Oral Talk #29
The EBV Glycoprotein BDLF2 is Essential for the Infection of
Stratified Epithelium
Ian Hayman1, Joshua Walston1, Mindy Gore2, Lindsey Hutt-Fletcher2, Clare E. Sample1
1Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, 2Department of Microbiology and Immunology, Louisiana State University Health Sciences Center
Epstein-Barr virus (EBV) is a widespread human pathogen associated with several neoplastic diseases of the epithelial compartment. Despite the global public health impact of EBV, there are significant unanswered questions regarding the lifecycle of EBV within the epithelium. We recently developed an organotypic culture model using primary keratinocytes that allows for efficient infection and spread of EBV. In this model, isolated productively replicating cells are observed as early as two days postinfection. The number of productively replicating cells increases very quickly over the next few days, suggesting that the virus may be spreading intercellularly throughout the epithelium. BDLF2, the most recently described of the eleven glycoproteins of EBV, is thought to form a complex with glycoprotein BMRF2. The complex is homologous to one encoded by murine gammaherpesvirus 68 that is essential for intercellular spread. Using a recombinant EBV deleted for BDLF2, we demonstrated that the loss of BDLF2 did not affect virion production or infection of primary B cells, B cell lines or 293 cells. In stratified epithelium, however, infection with virus lacking BDLF2 resulted in a limited number of isolated cells undergoing productive replication, whereas rafts infected with wildtype recombinant virus exhibited a broad expanse of productively replicating cells. These data support the hypothesis that BDLF2 is required for efficient infection of stratified epithelium, and that the loss of this glycoprotein results in reduced intercellular spread.
Stratified Epithelium
Ian Hayman1, Joshua Walston1, Mindy Gore2, Lindsey Hutt-Fletcher2, Clare E. Sample1
1Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, 2Department of Microbiology and Immunology, Louisiana State University Health Sciences Center
Epstein-Barr virus (EBV) is a widespread human pathogen associated with several neoplastic diseases of the epithelial compartment. Despite the global public health impact of EBV, there are significant unanswered questions regarding the lifecycle of EBV within the epithelium. We recently developed an organotypic culture model using primary keratinocytes that allows for efficient infection and spread of EBV. In this model, isolated productively replicating cells are observed as early as two days postinfection. The number of productively replicating cells increases very quickly over the next few days, suggesting that the virus may be spreading intercellularly throughout the epithelium. BDLF2, the most recently described of the eleven glycoproteins of EBV, is thought to form a complex with glycoprotein BMRF2. The complex is homologous to one encoded by murine gammaherpesvirus 68 that is essential for intercellular spread. Using a recombinant EBV deleted for BDLF2, we demonstrated that the loss of BDLF2 did not affect virion production or infection of primary B cells, B cell lines or 293 cells. In stratified epithelium, however, infection with virus lacking BDLF2 resulted in a limited number of isolated cells undergoing productive replication, whereas rafts infected with wildtype recombinant virus exhibited a broad expanse of productively replicating cells. These data support the hypothesis that BDLF2 is required for efficient infection of stratified epithelium, and that the loss of this glycoprotein results in reduced intercellular spread.
BX-1 BXLF1 replaced with GFP by HR
delta BDLF2 BX-1 backbone with BDLF2 deleted
BX-1 and delta BDLF2 Akata rEBVs induce similarly in vitro
Both rEBVs can transform primary B cells in vitro, no effects on replication, nor egress
BDLF2 KO negatively impacts intracellular infection
BDLF2 KO Negatively Impacts infection in Raft cultures
results in fewer and smaller BZLF1+Foci
Oral Talk #30
Kaposi Sarcoma-Associated Herpesvirus gH Is Essential for
Viral Infection, but Not for Virion Assembly or Egress from
Cells and is an Ideal Target for Vaccine Development
Murali Muniraju1, Joshua Nyagol1,3, Lorraine Z. Mutsvunguma1, Joslyn Foley1, David H. Mulama1,2, Felix Wussow1, Anne K. Barasa1,3, Javier G. Ogembo1
1Beckman Research Institute of City of Hope, 2Masinde Muliro University of Science and Technology, 3University of Nairobi
Introduction: Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and other malignancies. To date, there is no licensed KSHV vaccine. Development of an effective vaccine continues to be limited by a poor understanding of how KSHV initiates the acute primary infection. Although several KSHV-host interactions have been identified, all prior experiments were performed in vitro; to date, the critical complement of viral envelope glycoproteins required for KSHV entry in vivo remains unresolved.
Hypothesis and Significance: We hypothesized that surface glycoproteins K8.1, gB, and gH/gL are critical for virus entry in vivo and that inclusion of these key targets in a virus-like particle (VLP) would yield an effective prophylactic KSHV vaccine candidate.
Approach: Using a bacterial artificial chromosome containing a complete recombinant KSHV genome (rKSHV), we generated and characterized mutant viruses in which we deleted gH, K8.1, or gB. We assessed the infectivity of the three mutant viruses in vitro and in humanized mice. We generated VLPs incorporating K8.1, gB, gH/gL complex, and latent protein LANA1 (KSHV-LPs). We immunized wild-type rabbits and mice with KSHV-LPS as a multivalent vaccine against KSHV infection.
