose that the K14E7 mouse may be an appropriate model to further study the immunoregulatory effects of HPV E7 expression, and can facilitate development and testing of therapeutic vaccines. (PMID: 29807614)
Increased expression of chemokine ligand attract NKT and to E7 graft (2018 J Inves Dermatol. Kuo P)
J Invest Dermatol. 2018 Jun;138(6):1348-1359. doi: 10.1016/j.jid.2017.12.021. Epub 2017 Dec 23.
HPV16E7-Induced Hyperplasia Promotes CXCL9/10 Expression and Induces CXCR3+ T-Cell Migration to Skin.
Abstract
Chemokines regulate tissue immunity by recruiting specific subsets of immune cells. Mice expressing the E7 protein of human papilloma virus 16 as a transgene from a keratin 14 promoter (K14.E7) show increased epidermal and dermal lymphocytic infiltrates, epidermal hyperplasia, and suppressed local immunity. Here, we show that CXCL9 and CXCL10 are overexpressed in non-hematopoietic cells in skin of K14.E7 mice when compared with non-transgenic animals, and recruit CXCR3+ lymphocytes to the hyperplastic skin. Overexpression of CXCL9 and CXCL10 is not observed in E7 transgenic mice with mutated Rb gene whose protein product cannot interact with E7 (K14.E7xRbΔL/ΔL) and in consequence lack hyperplastic epithelium. CXCR3+ T cells are preferentially recruited by CXCL9 and CXCL10 in supernatants of K14.E7 but not K14.E7xRbΔL/ΔL skin cultures in vitro. CXCR3 signalling promotes infiltration of a subset of effector T lymphocytes that enables donor lymphocyte deficient, E7-expressing skin graft rejection. Taken together, this suggests that recruitment of CXCR3+ T cells can be an important factor in the rejection of precancerous skin epithelium providing they can overcome local immunosuppressive mechanisms driven by skin-resident lymphocytes. (PMID: 29277541)
Chandra J (unpublished)Turing K (unpublished)
Another reason for logical immunosuppression loss of UIL33 in E7 KC demonstrated by subtle cell RNAseq Kelvin Tuong : unpublished)
A further reason for failed immunotherapy
Endogenous antigen presentation by hyperproliferative keratinocytes is impaired (Paula Kuo, unpublished)
Conclusion: Hyperproliferative epithelial cells (induced by HPV16 E7), rather than E7 induced signaling patwhays Induced IFNG dependent local immune regulation, inhibiting effector T cell function
Oral Talk #103
A Novel Virus Counteraction Mechanism against APOBEC3-
Mediated Hypermutation and Restriction
Adam Cheng1, Jaime Yockteng-Melgar2, Lori Frappier2, Reuben Harris1
1University of Minnesota, 2University of Toronto
The APOBEC family of single-stranded DNA cytosine deaminases comprises a vital arm of human innate antiviral immunity. These enzymes are responsible for restricting the replication of a wide range of viruses and transposons. The best-studied mechanism is APOBEC3G-mediated restriction of HIV-1, which is counteracted by the viral accessory protein Vif by targeting APOBEC proteins for ubiquitination and proteasome-dependent degradation. Accordingly, most work to date has focused on APOBEC restriction of HIV-1 and related retroviruses and retrotransposons, which all have susceptible single-stranded cDNA replication intermediates. It is not clear whether large double-stranded DNA viruses may be subject to restriction by APOBEC enzymes and, if so, what counteraction mechanisms are employed to maintain viral genomic integrity during pathogenesis.
Here, we define the mechanism by which the large double-stranded DNA herpesvirus Epstein-Barr virus (EBV) counteracts APOBEC3B (A3B)-mediated mutagenesis using a two-pronged approach of direct enzymatic inhibition and subcellular relocalization. The large subunit of the EBV ribonucleotide reductase, BORF2, was discovered to interact with A3B in a proteomic screen. Immunoprecipitation experiments and binding assays using recombinant proteins confirm this interaction. Structure-guided mutagenesis experiments map the interaction to a conserved loop in the A3B catalytic domain. Biochemical studies demonstrate a direct stoichiometric inhibition of A3B DNA cytosine deaminase activity by BORF2. Immunofluorescence microscopy and live cell imaging reveal a BORF2-dependent relocalization of nuclear A3B to the endoplasmic reticulum. Interestingly, A3B/BORF2 aggregates are stabilized in the endoplasmic reticulum, which is in contrast to known degradation-dependent counteraction mechanisms. A targeted deletion of BORF2 from the EBV genome was achieved using CRISPR/Cas9. Upon reactivation from latency, EBVΔBORF2 viral genomes became sensitive to A3B-dependent restriction as evidenced by accumulation of hypermutated viral DNA, loss of viral genomic integrity, and impaired viral replication.
