Session Chairs: Hongyu Deng (a “She”) and Cliona Rooney
Oral Talk #90
The KSHV Protein ORF68 is a Proteasome-Manipulating
Nuclease Required for DNA Packaging
Matthew Gardner1, Stephanie Gates1, Andreas Martin1, Britt Glaunsinger1
1The University of California Berkeley
Six viral proteins are required for DNA packaging into nascent herpesviral capsids and for HSV, the majority of these proteins have well established functions as part of the viral terminase complex. However, one of these proteins, UL32 in HSV-1 and ORF68 in KSHV, has a largely undefined function. We generated an ORF68.stop mutant of KSHV and confirmed that ORF68 was not required for viral gene expression or DNA replication but was essential for infectious virion production. Additionally, KSHV ORF68.stop virus did not cleave or package viral DNA and exclusively accumulated immature B-capsids. Further biochemical characterization of ORF68 revealed several novel activities for this protein that significantly expand our understanding of its function. Unexpectedly, we found that ORF68 robustly binds the KSHV terminal repeats and exhibits metal-dependent nuclease activity and dsDNA cleavage in vitro. These observations suggest a role in binding and catalytically processing the viral genome during packaging. Finally, protein-protein interaction profiling of ORF68 revealed a striking association with multiple subunits of the 20S proteasome, the main protein degradation apparatus of the cell. We revealed that ORF68 contains a canonical gate-opening motif observed in proteasome-interacting proteins. Deletion or blocking of this gate-opening motif in ORF68 inhibited the degradation of a model substrate in transfected cells, suggesting that ORF68 may assemble on proteasomes to control substrate access. Electron microscopy of recombinant ORF68 showed that it forms pentameric rings with central pores, reminiscent of cellular proteasome activators. Notably, deletion of this gate-opening motif of ORF68 in the context of KSHV prevented progeny virion production, indicating that this function is central to its role in the viral replication cycle. Given that we observe proteasomes concentrated within replication compartments, we are currently exploring the possibility that ORF68 may control protein turnover in a manner important for viral DNA packaging.
Nuclease Required for DNA Packaging
Matthew Gardner1, Stephanie Gates1, Andreas Martin1, Britt Glaunsinger1
1The University of California Berkeley
Six viral proteins are required for DNA packaging into nascent herpesviral capsids and for HSV, the majority of these proteins have well established functions as part of the viral terminase complex. However, one of these proteins, UL32 in HSV-1 and ORF68 in KSHV, has a largely undefined function. We generated an ORF68.stop mutant of KSHV and confirmed that ORF68 was not required for viral gene expression or DNA replication but was essential for infectious virion production. Additionally, KSHV ORF68.stop virus did not cleave or package viral DNA and exclusively accumulated immature B-capsids. Further biochemical characterization of ORF68 revealed several novel activities for this protein that significantly expand our understanding of its function. Unexpectedly, we found that ORF68 robustly binds the KSHV terminal repeats and exhibits metal-dependent nuclease activity and dsDNA cleavage in vitro. These observations suggest a role in binding and catalytically processing the viral genome during packaging. Finally, protein-protein interaction profiling of ORF68 revealed a striking association with multiple subunits of the 20S proteasome, the main protein degradation apparatus of the cell. We revealed that ORF68 contains a canonical gate-opening motif observed in proteasome-interacting proteins. Deletion or blocking of this gate-opening motif in ORF68 inhibited the degradation of a model substrate in transfected cells, suggesting that ORF68 may assemble on proteasomes to control substrate access. Electron microscopy of recombinant ORF68 showed that it forms pentameric rings with central pores, reminiscent of cellular proteasome activators. Notably, deletion of this gate-opening motif of ORF68 in the context of KSHV prevented progeny virion production, indicating that this function is central to its role in the viral replication cycle. Given that we observe proteasomes concentrated within replication compartments, we are currently exploring the possibility that ORF68 may control protein turnover in a manner important for viral DNA packaging.