Results: In our mutant rKSHV studies, gH was essential for viral infection of diverse cell types in vitro and in vivo, and K8.1 and gB supported infectious entry. None of the glycoproteins was essential for virion assembly or egress from infected cells. Our KSHV-LP studies showed that sera from rabbits or mice immunized with KSHV-LPs elicited elevated titers of neutralizing antibodies that blocked KSHV infection in vitro.
Discussion: This is the first report that KSHV gH is essential for virus infection, but not for virion assembly or egress. Thus, it functions as a critical component of an effective prophylactic KSHV.
Viral Infection, but Not for Virion Assembly or Egress from
Cells and is an Ideal Target for Vaccine Development
Murali Muniraju1, Joshua Nyagol1,3, Lorraine Z. Mutsvunguma1, Joslyn Foley1, David H. Mulama1,2, Felix Wussow1, Anne K. Barasa1,3, Javier G. Ogembo1
1Beckman Research Institute of City of Hope, 2Masinde Muliro University of Science and Technology, 3University of Nairobi
Introduction: Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and other malignancies. To date, there is no licensed KSHV vaccine. Development of an effective vaccine continues to be limited by a poor understanding of how KSHV initiates the acute primary infection. Although several KSHV-host interactions have been identified, all prior experiments were performed in vitro; to date, the critical complement of viral envelope glycoproteins required for KSHV entry in vivo remains unresolved.
Hypothesis and Significance: We hypothesized that surface glycoproteins K8.1, gB, and gH/gL are critical for virus entry in vivo and that inclusion of these key targets in a virus-like particle (VLP) would yield an effective prophylactic KSHV vaccine candidate.
Approach: Using a bacterial artificial chromosome containing a complete recombinant KSHV genome (rKSHV), we generated and characterized mutant viruses in which we deleted gH, K8.1, or gB. We assessed the infectivity of the three mutant viruses in vitro and in humanized mice. We generated VLPs incorporating K8.1, gB, gH/gL complex, and latent protein LANA1 (KSHV-LPs). We immunized wild-type rabbits and mice with KSHV-LPS as a multivalent vaccine against KSHV infection.
Results: In our mutant rKSHV studies, gH was essential for viral infection of diverse cell types in vitro and in vivo, and K8.1 and gB supported infectious entry. None of the glycoproteins was essential for virion assembly or egress from infected cells. Our KSHV-LP studies showed that sera from rabbits or mice immunized with KSHV-LPs elicited elevated titers of neutralizing antibodies that blocked KSHV infection in vitro.
Discussion: This is the first report that KSHV gH is essential for virus infection, but not for virion assembly or egress. Thus, it functions as a critical component of an effective prophylactic KSHV.
Three goals
1. elucidate whether KSHV glycoproteins （gH， K8.1 and gB) are dispensable /indispensable for virion entry, assembly or egress
2. construct, purify, and characterise KSHV-like particle incorporating four glycoproteins as a prophylactic vaccine candidate
3. ....
New Zealand white rabbits
Marmosets
Construction of rKSHVdeltagH， deltaK8.1，
mutant rKSHVdeltagH-eGFP egressed normally from iSLK cells
rKSHVdeltagH-eGFP virions are defective in iSLK cell free virus infection
HeLa， Vero， HUVEC， ==》 deletion of K8.1 increases infectivity！！
Role of glycoproteins in in KSHV infection
gH is indispensable while K8.1 is dispensable for viral infection
Goal 2： hypothesis： immunisation with multivalent KSHV-LPs will generate potent neutralising antibodies
NDV-platform： K8.1-gB-gH/gL+/-HR2 F VLPs productioun
K8.1-gB-gH/gL+ or -HR2 F VLPs
UV-KSHV elicited higher antibody reposnes than KSHV-LPs
Previoulsy, BRRF1( Na protein) BGLF2 (tegulmetn protein) were found to feedback to regulate immediate-early protein
v-PK （BGLF4） enhances BZLF1 promoter activity by
Li et al, PLoS Pathogens 2017 and JV 2018 ==》 KAP1+ATM +... regulated ZEBRA promoter （methylation）
v-PK induces phosphorylation f KAP1 at S824， to enhance BZLF1 expression
v-PK =》 ATM =〉 p-KAP1 =》 BZLF1
v-PK induces p-S824 KAp1 independent of other EBV components （IFA asay）
v-PK induces p-S824 KAP1 via cellular kinase ATM
v-PK efficiently sustains EBV lytic cascade
Conclusion: in the EBV genome there are multiple KAP1 sites, thus not only BZLF1 maybe other viral proteins are also enhanced by v-PK
A safe viral vaccine: develop a safe replication-competent virus as a vaccine
lack of latency（oncogenic component), replace the latency locus with an constitutive RTA expression
highly attenuated lytci replication （remove immune evasion genes （IFN antagonists：
DIP: deficient in immune evasion and persistance (KO)
DIP produces no detectable viruses in vivo
DIP protects against latent infection, elicits durable protection time， elicits virus-specific CD9+ T cells (ORF6 and ORF61 D8 T cells), also elicits robust memory precursor effector cells.