Taken together, our studies define a novel host-pathogen interaction between cellular A3B and the BORF2 protein of EBV. Future strategies to disrupt the A3B/BORF2 interaction in vivo may have merit for compromising the genetic integrity of EBV and help to treat patients with infectious mononucleosis and, coupled to a reactivation strategy, potentially also patients with EBV-driven tumors.
Mediated Hypermutation and Restriction
Adam Cheng1, Jaime Yockteng-Melgar2, Lori Frappier2, Reuben Harris1
1University of Minnesota, 2University of Toronto
The APOBEC family of single-stranded DNA cytosine deaminases comprises a vital arm of human innate antiviral immunity. These enzymes are responsible for restricting the replication of a wide range of viruses and transposons. The best-studied mechanism is APOBEC3G-mediated restriction of HIV-1, which is counteracted by the viral accessory protein Vif by targeting APOBEC proteins for ubiquitination and proteasome-dependent degradation. Accordingly, most work to date has focused on APOBEC restriction of HIV-1 and related retroviruses and retrotransposons, which all have susceptible single-stranded cDNA replication intermediates. It is not clear whether large double-stranded DNA viruses may be subject to restriction by APOBEC enzymes and, if so, what counteraction mechanisms are employed to maintain viral genomic integrity during pathogenesis.
Here, we define the mechanism by which the large double-stranded DNA herpesvirus Epstein-Barr virus (EBV) counteracts APOBEC3B (A3B)-mediated mutagenesis using a two-pronged approach of direct enzymatic inhibition and subcellular relocalization. The large subunit of the EBV ribonucleotide reductase, BORF2, was discovered to interact with A3B in a proteomic screen. Immunoprecipitation experiments and binding assays using recombinant proteins confirm this interaction. Structure-guided mutagenesis experiments map the interaction to a conserved loop in the A3B catalytic domain. Biochemical studies demonstrate a direct stoichiometric inhibition of A3B DNA cytosine deaminase activity by BORF2. Immunofluorescence microscopy and live cell imaging reveal a BORF2-dependent relocalization of nuclear A3B to the endoplasmic reticulum. Interestingly, A3B/BORF2 aggregates are stabilized in the endoplasmic reticulum, which is in contrast to known degradation-dependent counteraction mechanisms. A targeted deletion of BORF2 from the EBV genome was achieved using CRISPR/Cas9. Upon reactivation from latency, EBVΔBORF2 viral genomes became sensitive to A3B-dependent restriction as evidenced by accumulation of hypermutated viral DNA, loss of viral genomic integrity, and impaired viral replication.
Taken together, our studies define a novel host-pathogen interaction between cellular A3B and the BORF2 protein of EBV. Future strategies to disrupt the A3B/BORF2 interaction in vivo may have merit for compromising the genetic integrity of EBV and help to treat patients with infectious mononucleosis and, coupled to a reactivation strategy, potentially also patients with EBV-driven tumors.
APOBEC3 restricts a number of viruses including …. but not DNA viruses
A3B is an essential DDR activator
EBV BORF2 specifically pulls down A3B loop 7
BORF2 aborages A3B demitasse activity
BORF2 relicalizes endogenous A3B
Transferred BORF2 is sufficient for A3B relicalization
A3B in EBVdeltaBORF2 goes to DNA replication sites
3D-PCR differentiated mutated vs WT sequences
EBV delta BORF2 is sensitive to A3B deamination
A3B-mediated mutation burden in lytic EBVdletaBORF2
Oral Talk #104
APOBEC3A is a Novel Restriction Factor Antagonizing Efficient
HAdV Replication
Julia Mai1, Christina Weiss1, Lilian Göttig1, Samuel Hofmann1, Daniela Stadler1, Ulrike Protzer1, Sabrina Schreiner1
1 Technische Universität München, Helmholtz Zentrum München
Apobec cytidine deaminases promote innate immune defense against many DNA-based parasites, such as exogenous viruses and endogenous transposable elements. Although the majority of information about Apobec-mediated viral inhibition pertains to retroviruses, Apobec has also been reported to restrict DNA viruses, such as HBV and HPV.