DNA packaging is a tightly controlled process
- ORF68 is responsible for cleavage of FNA
- KSHV lacking ORF68 cannot cleave the replicated genome
- Only B-capsizes are observed in the ORF68PTC virus, suggesting no problem in DNA replication but defective in packaging
- ORF68 bonds DNA in vitro with high affinity
- The KSHV ointeractome sows ORF68 associating with the proteasome
Mechanism of 20S gate
- ORF68 co-purifies with the host proteasome (beta 7)
- ORF68 contains a canonical HbYX gate ioenuing mooted so as (EBV BFLF1)
- ORF68 C-terminal modifications inhibit proteasome degradation EGFP+PEST
- DeltaLYA ORF68 loss the
- ORF68 exists a s a pentameric ring in solution
- Deletion of the HbYX motif prevents the production of visions (viral supernatant transfer)
- ORF68 is recruited to the replicating DNAs to chop off Pol II or enzymes like that.
Oral Talk #91
Epstein-Barr Virus Nuclear Antigen-1, Action and Reaction as
an Oncogene
Sana Alqarni1, Joanna Wilson1
1College of Medical, Veterinary and Life Sciences, University of Glasgow
Epstein-Barr virus (EBV) is associated with several tumours of B-cell and epithelia cell origin. EBV nuclear antigen 1 (EBNA1) is an essential viral protein, required for the maintenance, replication and mitotic segregation of viral genome episomes. EBNA1 is a DNA binding protein and also interacts with several cellular proteins, thereby affecting host cell-signalling pathways and in so doing may contribute to cell survival and proliferation. EBNA1 is the only latent protein that is expressed in all EBV-associated malignancies and indeed is the only viral protein consistently expressed in Burkitt lymphoma (BL) cells.
In order to explore any oncogenic mechanism attributable to EBNA1, we have studied our transgenic mouse model of BL; mice expressing EBNA1 in B-cells, which succumb to B- cell lymphoma, denoted EμEBNA1 mice. Selected cellular proteins potentially involved in the tumourigenic process were examined, including C-myc, Mdm2, E2F1, p53, Stat1, c- Fos, PTEN, Xiap and others.
Overexpression of specific Mdm2 isoforms were detected in all EBNA1 tumour samples, but not detected in the tumour samples from Eμc-Myc mice, or in non-tumour or transgene negative samples. In addition, C-Myc was overexpressed in EBNA1 tumours, supporting our previous observations regarding the cooperation of these two proteins in tumourigenesis. Using four Mdm2 inhibitors revealed that the EμEBNA1 tumour cells are dependant upon Mdm2 for survival. Additionally, Mdm2 inhibition led to loss of E2F1 expression, linking EBNA1 to Mdm2 and thence to the cell cycle. These data provide new insights into the consequences of EBNA1 expression.
an Oncogene
Sana Alqarni1, Joanna Wilson1
1College of Medical, Veterinary and Life Sciences, University of Glasgow
Epstein-Barr virus (EBV) is associated with several tumours of B-cell and epithelia cell origin. EBV nuclear antigen 1 (EBNA1) is an essential viral protein, required for the maintenance, replication and mitotic segregation of viral genome episomes. EBNA1 is a DNA binding protein and also interacts with several cellular proteins, thereby affecting host cell-signalling pathways and in so doing may contribute to cell survival and proliferation. EBNA1 is the only latent protein that is expressed in all EBV-associated malignancies and indeed is the only viral protein consistently expressed in Burkitt lymphoma (BL) cells.
In order to explore any oncogenic mechanism attributable to EBNA1, we have studied our transgenic mouse model of BL; mice expressing EBNA1 in B-cells, which succumb to B- cell lymphoma, denoted EμEBNA1 mice. Selected cellular proteins potentially involved in the tumourigenic process were examined, including C-myc, Mdm2, E2F1, p53, Stat1, c- Fos, PTEN, Xiap and others.
Overexpression of specific Mdm2 isoforms were detected in all EBNA1 tumour samples, but not detected in the tumour samples from Eμc-Myc mice, or in non-tumour or transgene negative samples. In addition, C-Myc was overexpressed in EBNA1 tumours, supporting our previous observations regarding the cooperation of these two proteins in tumourigenesis. Using four Mdm2 inhibitors revealed that the EμEBNA1 tumour cells are dependant upon Mdm2 for survival. Additionally, Mdm2 inhibition led to loss of E2F1 expression, linking EBNA1 to Mdm2 and thence to the cell cycle. These data provide new insights into the consequences of EBNA1 expression.