Both T cells and antibodies contribute to protectivity
DIP induces immune mediator expression， IFNbeta， IL1beta， IL12
DIP increases infiltrating ells in peritoneal cavity
DIP induces robust pDCs
Both endogenous EBV and its amplicon DNA increase by 100-1000 fold during its lytic phase (in very short time ～24h)
EBV amplification DNA is amplified 5-22 hours post induction
Meselsin-Stahl
M-S or density shift experiments measure changes in the density of DNAs resulting from the incorporation of heavy nucleotides during DNA synthesis
At 8pi, EBV DNA is rapidly synthesised HH DNA can be seen as early as 60 min
Both copies of oriLyt are likely used for EBV DNA synthesis (compare wild type P3HR1 and 4012 visible amplicon)
At 30hpi, the rate of duplication f EBV DNA has declined dramatically
Controlling DNA length provides evidence for discontinuois DNA synthesis late in its lytic phase (at late er on is characteristically slow and likely discontinuous)
PLPP， YASIL， YEEVL-cCOOH
Identification f K15 interacting proteins+/- reactivation （RTA+SB）
most of the interactions of K15
PI3K-C2a ， involved in primary cilium assembly， sonic hedgehog signaling， angiogenesis， receptor-mediated endocytosis and endocytic/exocytic trafficking
KO in mice is embryonic lethal
K15 recruits C2a in vesicular structures in the perinuclear area
C2a plays a role in K15 mediated signaling
C2a cointributs to KSHV lytic reactivation
C2a contributes to KSHV spontaneou lytic replication
Most of the interactions of K15 with cellular proteins occur during KSHV lytic replication
K15 associates with components of the endocytic machinery during lytic replication
K15 interacts and associates with the class-II PI3K， PI3K-C2a in vesicle like structures
recruitment of C2a is required for K15-mediates signaling
IE —》 E DNA replication enzymes/immune evasion
Late RNA from new DNA
3 things found （PLoS Pathogens Jun 2016）
Do all late genes depend on the pre-initiation complex （vPIC） for transcription
Overlapping EBV lytic transcripts
41 mRNAs in 16 nests
Cap Analysis of Gene Expression CAGE-seq
strand specific
50+ base pair tags
quantitative
Map TSS down to the nucleotide。（TSS peaks）
Experimental Sretup
p2089 EBV PBAC delta vPIC （delta BDLF）
delta DNA （=delta BALF2）
delta oriLyt
WT
no induction or induction+trans-complementation
CAGE-seq early gene examples （eg BGLF4， BGLF5）
CAGE-seq late gene examples （eg BFFR2， BFFR3）
BDLF2 appears to be non-canonical、、BLRF2 is a leaky gene
lytic gene dependencies
BLRF1， closer look
leaky genes —》 hybrid promoters of early and lytic （from newly synthesised DNA）
BGLF3 and BGLF4 regulate expression f EBV late genes
Current models for transcription of EBV late genes
Current topics in Mciro and Immunity 2015 McKenzie and El-Guindy
Does the BGLF4 protien kinase regulate thje function of vPLC
BGLF4 interacting ptoyerins nby Flag -Ip and MS/MS
BGLF3 co-immunopreciputates with BGLF4 and BGLF4（K/I）
BGLF3 is phosphrylated in vio at T42
BGLF3 phosphorylation sites are dispendible for viral DNA replication
BGLF3 T42 is essential for late gene expression
vIL10 and vDUB are expressed independent of BGLF3
Late gene regulators （aka vPIC）
BcRF1
BDLF4
BFRF2
BVLF1
Extopic expresion of vPIC components partially suppresses the phenotype of BGLF3（T42A）
BFRF2 and BVLF1 are sufficient to partialluy rescue thje defect in BGLF3（T42）
New Zealand white rabbits
Marmosets
Construction of rKSHVdeltagH， deltaK8.1，
mutant rKSHVdeltagH-eGFP egressed normally from iSLK cells
rKSHVdeltagH-eGFP virions are defective in iSLK cell free virus infection
HeLa， Vero， HUVEC， ==》 deletion of K8.1 increases infectivity！！
Role of glycoproteins in in KSHV infection
gH is indispensable while K8.1 is dispensable for viral infection
Goal 2： hypothesis： immunisation with multivalent KSHV-LPs will generate potent neutralising antibodies
NDV-platform： K8.1-gB-gH/gL+/-HR2 F VLPs productioun
K8.1-gB-gH/gL+ or -HR2 F VLPs
UV-KSHV elicited higher antibody reposnes than KSHV-LPs
Oral Talk #31
An Early Lytic Gene Regulates Immediate Early Gene BZLF1
Xiaofan Li1, Ayman El-Guindy2, Eric Burton3, Sumita Bhaduri-McIntosh1,4
1Division of Infectious Diseases, Department of Pediatrics, University of Florida, 2Division of Infectious Diseases, Department of Pediatrics, Yale University, 3Department of Molecular Genetics and Microbiology, Stony Brook University, 4Department of Molecular Genetics and Microbiology, University of Florida
DNA viruses replicate through tightly ordered expression of sets of temporally regulated genes. In herpesviruses, these sets of genes represent 3 kinetic classes: immediate-early, early, and late. Immediate-early genes regulate expression of early and late genes, with early genes regulating late genes. It is not clear whether this vectorially regulated viral gene expression also allows for retrograde regulation of immediate-early genes by kinetically downstream genes. Here, we report that the EBV viral protein kinase (vPK) encoded by an early lytic gene, BGLF4, regulates the expression of BZLF1, the immediate- early latency-to-lytic switch, in Burkitt lymphoma and lymphoblastoid cell lines. We find that vPK functions via ATM, the cellular PI3-kinase-related kinase, to cause phosphorylation of TRIM28/KAP1, the universal transcriptional co-repressor. When phosphorylated, TRIM28 derepresses BZLF1 encoded by the viral genome but not when encoded in an extra-viral context. To derepress BZLF1 in lytic cells, vPK directly interacts with ATM but not TRIM28. Notably, vPK can cause phosphorylation of TRIM28 in the absence of known lytic triggers or even viral genomes. Concurrent studies in the Bhaduri lab have shown that TRIM28 is recruited to the BZLF1 promoter through KRAB-ZFP DNA binding proteins. One outcome of this retrograde regulation of BZLF1 by vPK is to accelerate the completion of the EBV lytic cascade.