Human Adenoviruses (HAdV) are non-enveloped viruses and contain a linear, double- stranded DNA genome surrounded by an icosahedral capsid with type specific antigens. Our analyses of the Apobec family members show that Apobec3A (A3A), a DNA damage activating protein resulted in reduced adenoviral gene expression and decreased virus progeny production. A3A further blocks the efficient establishment of adenoviral replication centers marked by the HAdV E2A/DBP protein. Our data further indicate that A3A is not targeted by the viral E1B-55K/E4orf6 E3 ubiquitin ligase complex, and thus, not sequestered into the host proteasomal degradation pathway. However, A3A functions were modulated during HAdV infection by virus-mediated changes in A3A posttranslational modifications. Here, we provide findings provide evidence that A3A represents a novel host restriction factor that counteracts HAdV productive infection. Consequently, a better understanding of A3A-mediated regulation and processes to antagonize HAdV infection in human cells provide new perspectives for the development of antiviral agents and therapeutic strategies.
HAdV Replication
Julia Mai1, Christina Weiss1, Lilian Göttig1, Samuel Hofmann1, Daniela Stadler1, Ulrike Protzer1, Sabrina Schreiner1
1 Technische Universität München, Helmholtz Zentrum München
Apobec cytidine deaminases promote innate immune defense against many DNA-based parasites, such as exogenous viruses and endogenous transposable elements. Although the majority of information about Apobec-mediated viral inhibition pertains to retroviruses, Apobec has also been reported to restrict DNA viruses, such as HBV and HPV.
Human Adenoviruses (HAdV) are non-enveloped viruses and contain a linear, double- stranded DNA genome surrounded by an icosahedral capsid with type specific antigens. Our analyses of the Apobec family members show that Apobec3A (A3A), a DNA damage activating protein resulted in reduced adenoviral gene expression and decreased virus progeny production. A3A further blocks the efficient establishment of adenoviral replication centers marked by the HAdV E2A/DBP protein. Our data further indicate that A3A is not targeted by the viral E1B-55K/E4orf6 E3 ubiquitin ligase complex, and thus, not sequestered into the host proteasomal degradation pathway. However, A3A functions were modulated during HAdV infection by virus-mediated changes in A3A posttranslational modifications. Here, we provide findings provide evidence that A3A represents a novel host restriction factor that counteracts HAdV productive infection. Consequently, a better understanding of A3A-mediated regulation and processes to antagonize HAdV infection in human cells provide new perspectives for the development of antiviral agents and therapeutic strategies.
HAdenovirs (HAdV) efficiently suppresses host DDR
A3A is not targeted by the HAdV E3 unique tin ligase
Oral Talk #105
The Enzymatic Activities of the Viral dUTPase and Viral Uracil-
DNA Glycosylase Stabilize the Gammaherpesvirus Genome
and Promote Pathogenesis in the Host
Qiwen Dong1, Kyle R Smith1, Darby G Oldenburg2, Maxwell G Shapiro1, William R Schutt1, Joshua Plummer3, Yunxiang Mu3, Thomas MacCarthy1, Douglas W White2, Kevin M McBride3, Laurie T Krug1
1Stony Brook University, 2Gundersen Health System, 3MD Anderson Cancer Center
Misincorporation of uracil or spontaneous cytidine deamination are common mutagenic insults to DNA. Herpesviruses encode a viral uracil DNA glycosylase (vUNG) and a viral dUTPase (vDUT), each with enzymatic and non-enzymatic functions. However, the coordinated roles of these activities in gammaherpesvirus pathogenesis and genomic stability have not been defined. In addition, the potential compensation by the host UNG has not been examined in vivo. The genetic tractability of the murine gammaherpesvirus 68 (MHV68) pathogen system enabled us to delineate the contribution of host and viral factors that prevent uracilated DNA. Recombinant MHV68 lacking vUNG (ORF46.stop) was not further impaired for acute replication in the lungs of UNG-/- mice compared to WT mice, indicating the vUNG provides non-overlapping functions from host UNG. Next, we investigated the separate and combinatorial consequences of mutating the catalytic residues of the vUNG (ORF46.CM) and vDUT (ORF54.CM). ORF46.CM was not impaired for replication, while ORF54.CM had a slight transient defect in lung replication. However, disabling both the vUNG and the vDUT enzymatic activities led to a significant defect in acute expansion in the lungs, followed by impaired establishment of latency in the splenic reservoir. Upon serial passage of the double ORF46.CM/ORF54.CM mutant in either fibroblasts or the lungs of mice, we noted rapid loss of the non-essential YFP reporter gene from the viral genome, consistent with an increase in homologous recombination. Taken together, our data indicate that the vUNG and vDUT coordinate to promote viral genomic stability and enable viral expansion prior to colonization of latent reservoirs. We are currently examining potential additional mutations and genomic rearrangements in the double mutant virus by whole-genome sequencing of plaque-purified viruses derived from infected lungs. We propose that the vUNG and vDUT are required to prevent mutagenic insults due to uracil incorporation that are detrimental to herpesvirus fitness.