USP7 K447 IN THE DNA BINDING /DIMERIZATION domain of EBNA1
Wilson et al 1996 EMBO J 15:
- Line 59 : Transgenic EBNA1 EuEBNA1.59 (chr )
- Line 26: Transgenic EBNA1 EuEBNA1.26
Line 26 appears to be sunected to translation suppression compared to line 59.
The line showing translation suppression shows treated tumor penetrate
Translation suppression Nature (2017)
Linking GlyALa to
Induction / activation of cellular genes in
Eu EBNA1 lymphoma and IL2
EuEBNA1 lymphoma, p53 and mdm2
Mdm2 are multiply spliced pser186
MDM2 is essential for survival of the EBNA1 transgenic lymphomas
Wilson JB[Author] AND (EBV[tiab] or KSHV[tiab])
Oral Talk #92
Genome-Wide Identification of Direct RTA Targets Reveals Key
Host Factors for KSHV Lytic Reactivation
Bernadett Papp1, Naeem Motlagh2, Richard Smindak2, Seung-Jin Jang2, Aria Sharma2, Zsolt Toth3
1University of Florida College of Dentistry, Department of Oral Biology, UF Health Cancer Center, UF Genetics Institute, UF Informatics Institute, 2University of Florida College of Dentistry, Department of Oral Biology, 3University of Florida College of Dentistry, Department of Oral Biology, UF Health Cancer Center, UF Genetics Institute
Kaposi’s sarcoma-associated herpesvirus (KSHV) maintains dormancy in the body, but can reactivate which requires the essential replication and transcription activator (RTA). While it is established that RTA is required for the induction of lytic viral genes for viral reactivation, it is still unknown to what extent RTA alters the host transcriptome and epigenome in order to promote KSHV lytic cycle and pathogenesis. We hypothesized that RTA induces the expression of specific human factors, which may be critical for the subsequent virus production. Our genome-wide analysis using primary effusion lymphoma (PEL) cells showed that over one hundred human genes are directly and rapidly activated by RTA during KSHV reactivation. Furthermore, we found that RTA drives activation of cellular enhancers through epigenetic reprogramming and can also elevate transcription of active genes. We established that several of these human genes are also induced by RTA in other infected as well as uninfected cell types, highlighting them as core RTA-inducible human genes. Further investigation of one of these core RTA-induced human genes, Gamma-glutamyltransferase 6 (GGT6), established its critical role as a key player for KSHV production. GGT6 belongs to the membrane-bound GGT family of proteins, but its function remains uncharacterized. Importantly, shRNA knock down of GGT6 abrogated RTA expression and RTA-dependent lytic gene induction, KSHV production in various infected cell types such as BCBL1 and iSLKBAC16, but did not affect the latent gene LANA. Even though GGT6 depletion did not impair the induction of immediate-early EBV gene ZEBRA in KSHV/EBV co-infected JSC1 cells during viral reactivation, it’s depletion impaired KSHV lytic gene induction, revealing a highly specialized feed-forward loop between RTA and GGT6 for efficient KSHV reactivation. Our results demonstrate that host genes that RTA rapidly and directly induces can be pivotal for driving the KSHV lytic cycle, identifying them as future therapeutic targets.
