Source: OKLAHOMA STATE UNIVERSITY submitted to NRP
REGULATION OF LATENCY-REACTIVATION CYCLE BY ORF2 AND BETA-CATENIN/WNT SIGNALING PATHWAY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1012519
Grant No.
2017-67015-26674
Cumulative Award Amt.
$457,000.00
Proposal No.
2016-09370
Multistate No.
(N/A)
Project Start Date
Jul 15, 2017
Project End Date
Jul 14, 2021
Grant Year
2017
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Veterinary Pathobiology
Non Technical Summary
Bovine herpesvirus 1 (BoHV-1) is an important cofactor of bovine respiratory disease complex (BRDC) because productive infection affects the upper respiratory tract and suppresses host immune responses. Following acute infection, latency is established in the peripheral nervous systems. Stress stimulates reactivation from latency, which results in virus shedding and transmission. Consequently, BoHV-1 is a persistent BRDC threat to cattle. The only abundant BoHV-1 gene expressed during latency is the latency-related (LR) gene. Recent studies discovered that cellular factors that promote survival of latently infected neurons and allow them to perform their normal functions are expressed in latently infected TG neurons, but not neurons from uninfected calves or those undergoing reactivation from latency. Viral gene products expressed in latently infected neurons promote expression of these cellular products. The studies in this grant will define the mechanims by which viral gene products control the latency-reactivation cycle and how cellular products impact these steps. Completion of these studies will provide novel strategies to develop novel BoHV-1 vaccines that do not maintain latency: thus, stress-induced reactivation cannot occur.
Animal Health Component
0%
Research Effort Categories
Basic
90%
Applied
0%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31133101101100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1101 - Virology;
Goals / Objectives
Bovine herpesvirus 1 (BoHV-1) is an important cofactor of bovine respiratory disease complex because productive infection affects the upper respiratory tract and suppresses host immune responses. Following acute infection, latency is established in trigeminal ganglionic (TG) sensory neurons and stress stimulates reactivation from latency: thus, BoHV-1 is a persistent problem to cattle producers. The only abundant BoHV-1 gene expressed during latency is the latency-related (LR) gene. ORF2, a protein encoded by the LR gene, is important for the latency-reactivation cycle because it inhibits apoptosis and interacts with and inactivates a protein that inhibits neuronal differentiation (Notch). We recently discovered a cellular transcription factor (β-catenin) that is expressed in latently infected TG neurons, but not neurons from uninfected calves or those undergoing reactivation from latency. Interestingly, most beta-catenin+ neurons express ORF2. ORF2 interacts with beta-catenin, stimulates beta-catenin dependent transcription, and cooperates with beta-catenin to inhibit apoptosis. In neurons, the Wnt/beta-catenin signaling pathway promotes axonal growth and navigation to their synaptic targets: conversely disruption of the pathway results in neuro-degeneration and apoptosis. Proposed project activities are based on the following hypothesis: ORF2 and beta-catenin cooperate to promote repair of damaged neurons after infection: thus establishment and maintenance of latency is enhanced. The major goals of this grant are listed below.1. Identify how BoHV-1 regulates Wnt/beta-catenin signaling in TG neurons during latency-reactivation cycle.2. Examine the mechanism by which ORF2, a BoHV-1 expressed during latency, regulates beta-catenin dependent transcription. 3. Examine how ORF2 and Wnt/beta-catenin signaling pathway regulates the latency-reactivation cycle in calves.
Project Methods
For Specific Aim 1, we will use Next Generation Sequencing and immuno-hostochemistry approaches to identify cellular genes, including those that are regulated by the Wnt/beta-catenin signaling pathway, during the latency-reactivation cycle.For Specific Aim 2, we will utilize immuno-precipitation studies to provide further insight into how ORF interacts with cellualr proteins that regulate the Wnt/beta-catenin signaling pathway. Transient transfection assays will also examine the ability of ORF2 and ORF2 mutants to regulate beta-catenin dependent transcription.For Specific Aim 3, homologous recombination will be used to generate recombinant BoHV-1 LR mutant that expresses a stable beta-catenin protein in sensory neurons to determine whether beta-catenin can promote the maintenance of latency and restore stress-induced reactivation to the LR mutant. We will construct a LR mutant virus that will express ORF2 from the unique long region to test whether ORF2 expression can restore reacitvation from latency to this mutant. Calves will be used to examine these novel virus strains.

