Source: UNIVERSITY OF NEBRASKA submitted to
ANALYSIS OF VIRAL FACTORS THAT REGULATE THE BOVINE HERPESVIRUS 1 (BHV-1) LATENCY-REACTIVATION CYCLE.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0219185
Grant No.
2009-65119-05674
Project No.
NEB-39-154
Proposal No.
2009-01653
Multistate No.
(N/A)
Program Code
92522
Project Start Date
Sep 1, 2009
Project End Date
Aug 31, 2013
Grant Year
2014
Project Director
Jones, C. J.
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
Veterinary and Biomedical Sciences
Non Technical Summary
Bovine herpes virus 1 (BHV-1) is an important pathogen of cattle because it induces a variety of clinical disorders (conjunctivitus, tracheitis, and genital infections). Furthermore, BHV-1 is an initiator of bovine respiratory disease complex (BRDC). The ability of BHV-1 to induce immune-suppression enables secondary bacterial infections to cause pneumonia. BHV-1 establishes a latent infection in sensory neurons in trigeminal ganglia or lumbosacral ganglia. Periodically, BHV-1 reactivates from latency, and sheds infectious virus. During this time, BHV-1 can induce BRDC and is spread to uninfected hosts. The latency-reactivation cycle of BHV-1 is crucial for viral transmission and BRDC. Although modified live vaccines are available, these modified lieve vaccines also establish latency, can cause disease in calves, and are immuno-suppressive. Completion of the studies in this grant will enhance our understanding of the mechanism by which BHV-1 establishes, maintains, and reactivates from latency. This may lead to a better modified live vaccine.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113310104025%
3113410110125%
3114030110150%
Goals / Objectives
One goal of this project is to identify cellular proteins that interact with a protein (ORF2) encoded by the latency related (LR) gene encoded by bovine herpesvirus 1 (BHV-1). A second goal of this project is to determine whether ORF2 is important for the BHV-1 latency-reactivation cycle. A third goal of this project it to identify and characterize a small non-protein RNA encoded by the LR gene that inhibits expression of an important viral gene (bICP0). Collectively, these studies will help us understand the mechanism by which BHV-1 establishes, maintains, and reactivates from latency.
Project Methods
To identify cellular proteins that interact with ORF2, immunoprecipitation-Western blot assays will be performed. Functional assays will then evaluate whether the interaction between ORF2 and a given cellular protein inhibit viral transcription and productive infection. The ORF2 mutant will be generated using standard mutagenesis procedures. The ability of the ORF2 mutant to induce disease and test whether this mutant will reactivate from latency will be examined in calves. To identify small RNAs encoded by the LR gene, we will clone small RNAs from cells transfected with the LR gene or from trigeminal ganglia of latently infected calves. These RNAs will then be expressed in an appropriate vector and tested for their ability to inhibit bICP0 expression and productive infection.