Host Factors for KSHV Lytic Reactivation
Bernadett Papp1, Naeem Motlagh2, Richard Smindak2, Seung-Jin Jang2, Aria Sharma2, Zsolt Toth3
1University of Florida College of Dentistry, Department of Oral Biology, UF Health Cancer Center, UF Genetics Institute, UF Informatics Institute, 2University of Florida College of Dentistry, Department of Oral Biology, 3University of Florida College of Dentistry, Department of Oral Biology, UF Health Cancer Center, UF Genetics Institute
Kaposi’s sarcoma-associated herpesvirus (KSHV) maintains dormancy in the body, but can reactivate which requires the essential replication and transcription activator (RTA). While it is established that RTA is required for the induction of lytic viral genes for viral reactivation, it is still unknown to what extent RTA alters the host transcriptome and epigenome in order to promote KSHV lytic cycle and pathogenesis. We hypothesized that RTA induces the expression of specific human factors, which may be critical for the subsequent virus production. Our genome-wide analysis using primary effusion lymphoma (PEL) cells showed that over one hundred human genes are directly and rapidly activated by RTA during KSHV reactivation. Furthermore, we found that RTA drives activation of cellular enhancers through epigenetic reprogramming and can also elevate transcription of active genes. We established that several of these human genes are also induced by RTA in other infected as well as uninfected cell types, highlighting them as core RTA-inducible human genes. Further investigation of one of these core RTA-induced human genes, Gamma-glutamyltransferase 6 (GGT6), established its critical role as a key player for KSHV production. GGT6 belongs to the membrane-bound GGT family of proteins, but its function remains uncharacterized. Importantly, shRNA knock down of GGT6 abrogated RTA expression and RTA-dependent lytic gene induction, KSHV production in various infected cell types such as BCBL1 and iSLKBAC16, but did not affect the latent gene LANA. Even though GGT6 depletion did not impair the induction of immediate-early EBV gene ZEBRA in KSHV/EBV co-infected JSC1 cells during viral reactivation, it’s depletion impaired KSHV lytic gene induction, revealing a highly specialized feed-forward loop between RTA and GGT6 for efficient KSHV reactivation. Our results demonstrate that host genes that RTA rapidly and directly induces can be pivotal for driving the KSHV lytic cycle, identifying them as future therapeutic targets.
TREX-BCBL1-3XFLAG-KRTA
- RNA-seq at 0,6,12, 24 h pi
RTA on the genome prior to reactivation
- ChIP-seq
- 23 peaks identified at 12 hip on the KSHV genome
- De novo mixture under RTA peaks on KSHV best match is RBP-jk
- Viral chromatin changes during RTA expression
RTA bunting on the host genome during reactivation
- RTA binds to 11469 sites on the genome
- 80% are euchromatin-markers during reactivity
- RTA converts “unmarked” genomeiuc regions to H3K4me2+/H3K27ac+
- 20% if RTA binding sites are unmarked during latency
- 50@ of these unmarked sites
Deregulation;acted most gene expression in B cel lymphoma cells upon lytic KSHV reactivation
- induced 1280
- Repressed 1522
- 123 host genes are directly bound by RTA
- Characterization of the 123 core RTA bound and rapidly induced host genes
- Cellular component analysis
- Plasma membrane (30% belong to plasma membrane protein)
- GGT6
- HEY1, HEY2, APOBEC3C, 3H
Testing the role of RBP-JK in RTA induced cellular genes
RBP-JK is required for RTA driven reaction and rapid RTA driven induction of the majority of fire RTA target genes in uninflected cells, (iSLK cells KSKV-)
GGT6 is an characterized membrane protein
Gamma Glutamyl-transferase 6
Oral Talk #93
EBV Lytic Replication in mTOR-Inhibited Epithelial Cells is
Correlated with YY1 and Mnk1/2 Activities
Amy L. Adamson1, Dana Jeffus1, Steven Moran1, Alexis Davis1
1University of North Carolina at Greensboro
EBV lytic replication is dependent upon the expression of the immediate-early genes BZLF1 (Z) and BRLF1 (R). We found that the mTOR pathway plays a key role in regulating Z and R transcript levels in a cell type dependent manner. Here we describe our findings on how the transcription factor and target of mTOR, YY1, participates in the regulation of Z and R transcription under mTOR inhibition via rapamycin. We also demonstrate an association between rapamycin treatment and the increased presence of phosphorylated Mnk1/2 (MAPK interacting kinase 1/2), which persistently localizes with Z protein in EBV- positive epithelial cells, but not B cells. The persistent activation of Mnk under these conditions may facilitate continued, even increased, EBV lytic replication under conditions where translation should be halted.