Xiaofan Li1, Ayman El-Guindy2, Eric Burton3, Sumita Bhaduri-McIntosh1,4
1Division of Infectious Diseases, Department of Pediatrics, University of Florida, 2Division of Infectious Diseases, Department of Pediatrics, Yale University, 3Department of Molecular Genetics and Microbiology, Stony Brook University, 4Department of Molecular Genetics and Microbiology, University of Florida
DNA viruses replicate through tightly ordered expression of sets of temporally regulated genes. In herpesviruses, these sets of genes represent 3 kinetic classes: immediate-early, early, and late. Immediate-early genes regulate expression of early and late genes, with early genes regulating late genes. It is not clear whether this vectorially regulated viral gene expression also allows for retrograde regulation of immediate-early genes by kinetically downstream genes. Here, we report that the EBV viral protein kinase (vPK) encoded by an early lytic gene, BGLF4, regulates the expression of BZLF1, the immediate- early latency-to-lytic switch, in Burkitt lymphoma and lymphoblastoid cell lines. We find that vPK functions via ATM, the cellular PI3-kinase-related kinase, to cause phosphorylation of TRIM28/KAP1, the universal transcriptional co-repressor. When phosphorylated, TRIM28 derepresses BZLF1 encoded by the viral genome but not when encoded in an extra-viral context. To derepress BZLF1 in lytic cells, vPK directly interacts with ATM but not TRIM28. Notably, vPK can cause phosphorylation of TRIM28 in the absence of known lytic triggers or even viral genomes. Concurrent studies in the Bhaduri lab have shown that TRIM28 is recruited to the BZLF1 promoter through KRAB-ZFP DNA binding proteins. One outcome of this retrograde regulation of BZLF1 by vPK is to accelerate the completion of the EBV lytic cascade.
Previoulsy, BRRF1( Na protein) BGLF2 (tegulmetn protein) were found to feedback to regulate immediate-early protein
v-PK （BGLF4） enhances BZLF1 promoter activity by
Li et al, PLoS Pathogens 2017 and JV 2018 ==》 KAP1+ATM +... regulated ZEBRA promoter （methylation）
v-PK induces phosphorylation f KAP1 at S824， to enhance BZLF1 expression
v-PK =》 ATM =〉 p-KAP1 =》 BZLF1
v-PK induces p-S824 KAp1 independent of other EBV components （IFA asay）
v-PK induces p-S824 KAP1 via cellular kinase ATM
v-PK efficiently sustains EBV lytic cascade
Conclusion: in the EBV genome there are multiple KAP1 sites, thus not only BZLF1 maybe other viral proteins are also enhanced by v-PK
Immune Mechanisms of Protection Against Latency from
Vaccination of a Genetically Engineered Gamma-Herpesvirus
Ting-Ting Wu1, Gurpreet Brar1, Nisar Farhat1, Alisa Sukhina1, Yong Hoon Kim1, Tiffany Hsu1, Marcia Blackman2, Ren Sun1
1University of California Los Angeles, 2The Trudeau Institute
Significant efforts have been made to develop vaccines against EBV with the focus on gp350. The gp350 vaccine reduces infectious mononucleosis but is unable to prevent infection in humans. In our laboratory, we investigate alternative strategies of vaccines to prevent gammaherpesvirus latent infection using MHV-68 as a model system. We constructed a recombinant virus by removing the immune evasion genes as a vaccine. Such a recombinant virus is able to induce the expression of multiple inflammatory genes; moreover, its replication in mice is severely attenuated and undetectable. Despite undetectable replication, the recombinant virus elicits similar levels of viral specific T cells and antibodies as the replicating wild-type virus. Immunization of this in vivo replication- deficient virus induces long-lasting protective immunity against latent infection from challenge of the wild-type virus. Here we present the results from the studies to characterize the mechanism of protection. Adoptive transfer of total T cells reduces latent infection in recipient mice. However, depletion of total T cells does not abolish vaccine- induced protection. This finding indicates that vaccine-induced antibodies alone are sufficient to provide protection against latent infection. Studies are underway to characterize the antibody responses.