Progress 09/01/09 to 08/31/13

Outputs
Target Audience: This target of this grant is veterinarians, herpes virologists and other virologists. Changes/Problems: Stress-induced reactivation from latencty is being pursued because a new grant was obtained. What opportunities for training and professional development has the project provided? During the course of these studies, 3 graduate students did most of the work. Two of these students graduated and are now working in industry (CEVA). The other graduate student is still working on her dissertation project. My laboratory has also had a couple of undergraduate students who have assisted with these projects. How have the results been disseminated to communities of interest? The results were published in peer-reviewed journals and we have presented the data at the International Herpesvirus Workshop. In addition, I have presented this data at seminars at several other universities and for a continuing education program for veterinarians that is sponsored by Novartus. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A yeast two-hybrid screen was performed using as bait ORF2. Among the cellular proteins that interacted with ORF2, Notch3 and Notch1 were identified. The Notch family of proteins are transcription factors that regulate embryogenesis, cell survival, differentiation, and when inappropriately expressed contribute to a growing number of tumors. Additional studies were performed to understand why ORF2 would interact with Notch3 and Notch1. Notch1, but not Notch3, stimulated productive infection and the promoter of a viral regulatory protein, bICP0. Notch3 and Notch1 stimulated the glycoprotein C promoter. ORF2 interfered with the ability of Notch family members to trans-activate viral promoters and cellular promoters activated by Notch. Notch family members interfere with neuronal differentiation by blocking neurite and axon formation. During embryogenesis, this is important because Notch maintains a pool of neuro-progenitor cells. In transient transfection assays, we confirmed that Notch1 or Notch3 expression in mouse neuroblastoma cells inhibit neurite sprouting. However, ORF2 blocked Notch signaling pathways and promoted neurite sprouting. Mutagenesis studies were performed to localize the domains in ORF2 that inhibit Notch signaling and apoptosis. Two distinct and separate domains in ORF2 are responsible for inhibiting apoptosis and Notch signaling. ORF2 contains 20% basic amino acids suggesting that it interacts with nucleic acids. Additional studies revealed that ORF2 interacts preferentially with single stranded DNA versus double stranded DNA; but not RNA. Mutation of the six-consensus protein kinase A/protein kinase C phosphorylation sites in ORF2 resulted in a protein that preferentially interacted with double stranded DNA. Additional mutagenesis studies revealed that the N-terminus of ORF2 was important for DNA binding, which over-lapped the domain of ORF2 necessary for inhibiting Notch signaling. Additional studies are testing whether ORF2 specifically binds DNA. Based on these observations, we predict that the ability of ORF2 to inhibit apoptosis and Notch signaling promotes survival of infected neurons. Stress, due to deprivation of food and water during shipping, can trigger BHV-1 reactivation from latency and BRDC, reviewed in. The early steps during reactivation from latency, which precede virus shedding, are operationally defined as the escape from latency. A single intravenous injection of the synthetic corticosteroid dexamethasone (DEX) to latently infected calves mimics stress, suppresses expression of all LR gene products, stimulates lytic cycle viral gene expression, and induces reactivation from latency. From 1.5 to three hours after DEX treatment of latently infected calves, certain cellular transcription factors are stimulated more than tenfold in TG neurons. Several Krüppel like transcription factors (KLF4, KLF6, KLF15, and PLZF) were stimulated by DEX in sensory neurons. The DEX inducible cellular transcription factors stimulate productive infection by trans-activating key viral promoters. KLF4 and KLF15 trans-activated the bICP0 early promoter more than 100 fold. KLF family members may play an important role in regulating viral transcription because they specifically bind GC and CAAC sequences, and the BHV-1 genome is more than 70% GC rich. The BHV-1 genome also contains glucocorticoid receptor (GR) binding sites in 24 promoters suggesting that activation of the GR by stress hormones directly stimulates viral transcription. Recent studies revealed that two viral regulatory proteins, bICP0 and VP16, but not glycoprotein C or glycoprotein D are expressed in neurons within 1.5 hours after calves latently infected with BHV-1 are treated with DEX. These studies have provided insight into the early events that lead to reactivation from latency.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Sinani, D., L. Frizzo da Silva, and C. Jones. 2013. A bovine herpesvirus 1 protein expressed in latently infected neurons (ORF2) promotes neurite sprouting in the presence of activated Notch1 or Notch3. J Virol:1183-1192.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Pittayakhajonwut, D., D. Sinani, and C. Jones. 2013. A protein (ORF2) encoded by the latency related gene of bovine herpesvirus 1 interacts with DNA. J Virol 87:5943-5501.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Devis Sinani, Ethan Cordes, Aspen Workman, Prasanth Thunuguntia, and Clinton Jones. 2013. Stress-induced cellular transcription factors expressed in trigeminal ganglionic neurons stimulate the herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) promoter. J of Virology, 87:1183-1192.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Frizzo da Silva, L. I. Kook, A. Doster, and C. Jones. Bovine herpesvirus 1 regulatory proteins, bICP0 and VP16, are readily detected in trigeminal ganglionic neurons expressing the glucocorticoid receptor during the early stages of reactivation from latency. J of Virology, 87: 11214-11222.
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Jones, C. 2013. Bovine herpesvirus 1 (BHV-1) and herpes simplex virus type 1 (HSV-1) promote survival of latently infected sensory neurons, in part by inhibiting apoptosis. J of Cell Death, 6:1-16.


Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: Studies were performed to identify cellular transcription factors induced during bovine herpesvirus 1 (BHV-1) reactivation from latency. These cellular transcription factors were shown to induce productive infection and to activate certain BHV-1 transcriptional promoters. We believe these cellular transcripts are important for inducing reactivation from latency. A viral protein (ORF2) that is expressed in latently infected sensory neurons was shown to interact with Notch1 and Notch3, which are two cellular transcription factors that regulate neuronal development and activate viral transcription. Recent studies demonstrated that ORF2 induces neurite sprouting in the presence of Notch1 or Notch3. This may be important for live long latency because Notch family members interfere with axonal repair, and this leads to neuronal degeneration. Neurite sprouting is a precursor for axonal repair. Tow viral micro-RNAs were identified that are expressed during latency. These micro-RNAs interfere with bICP0 protein expression and promote cell survival suggesting they play a role in latency. These studies have been discussed at the International Herpesvirus Work Shop and were published in peer-reviewed virology journals. BHV-1 contributes to this poly-microbial disease. These studies were discussed at the International Herpesvirus meeting and at two seminars that I presented at MS State University and the Brazilian Virology meeting. PARTICIPANTS: Graduate students that performed these studies include Devis Sinani, Leticia Frizzo da Silva, Aspen Workman, and Natasha Gadreault. I have also collaborated with Shafiq Chowdhury (LSU Vet School). TARGET AUDIENCES: Cattle producers, herpes virologists, veterinarians, and the Animal Health Industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
These studies have provided insight into how BHV-1 regulates latency in sensory neurons. In addition, the finding that ORF2 interacts with Notch may also be used as a tool to understand the role that Notch plays in cancer and developmental biology. Finally, these studies will help develop novel strategies to develop new vaccines and understand how BHV-1 contributes to bovine respiratory disease complex.