Correlated with YY1 and Mnk1/2 Activities
Amy L. Adamson1, Dana Jeffus1, Steven Moran1, Alexis Davis1
1University of North Carolina at Greensboro
EBV lytic replication is dependent upon the expression of the immediate-early genes BZLF1 (Z) and BRLF1 (R). We found that the mTOR pathway plays a key role in regulating Z and R transcript levels in a cell type dependent manner. Here we describe our findings on how the transcription factor and target of mTOR, YY1, participates in the regulation of Z and R transcription under mTOR inhibition via rapamycin. We also demonstrate an association between rapamycin treatment and the increased presence of phosphorylated Mnk1/2 (MAPK interacting kinase 1/2), which persistently localizes with Z protein in EBV- positive epithelial cells, but not B cells. The persistent activation of Mnk under these conditions may facilitate continued, even increased, EBV lytic replication under conditions where translation should be halted.
EBV and MTOR
- MTOR as a modifier of Z and R
- Rapamycin effects on lytic replication are cell-type specific
- Up in epithelial cells
- Down in B cells
Is early lytic replication associated with activation of mTORC1 pathways?
- Different in B cos epithelial cells?
- Change when mTORC1 inhibited?
- P70
- Mnk1/2: highly responsive to EBV replication and is rapamycin dependent (AGS-BDneo)
- P-Mnl expression correlates with Z expression
- In epithelial cells, p-Mnk presence correlates with p-ERK
- In B cells, Z/p-Mnk presenc ecorrelated more with p-28
- P-eIF4E also associated with early lytic replication
- Time course of Z and p-Mnk (Z seems to appear first
- B: Rap or MEK inhibitor -> Z down and p-Mnk down (but not PML bodies)
- Preliminary: Mnk1/2 especially Mnk1 levels impact lytic
YY1 is an abundant TF, known to be an activator or repoessor , phosphorylation by MTOR
Generally a respressor of viral gene transcription
In response to mTORC1 inhibitorpm with rapamycin
YY1 inhibits X expression in B cells
Oral Talk #94
Interferon-Stimulated Gene Product RNF213 Modulates KSHV
Lytic Replication and Latency via Targeting RTA
Huabin Tian1, Kuai Yu1,2, Liang He1,2, Hongtao Xu1, Chuanhui Han1, Liguo Zhang1, Guangxia Gao1, Hongyu Deng1,2,3
1CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, 2University of the Chinese Academy of Sciences, 3CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
Kaposi’s sarcoma–associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus associated with several human malignancies, including Kaposi’s sarcoma and primary effusion lymphoma. The life cycle of KSHV infection consists of two phases: latency and lytic replication. The mechanisms by which host innate immune responses regulate KSHV latency remain largely unknown. We previously reported that RNF213, an interferon- stimulated gene product, inhibits the lytic replication of murine gammaherpesvirus-68 (MHV-68) through targeting MHV-68 RTA, the viral “molecular switch”. In this study, we further investigated the role of RNF213 in KSHV de novo infection and reactivation. We report that KSHV de novo infection induced RNF213 expression. Overexpression of RNF213 remarkably suppressed KSHV de novo infection as well as reactivation, whereas knock-down of endogenous RNF213 significantly promoted KSHV lytic infection and reactivation. Further analysis revealed that RNF213 interacted with KSHV RTA and served as an E3 ligase to ubiquitinate and degrade KSHV RTA through the proteasome pathway. Taken together, our results indicated that RNF213 plays an important role in modulating KSHV life cycle.
Lytic Replication and Latency via Targeting RTA
Huabin Tian1, Kuai Yu1,2, Liang He1,2, Hongtao Xu1, Chuanhui Han1, Liguo Zhang1, Guangxia Gao1, Hongyu Deng1,2,3
1CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, 2University of the Chinese Academy of Sciences, 3CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
Kaposi’s sarcoma–associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus associated with several human malignancies, including Kaposi’s sarcoma and primary effusion lymphoma. The life cycle of KSHV infection consists of two phases: latency and lytic replication. The mechanisms by which host innate immune responses regulate KSHV latency remain largely unknown. We previously reported that RNF213, an interferon- stimulated gene product, inhibits the lytic replication of murine gammaherpesvirus-68 (MHV-68) through targeting MHV-68 RTA, the viral “molecular switch”. In this study, we further investigated the role of RNF213 in KSHV de novo infection and reactivation. We report that KSHV de novo infection induced RNF213 expression. Overexpression of RNF213 remarkably suppressed KSHV de novo infection as well as reactivation, whereas knock-down of endogenous RNF213 significantly promoted KSHV lytic infection and reactivation. Further analysis revealed that RNF213 interacted with KSHV RTA and served as an E3 ligase to ubiquitinate and degrade KSHV RTA through the proteasome pathway. Taken together, our results indicated that RNF213 plays an important role in modulating KSHV life cycle.