Vaccination of a Genetically Engineered Gamma-Herpesvirus
Ting-Ting Wu1, Gurpreet Brar1, Nisar Farhat1, Alisa Sukhina1, Yong Hoon Kim1, Tiffany Hsu1, Marcia Blackman2, Ren Sun1
1University of California Los Angeles, 2The Trudeau Institute
Significant efforts have been made to develop vaccines against EBV with the focus on gp350. The gp350 vaccine reduces infectious mononucleosis but is unable to prevent infection in humans. In our laboratory, we investigate alternative strategies of vaccines to prevent gammaherpesvirus latent infection using MHV-68 as a model system. We constructed a recombinant virus by removing the immune evasion genes as a vaccine. Such a recombinant virus is able to induce the expression of multiple inflammatory genes; moreover, its replication in mice is severely attenuated and undetectable. Despite undetectable replication, the recombinant virus elicits similar levels of viral specific T cells and antibodies as the replicating wild-type virus. Immunization of this in vivo replication- deficient virus induces long-lasting protective immunity against latent infection from challenge of the wild-type virus. Here we present the results from the studies to characterize the mechanism of protection. Adoptive transfer of total T cells reduces latent infection in recipient mice. However, depletion of total T cells does not abolish vaccine- induced protection. This finding indicates that vaccine-induced antibodies alone are sufficient to provide protection against latent infection. Studies are underway to characterize the antibody responses.
A safe viral vaccine: develop a safe replication-competent virus as a vaccine
lack of latency（oncogenic component), replace the latency locus with an constitutive RTA expression
highly attenuated lytci replication （remove immune evasion genes （IFN antagonists：
DIP: deficient in immune evasion and persistance (KO)
DIP produces no detectable viruses in vivo
DIP protects against latent infection, elicits durable protection time， elicits virus-specific CD9+ T cells (ORF6 and ORF61 D8 T cells), also elicits robust memory precursor effector cells.
Both T cells and antibodies contribute to protectivity
DIP induces immune mediator expression， IFNbeta， IL1beta， IL12
DIP increases infiltrating ells in peritoneal cavity
DIP induces robust pDCs
Oral Talk #33
How is EBV's Genome Amplified during its Lytic Cycle?
Thejaswi Nagaraju1, Bill Sugden1
1University of Wisconsin-Madison
We are examining how EBV amplifies its DNA during its lytic cycle. qPCR measurements of EBV DNA indicate that EBV synthesizes up to 50% of its DNA during the first 10-20 hrs of its lytic cycle. Live-cell imaging has shown that EBV amplifies its DNA in factories that grow 20-fold in volume in the nucleus towards the nuclear membrane over the course of 20 hours post induction (hpi). The nuclear volume increases modestly during this time, by 1.6- fold. We mapped the rate of viral DNA synthesis per cell using EdU pulse-labelling and found that the rate of viral DNA synthesis rises 3-4 –fold during the first 10-15hrs of its lytic cycle. This rate subsequently remains approximately constant from 15-40 hpi before sharply dropping at ~ 50 hpi. This time course further supports viral DNA accumulation being rapid early and then slowing. We have analyzed the rate of DNA synthesis using Meselson-Stahl analysis which indicates that the rate of EBV DNA synthesis slows between 20-30hrs following entry into its lytic cycle. Two color live-cell imaging of EBV DNA and its capsids show that the capsids begin to accumulate when DNA accumulation peaks about 20 hours into the lytic cycle. Our measurements indicate that EBV accumulates most of its DNA early in its lytic cycle and that it is the rate of elongation that limits its synthesis. We are now testing whether DNA encapsidation also contributes to the regulation of DNA accumulation.
Thejaswi Nagaraju1, Bill Sugden1
1University of Wisconsin-Madison
We are examining how EBV amplifies its DNA during its lytic cycle. qPCR measurements of EBV DNA indicate that EBV synthesizes up to 50% of its DNA during the first 10-20 hrs of its lytic cycle. Live-cell imaging has shown that EBV amplifies its DNA in factories that grow 20-fold in volume in the nucleus towards the nuclear membrane over the course of 20 hours post induction (hpi). The nuclear volume increases modestly during this time, by 1.6- fold. We mapped the rate of viral DNA synthesis per cell using EdU pulse-labelling and found that the rate of viral DNA synthesis rises 3-4 –fold during the first 10-15hrs of its lytic cycle. This rate subsequently remains approximately constant from 15-40 hpi before sharply dropping at ~ 50 hpi. This time course further supports viral DNA accumulation being rapid early and then slowing. We have analyzed the rate of DNA synthesis using Meselson-Stahl analysis which indicates that the rate of EBV DNA synthesis slows between 20-30hrs following entry into its lytic cycle. Two color live-cell imaging of EBV DNA and its capsids show that the capsids begin to accumulate when DNA accumulation peaks about 20 hours into the lytic cycle. Our measurements indicate that EBV accumulates most of its DNA early in its lytic cycle and that it is the rate of elongation that limits its synthesis. We are now testing whether DNA encapsidation also contributes to the regulation of DNA accumulation.