Progress 07/15/17 to 07/14/21

Outputs
Target Audience:Our target audience includes herpes virologists, microbiologists, cattle producers, veterinarians, and vaccine companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students and a former postdoc performed the studies described above as well as contributed to the publications for this project last year. How have the results been disseminated to communities of interest?These studies were presented at the 2021 International herpesvirus workshop and the American Society for Virology. Due to the COVID-19 pandemic, we did not attend any meetings this year. Our results were also published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?This was the last year of this grant.

Impacts
What was accomplished under these goals? 1. We have demonstrated that the Akt1 and Akt2 inhibit stress-induced transcription using a BoHV-1 promoter, immediate early transcription unit 1 (IEtu1), that drive expression of two key viral regulatory proteins, infected protein 0 (ICP0) and ICP4. Since we demonstrated that the Akt pathway is expressed at higher levels during latency versus reactivation, we predict this finding is important for maintaining a latent infection. 2. Recent studies also demonstrated that Akt3 promotes neuronal survival and differentiation. Akt3 expression is significantly higher during latency than during reactivation. it is also known that the Wnt signaling pathway activates Akt3 and Wnt is expressed at higher levels during latency. Hence, we predict Akt 3 promotes the maintenance of latency. 3. Recently published studies demonstrated that a non-coding viral RNA expressed during latency and beta-catenin also inhibits stress-induced activation of the IEtu1 promoter. These results indicate that viral products expressed during latency actively maintain latency. 4. Recently published studies have determined that two other Type I Nuclear Hormone Receptors, androgen receptor (AR) and progesterone receptor (PR) transactivate the bICP0 E promoter. Additional studies demonstrated AR and PR cooperate with the pioneer factor, Krüppel Like Factor 4 (KLF4), to stimulate bICP0 E promoter activity. Newly published studies demonstrated that KLF4/Sp1 binding sires are crucial for the cooperative transactivation of the bICP0 E promoter.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Sawant, L. N. Wijesekera, and C. Jones. 2020. Pioneer transcription factors, progesterone receptor and Kruppel like transcription factor 4, cooperatively stimulate the bovine herpesvirus 1 ICP0 early promoter and productive late protein expression. Virus Research 288:198115.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Sawant, L, P. Thunuguntla, and C. Jones. 2021. Cooperative activation of bovine herpesvirus 1 productive infection and viral regulatory promoters by androgen receptor and Kr�ppel-like transcription factors 4 and 15. Virology 552:63-72
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhao, J., N. Wijesekera, and C. Jones. 2021. Inhibition of stress induced viral promoters by a bovine herpesvirus 1 non-coding RNA and the cellular transcription factor, ?-catenin. International Journal of Molecular Sciences. 22:519. https://doi.org/10.3390/
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: El mayet F., K.S. Harrison, and Clinton Jones. 2021. Regulation of Kruppel?Like Factor 15 Expression by Herpes Simplex Virus Type 1 or Bovine Herpesvirus 1 Productive Infection. Viruses. 13, 1148. https://doi.org/10.3390/v13061148.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: Sawant L, J.B. Ostler, and C. Jones. 2021. A pioneer transcription factor and type I nuclear hormone receptors synergistically activate the bovine herpesvirus 1 infected cell protein 0 (ICP0) early promoter. IN PRESS, J Virology.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2021 Citation: Regulation of neurotropic herpesvirus productive infection and latency-reactivation cycle by glucocorticoid receptor and stress-induced transcription factors. 2021. Ostler, JB L. Sawant, K. Harrison, and C. Jones. In, HORMONES, REGULATORS AND VIRUSES. Elsiever.