Cytotoxic nuclei acid pattern recognition and activation of ISGs
RNF213
- 58.5 kD
- Expressed in all tissues, higher mRNA levels in immune tissues
- GWAS identified RNF213 as a susceptibility gene
De novo infection of KSHV in HUVEC cells increases RNA213 expression
Inhibition of KSHV de novo infection by RNA213 expression
RNA213 suppresses transcriptional activation by RTA
- Treatment of proteasome inhibitor MG132
- RNF213 promotes poly ubiquity national RTA via K48 linkage
Oral Talk #95
An Analog Sensitive Derivative of the Epstein Barr Virus v-Cdk
Permits the Direct Identification of Substrates Participating in
Multiple Stages of Gene Expression
Angie Umaña1, Robert F. Kalejta1
1University of Wisconsin-Madison
Viral cyclin-dependent kinases (v-Cdks) functionally emulate their cellular Cdk counterparts. Such viral mimicry is an established phenomenon that we extend here through chemical genetics. Kinases contain gatekeeper residues that limit the size of molecules that can be accommodated within the enzyme active site. Mutating gatekeeper residues to smaller amino acids allows larger molecules access to the active site. Such mutants can utilize bio-orthoganol ATPs for phosphate transfer and are inhibited by compounds ineffective against the wild type protein, and thus are referred to as analog- sensitive (AS) kinases. We identified the gatekeeper residue of the v-Cdk encoded by the Epstein-Barr Virus (EBV) and mutated it to generate an active AS kinase. This AS-v-Cdk was used to transfer a thiolated phosphate group to targeted proteins which were then purified through covalent capture and identified by mass spectrometry. Pathway analysis of these newly identified direct substrates of the EBV v-Cdk extends the potential influence of this kinase into all stages of gene expression (transcription, splicing, mRNA export, and translation). Our work demonstrates the biochemical similarity of the cellular and viral CDKs, as well as the utility of AS v-Cdks for substrate identification to increase our understanding of Cdk biology, viral infections and cancer.
Identifying direct substrate of BGLF4
Permits the Direct Identification of Substrates Participating in
Multiple Stages of Gene Expression
Angie Umaña1, Robert F. Kalejta1
1University of Wisconsin-Madison
Viral cyclin-dependent kinases (v-Cdks) functionally emulate their cellular Cdk counterparts. Such viral mimicry is an established phenomenon that we extend here through chemical genetics. Kinases contain gatekeeper residues that limit the size of molecules that can be accommodated within the enzyme active site. Mutating gatekeeper residues to smaller amino acids allows larger molecules access to the active site. Such mutants can utilize bio-orthoganol ATPs for phosphate transfer and are inhibited by compounds ineffective against the wild type protein, and thus are referred to as analog- sensitive (AS) kinases. We identified the gatekeeper residue of the v-Cdk encoded by the Epstein-Barr Virus (EBV) and mutated it to generate an active AS kinase. This AS-v-Cdk was used to transfer a thiolated phosphate group to targeted proteins which were then purified through covalent capture and identified by mass spectrometry. Pathway analysis of these newly identified direct substrates of the EBV v-Cdk extends the potential influence of this kinase into all stages of gene expression (transcription, splicing, mRNA export, and translation). Our work demonstrates the biochemical similarity of the cellular and viral CDKs, as well as the utility of AS v-Cdks for substrate identification to increase our understanding of Cdk biology, viral infections and cancer.
Identifying direct substrate of BGLF4
- Bump ATP PNBM
- Bio-orhtoganol ATP
- ATP analog (AD) gatekeeper residue
- By structural homologous Cdk2 to identify gatekeeper residue —> analog sensitive
- CHALFMPQFRC SLQD
- Identifying AS-kinases through selective inhibition
- Labeling and capturing substrates with AS-kinases
- AS1 and AS2 : 21 proteins common