Both endogenous EBV and its amplicon DNA increase by 100-1000 fold during its lytic phase (in very short time ～24h)
EBV amplification DNA is amplified 5-22 hours post induction
Meselsin-Stahl
M-S or density shift experiments measure changes in the density of DNAs resulting from the incorporation of heavy nucleotides during DNA synthesis
At 8pi, EBV DNA is rapidly synthesised HH DNA can be seen as early as 60 min
Both copies of oriLyt are likely used for EBV DNA synthesis (compare wild type P3HR1 and 4012 visible amplicon)
At 30hpi, the rate of duplication f EBV DNA has declined dramatically
Controlling DNA length provides evidence for discontinuois DNA synthesis late in its lytic phase (at late er on is characteristically slow and likely discontinuous)
Oral Talk #34
The Kaposi's Sarcoma Herpesvirus Non-Structural Membrane
Protein pK15 Recruits the Class II PI3K Phosphatidylinositol 4-
phosphate 3-kinase C2 alpha to Activate Productive Viral
Replication
Bizunesh Abere1,2, Naira Samarina1,2, Silvia Gramolelli1,2, Jessica Rückert1,2, Gisa Gerold3, Andreas Pich4, Thomas F. Schulz1,2
1Institute of Virology, Hannover Medical School, 2German Centre for Infection Research, Hannover–Braunschweig Site, 3Institute for Experimental Virology, TWINCORE -Center for Experimental and Clinical Infection Research, 4Research Core Unit Proteomics, Institute of Toxicology, Hannover Medical School
Kaposi’s sarcoma-associated herpesvirus (KSHV)/Human herpesvirus-8 (HHV-8) causes the angiogenic tumor Kaposi’s Sarcoma (KS) and two B-cell malignancies. The KSHV non- structural membrane protein encoded by open reading frame (orf) K15 recruits and activates several cellular proteins including PLCγ1, components of the NF-κB pathway as well as members of the Src family of non-receptor tyrosine kinases and thereby plays an important role in the activation of angiogenic and inflammatory pathways that contribute to the pathogenesis of KS as well as KSHV productive (lytic) replication. In order to identify novel cellular components involved in the biology of pK15, we immunoprecipitated pK15 from KSHV-infected endothelial cells and identified associated proteins by label free quantitative mass spectrometry. Cellular proteins interacting with pK15 point to previously unappreciated cellular processes such as the endocytic pathway that could be involved in the function of pK15. We found the class II PI3K, PI3K-C2α, which is involved in the endocytosis of activated receptor tyrosine kinases and their signaling from intracellular organelles, to interact and co-localize with pK15 in vesicular structures abundant in the perinuclear area. Further functional analysis revealed that PI3K-C2α contributes to the pK15-dependent phosphorylation of PLCγ1 and Erk1/2. PI3K-C2α also plays a role in KSHV lytic replication as evidenced by the reduced expression of viral lytic genes K-bZIP and ORF 45 as well as the reduced release of infectious virus in PI3K-C2α -depleted KSHV-infected endothelial cells. Taken together, our results suggest a role of the cellular PI3K-C2α protein in the functional properties of the KSHV K15 protein.
Replication
Bizunesh Abere1,2, Naira Samarina1,2, Silvia Gramolelli1,2, Jessica Rückert1,2, Gisa Gerold3, Andreas Pich4, Thomas F. Schulz1,2
1Institute of Virology, Hannover Medical School, 2German Centre for Infection Research, Hannover–Braunschweig Site, 3Institute for Experimental Virology, TWINCORE -Center for Experimental and Clinical Infection Research, 4Research Core Unit Proteomics, Institute of Toxicology, Hannover Medical School
Kaposi’s sarcoma-associated herpesvirus (KSHV)/Human herpesvirus-8 (HHV-8) causes the angiogenic tumor Kaposi’s Sarcoma (KS) and two B-cell malignancies. The KSHV non- structural membrane protein encoded by open reading frame (orf) K15 recruits and activates several cellular proteins including PLCγ1, components of the NF-κB pathway as well as members of the Src family of non-receptor tyrosine kinases and thereby plays an important role in the activation of angiogenic and inflammatory pathways that contribute to the pathogenesis of KS as well as KSHV productive (lytic) replication. In order to identify novel cellular components involved in the biology of pK15, we immunoprecipitated pK15 from KSHV-infected endothelial cells and identified associated proteins by label free quantitative mass spectrometry. Cellular proteins interacting with pK15 point to previously unappreciated cellular processes such as the endocytic pathway that could be involved in the function of pK15. We found the class II PI3K, PI3K-C2α, which is involved in the endocytosis of activated receptor tyrosine kinases and their signaling from intracellular organelles, to interact and co-localize with pK15 in vesicular structures abundant in the perinuclear area. Further functional analysis revealed that PI3K-C2α contributes to the pK15-dependent phosphorylation of PLCγ1 and Erk1/2. PI3K-C2α also plays a role in KSHV lytic replication as evidenced by the reduced expression of viral lytic genes K-bZIP and ORF 45 as well as the reduced release of infectious virus in PI3K-C2α -depleted KSHV-infected endothelial cells. Taken together, our results suggest a role of the cellular PI3K-C2α protein in the functional properties of the KSHV K15 protein.