Progress 07/15/19 to 07/14/20

Outputs
Target Audience:Our target audience includes herpesvirologists, cattle ranchers,veterinarians, and vaccine companies. Changes/Problems:The studies are making progress. Consequently, I do not feel that there is a need to make significant changes in the approach we are taking. What opportunities for training and professional development has the project provided?Graduate students and a former postdoc performed the studies described above as well as contributed to the publications for this project last year. How have the results been disseminated to communities of interest?These studies were presented at the 2019 International herpesvirus workshop. Due to the COVID-19 pandemic, we did not attend any meeting this year. A Ph.D. student in my lab, a graduate student in my lab, presented his dissertation defense this spring. Our results will be published in peer-reviewed journals and presented at professionalmeetings (American Society of Virology and the International Herpesvirus Workshop). The results of these studies will also bepresented at meetings sponsored by vaccine company meetings (Boehringer Manneim and Pfizer for example) and commoditygroups. This information will also be disseminated by giving seminars at universities and in a classroom setting. A web siteproviding updates of this research will also be included in my OSU web page. What do you plan to do during the next reporting period to accomplish the goals?1. We are completing the studies demonstrating that a viral non-coding RNA impairs stress-induced transcription. We will then write a manuscript and submit it to a peer-reviewed journal. 2. We are also examining in detail how the glucocorticoid receptor activates reactivation from latency and identify genes that are expressed at higher levels during latency.

Impacts
What was accomplished under these goals? 1. We have demonstrated that the Akt1 and Akt2 inhibit stress-induced transcription using a BoHV-1 promoter,immediate early transcription unit 1,that drive expression of two key viral regulatory proteins, infected protein 0 (ICP0) and ICP4. Since we demonstrated that the Akt pathway is expressed at higher levels during latency versus reactivation, we predict this finding is important for maintaining a latent infection. 2. Recent studies also demonstrated that Akt3 promotes neuronal survival and differentiation. Akt3 expression is significantly higher during latency than during reactivation. it is also known that the Wnt signaling pathway activates Akt3 and Wnt is expressed at higher levels during latency. Hence, we predict Akt 3 promotes the maintenance of latency. 3. Preliminary studies suggest that a non-coding viral RNA expressed during latency also inhibits stress-induced reactivation. These results indicate that viral products expressed during latency actively maintain latency.

Publications


    Progress 07/15/18 to 07/14/19

    Outputs
    Target Audience:Our target audience includes herpes virologists, cattle ranchers, veterinarians, and companies that are interested in developing novel vaccines for cattle. These results will be published in peer-reviewed journals and presented at professional meetings: for example, nternational Herpesvirus Workshop and/or American Society of Virology. The results of these studies will also be presented at meetings sponsored by vaccine company meetings and commodity groups. Finally, the information will be disseminated by presenting seminars at universities and in a classroom setting. A web site providing updates of this research will also be included in my OSU web page. Changes/Problems:The studies are making progress, and no major problems have been encountered. Hence, no major changes in the proposed studies are necessary. What opportunities for training and professional development has the project provided?The studies published in 2018 related to this grant were performed by 2 graduate students and a postdoctoral fellow. These individuals leanred new scientific techniques and gained experience performing detailed studies related to BoHV-1 latency. How have the results been disseminated to communities of interest? Publications described above One of the graduate students (Dr. Laximan Sawant) gave a seminar at Oklahoma State Unversity. Dr. Fouad El-Mayet defended his PhD dissertation and is now an assistant professor in Egypt. I gave a keynote lecture at the International Herpesvirus Workshop this year and a talk at CRAWAD describing these studies, What do you plan to do during the next reporting period to accomplish the goals?1. Identify how BoHV-1 regulates Wnt/beta-catenin signaling in TG neurons during latency-reactivation cycle. The RNA-Sequencing data is being mined further, whcih will provide insight into events that regulate latency and reactivation from latency. 2. Examine the mechanism by which ORF2, a BoHV-1 expressed during latency, regulates beta-catenin dependent transcription. Studies designed to compare how ORF2 stimulates beta-catenin dependent transcription but impairs stress-induced transcription will be exmained. 3. Examine how ORF2 and Wnt/beta-catenin signaling pathway regulates the latency-reactivation cycle in calves. We are testing whether ORF2 regulates Akt pathways, which are activated by the Wnt/beta-catenin pathway.

