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