PLPP， YASIL， YEEVL-cCOOH
Identification f K15 interacting proteins+/- reactivation （RTA+SB）
most of the interactions of K15
PI3K-C2a ， involved in primary cilium assembly， sonic hedgehog signaling， angiogenesis， receptor-mediated endocytosis and endocytic/exocytic trafficking
KO in mice is embryonic lethal
K15 recruits C2a in vesicular structures in the perinuclear area
C2a plays a role in K15 mediated signaling
C2a cointributs to KSHV lytic reactivation
C2a contributes to KSHV spontaneou lytic replication
Most of the interactions of K15 with cellular proteins occur during KSHV lytic replication
K15 associates with components of the endocytic machinery during lytic replication
K15 interacts and associates with the class-II PI3K， PI3K-C2a in vesicle like structures
recruitment of C2a is required for K15-mediates signaling
Oral Talk #35
CAGE-seq Analysis of Epstein-Barr Virus Lytic Gene
Transcription: 3 Kinetic Classes from 2 Mechanisms
Reza Djavadian1,2, Mitchell Hayes2, Eric Johannsen1,2
1Department of Medicine (McArdle Laboratory for Cancer Research), University of Wisconsin School of Medicine and Public Health, 2Department of Oncology (McArdle Laboratory for Cancer Research), University of Wisconsin School of Medicine and Public Health
PLoS Pathog. 2018 Jun 4;14(6):e1007114. doi: 10.1371/journal.ppat.1007114. eCollection 2018 Jun.
Transcription: 3 Kinetic Classes from 2 Mechanisms
Reza Djavadian1,2, Mitchell Hayes2, Eric Johannsen1,2
1Department of Medicine (McArdle Laboratory for Cancer Research), University of Wisconsin School of Medicine and Public Health, 2Department of Oncology (McArdle Laboratory for Cancer Research), University of Wisconsin School of Medicine and Public Health
PLoS Pathog. 2018 Jun 4;14(6):e1007114. doi: 10.1371/journal.ppat.1007114. eCollection 2018 Jun.
CAGE-seq analysis of Epstein-Barr virus lytic gene transcription: 3 kinetic classes from 2 mechanisms.
Abstract
Epstein-Barr virus (EBV) lytic replication proceeds through an ordered cascade of gene expression that integrates lytic DNA amplification and late gene transcription. We and others previously demonstrated that 6 EBV proteins that have orthologs in β- and γ-, but not in α-herpesviruses, mediate late gene transcription in a lytic DNA replication-dependent manner. We proposed a model in which the βγ gene-encoded viral pre-initiation complex (vPIC) mediates transcription from newly replicated viral DNA. While this model explains the dependence of late gene transcription on lytic DNA replication, it does not account for this dependence in α-herpesviruses nor for recent reports that some EBV late genes are transcribed independently of vPIC. To rigorously define which transcription start sites (TSS) are dependent on viral lytic DNA replication or the βγ complex, we performed Cap Analysis of Gene Expression (CAGE)-seq on cells infected with wildtype EBV or EBV mutants defective for DNA replication, βγ function, or lacking an origin of lytic replication (OriLyt). This approach identified 16 true-late, 32 early, and 16 TSS that are active at low levels early and are further upregulated in a DNA replication-dependent manner (leaky late). Almost all late gene transcription is vPIC-dependent, with BCRF1 (vIL10), BDLF2, and BDLF3 transcripts being notable exceptions. We present evidence that leaky late transcription is not due to a distinct mechanism, but results from superimposition of the early and late transcription mechanisms at the same promoter. Our results represent the most comprehensive characterization of EBV lytic gene expression kinetics reported to date and suggest that most, but not all EBV late genes are vPIC-dependent.
- PMID:
- 29864140
IE —》 E DNA replication enzymes/immune evasion
Late RNA from new DNA
3 things found （PLoS Pathogens Jun 2016）
PLoS Pathog. 2016 Jun 27;12(6):e1005718. doi: 10.1371/journal.ppat.1005718. eCollection 2016 Jun.
An Epstein-Barr Virus-Encoded Protein Complex Requires an Origin of Lytic Replication In Cis to Mediate Late Gene Transcription.