    Impacts
    What was accomplished under these goals? 1. Identify how BoHV-1 regulates Wnt/beta-catenin signaling in TG neurons during latency-reactivation cycle. We published a paper in Journal of Virology in 2018 demonstrating that the Wnt/beta-catenin pathway is active during latency, but is repressed during dexamethasone induced reactivation from latency. 2. Examine the mechanism by which ORF2, a BoHV-1 expressed during latency, regulates beta-catenin dependent transcription. We have demonstrated that ORF2 stimulates beta-catenin dependent transcription, whcih we believe plays a crucial role in maintaining a latent infection, We also demosntrated that ORF2 interacted with a cellular protein kinase (Akt3), which enhances cell survivial of sensory neurons. 3. Examine how ORF2 and Wnt/beta-catenin signaling pathway regulates the latency-reactivation cycle in calves. Recent studies have demonstrated that RNA sequences encompassing ORF2 interfere with stress-induced transcription. In addition, we demonstrated that the viral protein VP16, which stimulates immediate early transcription, stimulates beta-catenin dependent transcrition during late stages of productive infection.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Workman, A. L. Zhu, B.N. Keel, T.P.L. Smith, and C. Jones. 2018. The Wnt signaling pathway is differentially expressed during the bovine herpesvirus 1 latency-reactivation cycle: evidence that two protein kinases associated with neuronal survival (Akt3 and bone morphogenetic protein receptor 2) are expressed at higher levels during latency. J of Virology 92: e01937-17
    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Sawant, L., I. Kook, J.L. Vogel, T.M. Kristie, and C. Jones. 2018. The cellular coactivator HCF-1 is required for glucocorticoid receptor-mediated transcription of bovine herpesvirus 1 immediate early genes. J of Virology, 92: e00987-18.
    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Jefferson, V.A., K.A. Barber, F.S. El-mayet, C. Jones, B. Nanduri, and Florencia Meyer. 2018. Proteogenomic Identification of a novel bovine herpesvirus 1 gene that expresses a protein readily detected during productive infection. Viruses, 10,499; doi:10.3390/v10090499.
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: El-mayet, F.S., A.S. El-Habbaa, J. DOffay, and C. Jones. 2019. Synergistic activation of bovine herpesvirus 1 productive infection and viral regulatory promoters by the progesterone receptor and Kr�ppel-like transcription factor 15. J of Virology, 93: e01519-18.
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Guo, J., Q. Li, and C. Jones. 2019. The bovine herpesvirus 1 regulatory proteins, bICP4 and bICP22, are expressed during the escape from latency. J. of Neurovirology, 25: 42-49.
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Silvestro, C., C. Jones, A. Bratanich. 2019. Functional analysis of the latency related gene of bovine herpesvirus type 1 and 5. J Neurovirology, 25: 1-8.
    • Type: Book Chapters Status: Published Year Published: 2018 Citation: El-mayet, F.S., A.S. El-Habbaa, G.F. El-Bagoury, S.S.A. Sharawi, E.M. El-Nahas, and C. Jones. 2018. The Glucocorticoid Receptor and Certain KR�PPEL-Like Transcription Factors have the Potential to Synergistically Stimulate Bovine Herpesvirus 1 Transcription and Reactivation from Latency. In Transcriptional and Post-transcriptional Regulation, 978-1-78923-792-4. http://dx.doi.org/10.5772/intechopen.7545153
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Jones, C. 2019. Bovine herpesvirus 1 counteracts immune responses and immune-surveillance to enhance pathogenesis and virus transmission. Frontiers in Immunology, section Viral Immunology. doi:10.3389/fimmu.2019.01008.


    Progress 07/15/17 to 07/14/18

    Outputs
    Target Audience:Our target audience is wide because it reaches basic scientists and producers. This group of people includes cattle ranchers, dairy cattle producers, bovine health specialists, herpes virologists, and virologists were reached during the last year. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Many of these studies have been performed by a post-doctoral fellow and graduate student in my lab. These studies are very important for their training and professional development. How have the results been disseminated to communities of interest?Publications in peer-reviewed journals, as summarized above, are an important means of disseminating findings from ourstudies. The postdoc and I gave talks at the International Herpesvirus workshop last year. I also gave a talk at the NebraskaCenter for Virology Intercampus Meeting in 2017 (March 2017), University of California-Irvine (April 2017), and Coloradoalpha-herpesvirus Symposium (May 2017). I also presented talks at the Viral Pathogenesis Symposium, Eastern Virginia Medical School (June 24th, 2017), European Veterinary Herpesvirus Symposium (July 29, 2017 in Ghent, Belgium), and at the International Herpesvirus Workshop (August 1, 2017 in Ghent, Belgium). What do you plan to do during the next reporting period to accomplish the goals? Test whether ORF2 is stably associated with a beta-catenin-dependent promoter. Test whether beta-catenin influences stress-induced transcription. Examine additional cellular signaling pathways that are differentially regulated during the latency-reactivation cycle. Develop the mutants described in Objective 3.