DNA Glycosylase Stabilize the Gammaherpesvirus Genome
and Promote Pathogenesis in the Host
Qiwen Dong1, Kyle R Smith1, Darby G Oldenburg2, Maxwell G Shapiro1, William R Schutt1, Joshua Plummer3, Yunxiang Mu3, Thomas MacCarthy1, Douglas W White2, Kevin M McBride3, Laurie T Krug1
1Stony Brook University, 2Gundersen Health System, 3MD Anderson Cancer Center
Misincorporation of uracil or spontaneous cytidine deamination are common mutagenic insults to DNA. Herpesviruses encode a viral uracil DNA glycosylase (vUNG) and a viral dUTPase (vDUT), each with enzymatic and non-enzymatic functions. However, the coordinated roles of these activities in gammaherpesvirus pathogenesis and genomic stability have not been defined. In addition, the potential compensation by the host UNG has not been examined in vivo. The genetic tractability of the murine gammaherpesvirus 68 (MHV68) pathogen system enabled us to delineate the contribution of host and viral factors that prevent uracilated DNA. Recombinant MHV68 lacking vUNG (ORF46.stop) was not further impaired for acute replication in the lungs of UNG-/- mice compared to WT mice, indicating the vUNG provides non-overlapping functions from host UNG. Next, we investigated the separate and combinatorial consequences of mutating the catalytic residues of the vUNG (ORF46.CM) and vDUT (ORF54.CM). ORF46.CM was not impaired for replication, while ORF54.CM had a slight transient defect in lung replication. However, disabling both the vUNG and the vDUT enzymatic activities led to a significant defect in acute expansion in the lungs, followed by impaired establishment of latency in the splenic reservoir. Upon serial passage of the double ORF46.CM/ORF54.CM mutant in either fibroblasts or the lungs of mice, we noted rapid loss of the non-essential YFP reporter gene from the viral genome, consistent with an increase in homologous recombination. Taken together, our data indicate that the vUNG and vDUT coordinate to promote viral genomic stability and enable viral expansion prior to colonization of latent reservoirs. We are currently examining potential additional mutations and genomic rearrangements in the double mutant virus by whole-genome sequencing of plaque-purified viruses derived from infected lungs. We propose that the vUNG and vDUT are required to prevent mutagenic insults due to uracil incorporation that are detrimental to herpesvirus fitness.
Consequences of uracil in DNA
- Gammaherpesviral UNG
- ORF46.CM is expressed but not enzymatically active, no growth defect in cell culture
- No latency defect in spleen
- No additional defect in UNG-/- mice
- Compensation of single enzymatic bUNG mutant by viral dUTPase
ORF54 (dUTPas) KO mice
- Diminish latency in spleen
ORF46 and ORF54 KO showed decreased ??