Abstract
Epstein-Barr virus lytic replication is accomplished by an intricate cascade of gene expression that integrates viral DNA replication and structural protein synthesis. Most genes encoding structural proteins exhibit "true" late kinetics-their expression is strictly dependent on lytic DNA replication. Recently, the EBV BcRF1 gene was reported to encode a TATA box binding protein homolog, which preferentially recognizes the TATT sequence found in true late gene promoters. BcRF1 is one of seven EBV genes with homologs found in other β- and γ-, but not in α-herpesviruses. Using EBV BACmids, we systematically disrupted each of these "βγ" genes. We found that six of them, including BcRF1, exhibited an identical phenotype: intact viral DNA replication with loss of late gene expression. The proteins encoded by these six genes have been found by other investigators to form a viral protein complex that is essential for activation of TATT-containing reporters in EBV-negative 293 cells. Unexpectedly, in EBV infected 293 cells, we found that TATT reporter activation was weak and non-specific unless an EBV origin of lytic replication (OriLyt) was present in cis. Using two different replication-defective EBV genomes, we demonstrated that OriLyt-mediated DNA replication is required in cis for TATT reporter activation and for late gene expression from the EBV genome. We further demonstrate by fluorescence in situ hybridization that the late BcLF1 mRNA localizes to EBV DNA replication factories. These findings support a model in which EBV true late genes are only transcribed from newly replicated viral genomes. PMID: 27348612
Overlapping EBV lytic transcripts
41 mRNAs in 16 nests
Cap Analysis of Gene Expression CAGE-seq
strand specific
50+ base pair tags
quantitative
Map TSS down to the nucleotide。（TSS peaks）
Experimental Sretup
p2089 EBV PBAC delta vPIC （delta BDLF）
delta DNA （=delta BALF2）
delta oriLyt
WT
no induction or induction+trans-complementation
CAGE-seq early gene examples （eg BGLF4， BGLF5）
CAGE-seq late gene examples （eg BFFR2， BFFR3）
BDLF2 appears to be non-canonical、、BLRF2 is a leaky gene
lytic gene dependencies
BLRF1， closer look
leaky genes —》 hybrid promoters of early and lytic （from newly synthesised DNA）
Oral Talk #36
Phosphorylation Regulates the Function of the Epstein-Barr
Virus Pre-Initiation Complex in Transcription of Late Genes
Jinlin Li1, Ann W. Walsh1, Ayman S. El-Guindy1
1Yale School of Medicine
Epstein-Barr virus (EBV) encodes approximately 36 late genes that are crucial for many events in the life cycle of the virus. Some of these events include: capsid assembly, viral attachment, and immune suppression during de novo infection. Despite the importance of EBV late proteins, there is a fundamental gap in understanding the mechanisms that regulate their expression. Using genetic and genomic approaches, we previously identified two EBV-encoded regulators that are essential for transcription of late genes encoding structural proteins. These are BGLF3, an early lytic protein that has no cellular homologs or identifiable domains, and BGLF4, the viral Ser/Thr protein kinase. BGLF3 and BGLF4 are part of the viral pre-initiation complex (vPIC) that mediates transcription of late genes. The exact role of BGLF3 and BGLF4 in the process of late gene transcription is not known. Studying the interactome of BGLF4 during lytic infection, we found that BGLF4 interacts with BGLF3. In vitro phosphorylation experiments demonstrated that BGLF3 is a substrate for phosphorylation by BGLF4. Abolishing phosphorylation of BGLF3 disrupted transcription of late genes without affecting expression of early genes or amplification of the viral genome. Co-immunoprecipitation experiments suggest that phosphorylation of BGLF3 is necessary for the protein to form proper interactions with the transcription machinery. Our results provide novel insights into the mechanism that regulates the function of vPIC in transcription of late genes. Phosphorylation of BGLF3 by the BGLF4 protein kinase might serve as a checkpoint necessary for transcription of late genes. Drugs that target the ability of BGLF4 to phosphorylate BGLF3 could aid in the development of anti-EBV drugs that block virus propagation.
Virus Pre-Initiation Complex in Transcription of Late Genes
Jinlin Li1, Ann W. Walsh1, Ayman S. El-Guindy1
1Yale School of Medicine
Epstein-Barr virus (EBV) encodes approximately 36 late genes that are crucial for many events in the life cycle of the virus. Some of these events include: capsid assembly, viral attachment, and immune suppression during de novo infection. Despite the importance of EBV late proteins, there is a fundamental gap in understanding the mechanisms that regulate their expression. Using genetic and genomic approaches, we previously identified two EBV-encoded regulators that are essential for transcription of late genes encoding structural proteins. These are BGLF3, an early lytic protein that has no cellular homologs or identifiable domains, and BGLF4, the viral Ser/Thr protein kinase. BGLF3 and BGLF4 are part of the viral pre-initiation complex (vPIC) that mediates transcription of late genes. The exact role of BGLF3 and BGLF4 in the process of late gene transcription is not known. Studying the interactome of BGLF4 during lytic infection, we found that BGLF4 interacts with BGLF3. In vitro phosphorylation experiments demonstrated that BGLF3 is a substrate for phosphorylation by BGLF4. Abolishing phosphorylation of BGLF3 disrupted transcription of late genes without affecting expression of early genes or amplification of the viral genome. Co-immunoprecipitation experiments suggest that phosphorylation of BGLF3 is necessary for the protein to form proper interactions with the transcription machinery. Our results provide novel insights into the mechanism that regulates the function of vPIC in transcription of late genes. Phosphorylation of BGLF3 by the BGLF4 protein kinase might serve as a checkpoint necessary for transcription of late genes. Drugs that target the ability of BGLF4 to phosphorylate BGLF3 could aid in the development of anti-EBV drugs that block virus propagation.
BGLF3 and BGLF4 regulate expression f EBV late genes
Current models for transcription of EBV late genes
Current topics in Mciro and Immunity 2015 McKenzie and El-Guindy
Does the BGLF4 protien kinase regulate thje function of vPLC
BGLF4 interacting ptoyerins nby Flag -Ip and MS/MS
BGLF3 co-immunopreciputates with BGLF4 and BGLF4（K/I）
BGLF3 is phosphrylated in vio at T42
BGLF3 phosphorylation sites are dispendible for viral DNA replication
BGLF3 T42 is essential for late gene expression
vIL10 and vDUB are expressed independent of BGLF3
Late gene regulators （aka vPIC）
BcRF1
BDLF4
BFRF2
BVLF1
Extopic expresion of vPIC components partially suppresses the phenotype of BGLF3（T42A）
BFRF2 and BVLF1 are sufficient to partialluy rescue thje defect in BGLF3（T42）