    Impacts
    What was accomplished under these goals? Objective 1: We have identified more than 100 Wnt-responsive genes by RNA-seq studies that are differentially expressed in trigeminal calves (TG) of calves that are latently infected versus the same tissue from uninfected calves or during early stages of reactivation fromlatency. These studies revealed that Wnt agonists are expressed at higher levels during latency relative to uninfected calvesor during reactivation from latency. During stress-induced reactivation from latency, we found that Wnt agonists are inducedin TG. Strikingly, several of these Wnt antagonists are also expressed at higher levels in neurons of patients suffering fromAlzheimer's Disease or Parkinsons Disease adding support to our conclusions that expression of these proteins duringreactivation from latency is detrimental to maintaining a latent infection. These studies are paradigm shifting because it isknown that the Wnt signaling pathway is important for axonal growth, neuronal survival, and repair of damaged axons, allfeatures crucial for maintaining a life-long latent infection. Interestingly, these studies also determined that the mTORpathway, which is involved with HSV-1 reactivation from latency, is tightly regulated during the bovine herpesvirus 1 (BoHV-1)latency-reactivation cycle. We are still analyzing the RNA-Seq data to uncover other signaling pathways that are differentiallyregulated during the latency-reactivation cycle. Interestingly, we also found that the Wnt pathway is important for efficientproductive infection. Objective 2: We are testing whether ORF2 is associated with beta-catenin and DNA using chromatin immunoprecipitation assays. Preliminary studies suggest ORF2 is not stably bound to a beta-catenin-dependent promoter when beta-catenin is. We are confirming these studies. Objective 3: Mutant virus strains are being prepared to test whether ORF2 is required for inducing the Wnt-beta-catenin pathway during latency and whether a Wnt antagonist will interfere with this process.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhu, L, P. Thunuguntla , Y. Liu, M. Hancock, and C. Jones. 2017. The beta-catenin signaling pathway stimulates bovine herpesvirus 1 productive infection. Virology, 500: 91-95.
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Thunuguntla, P., F. S. El-mayet, and C. Jones. 2017. Bovine herpesvirus 1 can efficiently infect the human (SH-SY5Y) but not the mouse neuroblastoma cell line (Neuro-2A). 232:1-5 Virus Res
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhu, L, J. Thompson, F. Ma, J. Eudy, and C. Jones. 2017. Effects of the synthetic corticosteroid dexamethasone on bovine herpesvirus 1 productive infection. Virology:, 505: 71-79.
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhu, L., A. Workman, and C. Jones. 2017. A potential role for a beta-catenin coactivator (high mobility group AT-hook 1 protein) during the latency-reactivation cycle of bovine herpesvirus 1. J of Virology: 91: e02132-16
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhu, L. and C. Jones. The high mobility group AT-hook 1 protein stimulates bovine herpesvirus 1 productive infection. IN PRESS, Virus Res
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: El-mayet, F.S., Laximan Sawant, Prasanth Thunuguntla, and Clinton Jones. Combinatorial effects of the glucocorticoid receptor and Kr�ppel-like transcription factor 15 on bovine herpesvirus 1 transcription and productive infection. J of Virology 91: 91:e00904-17.
    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Workman, A. L. Zhu, B.N. Keel, T.P.L. Smith, and C. Jones. The Wnt signaling pathway is differentially expressed during the bovine herpesvirus 1 latency-reactivation cycle: evidence that two protein kinases associated with neuronal survival (Akt3 and bone morphogenetic protein receptor 2) are expressed at higher levels during latency. 2018. J of Virology 92: e01937-17