Impaired replication and destabilized viral genome
- Coordinated activities protects viral genome from hist repair
- Enzymatic independent functions for vUNG
- Nucleotide metabolism is a critical factor in pathogenesis
- Misincorporation uracils is deleterious to replication vUNG has biochemical and functional properties distinct from host (McBride)
Oral Talk #106
Epstein-Barr Virus Deubiquitinase BPLF1 Targets the RIG-I
Signalosome to Inhibit the IFN Response
Soham Gupta1, Päivi Ylä-Anttila1, Maria Grazia Masucci1
1Karolinska Institutet
As part of the host innate antiviral response, viral nucleic acids are recognized by pattern- recognition receptors (PRRs) and cytosolic retinoic acid-inducible gene-I (RIG-I)-like helicases (RLHs), which activates signaling pathways resulting in the production of type I IFN and pro-inflammatory cytokines. The activation RIG-I promotes the formation of the RIG-I signalosome where the 14-3-3 molecule scaffolds the interaction of RIG-I with the TRIM25 ubiquitin ligase, leading to RIG-I ubiquitination and translocation of the complex to the mitochondrial anti-viral signaling proteins (MAVS) for downstream signaling. Using a co-immunoprecipitation and mass-spectrometry approach we found that the BPLF1 N- terminal cysteine protease binds to all members of the 14-3-3-family of molecular scaffolds. BPLF1 and 14-3-3 form a tri-molecular complex with TRIM25, which results in activation and autoubiquitination of the ligase. In the presence of catalytically active BPLF1 polyubiquitin chains are trimmed to mono and di-ubiquitin species. RIG-I is still recruited to the complex but, in the presence of the viral enzyme its ubiquitination is considerably reduced, which effectively inhibits downstream signaling including phosphorylation and nuclear translocation of the interferon regulatory factor 3 (IRF3) and transcriptional upregulation of type I IFN. These findings illustrate a new strategy used by the virus to bypass cellular and immune control mechanisms. The large tegument proteins of herpesviruses are expressed during virus replication and are packaged into virus particles for delivery to newly infected cells Thus, their enzymatic activity may play a double role in the virus life cycle by extending the time of unchecked productive infection, and by participating in the cellular reprogramming that allows the establishment of latent infections.
Signalosome to Inhibit the IFN Response
Soham Gupta1, Päivi Ylä-Anttila1, Maria Grazia Masucci1
1Karolinska Institutet
As part of the host innate antiviral response, viral nucleic acids are recognized by pattern- recognition receptors (PRRs) and cytosolic retinoic acid-inducible gene-I (RIG-I)-like helicases (RLHs), which activates signaling pathways resulting in the production of type I IFN and pro-inflammatory cytokines. The activation RIG-I promotes the formation of the RIG-I signalosome where the 14-3-3 molecule scaffolds the interaction of RIG-I with the TRIM25 ubiquitin ligase, leading to RIG-I ubiquitination and translocation of the complex to the mitochondrial anti-viral signaling proteins (MAVS) for downstream signaling. Using a co-immunoprecipitation and mass-spectrometry approach we found that the BPLF1 N- terminal cysteine protease binds to all members of the 14-3-3-family of molecular scaffolds. BPLF1 and 14-3-3 form a tri-molecular complex with TRIM25, which results in activation and autoubiquitination of the ligase. In the presence of catalytically active BPLF1 polyubiquitin chains are trimmed to mono and di-ubiquitin species. RIG-I is still recruited to the complex but, in the presence of the viral enzyme its ubiquitination is considerably reduced, which effectively inhibits downstream signaling including phosphorylation and nuclear translocation of the interferon regulatory factor 3 (IRF3) and transcriptional upregulation of type I IFN. These findings illustrate a new strategy used by the virus to bypass cellular and immune control mechanisms. The large tegument proteins of herpesviruses are expressed during virus replication and are packaged into virus particles for delivery to newly infected cells Thus, their enzymatic activity may play a double role in the virus life cycle by extending the time of unchecked productive infection, and by participating in the cellular reprogramming that allows the establishment of latent infections.
PLoS Pathogens 2018
BPLF1, the largest tegument protein of EBV
72 BPLF1 interacting proteins identified, highest hit
YWHA contains 14-3-3 proteins
Highly conserved ubiquitous proteins
Seven members: b, epsilon, gamma..
E3 ligase: TRIM25
BPLF1 promotes auto-ubiquitination of TRIM25
BPLF2 inhibits RIG-1 ubiquitination
BPLF1 BM does not promote autoubiquitination of TRIM25
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