Publications

  • Frizzo da Silva, L. and C. Jones. 2011. Infection of cultured bovine cells with bovine herpesvirus 1 (BHV-1) or Sendai virus induces different beta interferon subtypes. Virus Research, 157: 54-60.
  • Workman, A. and C. Jones. 2011. Analysis of the cell cycle regulatory protein (E2F1) after infection of cultured cells with bovine herpesvirus 1 (BHV-1) or herpes simplex virus type 1 (HSV-1). Virus Research 160: 66-73.
  • Frizzo da Silva, L.F., N. Gaudreault, and C. Jones. 2011. Cytoplasmic localized infected cell protein 0 (bICP0) encoded by bovine herpesvirus 1 inhibits beta interferon promoter activity and reduces IRF3 (interferon response factor 3) protein levels. Virus Research 169:143-149.
  • Chowdhury, S.I., M.C.S. Brum, C. Coats, A. Doster, H. Wei, C. Jones. 2011. Bovine Herpesvirus Type 1 envelope protein Us9 acidic domain is crucial for anterograde axonal transport. Vet Micro 28:270-279.
  • Sinani, D. and C. Jones. 2011. Localization of sequences in a protein encoded by the latency related gene of bovine herpesvirus 1 (ORF2) that inhibits apoptosis and interferes with Notch1 mediated trans-activation of the bICP0 promoter. J Virol 85: 12124-12133.
  • Da Silva, L.F. and C. Jones. 2012. Two micro-RNAs encoded within the BHV-1 latency related (LR) gene promote cell survival by interacting with RIG-I and stimulating nuclear factor-kappa B (NF-kB) dependent transcription and beta-interferon signaling pathways. J Virol, 86:1670-1682.
  • Frizzo da Silva, L., D. Sinani, and C. Jones. 2012. The ICP27 protein encoded by bovine herpesvirus type 1 (bICP27) interferes with promoter activity of the bovine genes encoding beta interferon 1 (IFN-β1) and IFN-β3. Virus Research, 169:162-168.
  • Workman, A., J. Eudy, L. Smith, L. Frizzo da Silva, D. Sinani, H. Bricker, E. Cook, A. Doster, and C. Jones. 2012. Cellular transcription factors induced in trigeminal ganglia during dexamethasone-induced reactivation from latency stimulate bovine herpesvirus 1 productive infection and certain viral promoters. J Virol 86: 2459-2473.


Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: Like other members of the alpha-herpesvirus subfamily, a latent infection is established in sensory neurons following acute infection. However, the virus can reactivate and spread to other cattle. Reactivation from latency is the mechanism by which the virus survives in nature and is thus an important property of pathogenesis. During a latent infection, the viral latency related RNA (LR-RNA) is abundantly expressed. Expression of LR proteins is necessary for reactivation from latency and protecting infected neurons from apoptosis. We have demonstrated that a LR encoded protein (ORF-2) or ORF-2 fusion proteins encoded by alternatively spliced LR transcripts inhibit apoptosis in mouse neuroblastoma cells (neuro-2A). Frame shift mutants of ORF-2 do not inhibit apoptosis suggesting that protein expression, not LR-RNA expression, mediates the anti-apoptosis activity of the LR gene in transfected neuro-2A cells. Our results focused on the latency project are summarized below. PARTICIPANTS: Aspen Workman, graduate students. Devis Sinani, graduate students Leticiea da Silva, graduate student, Natasha Gaudrealt TARGET AUDIENCES: Cattle producers, scientists, and virologists. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Yeast two-hybrid analysis has revealed that ORF-2 sequences specifically interact with two cellular proteins that regulate cell survival and differentiation (Notch 1 and Notch 3). We have confirmed that ORF2 interacts with Notch 1 and 3 by confocal microscopy and pull-down assays. Interestingly, ORF2 inhibits the ability of Notch 1 to trans-activate promoters. A manuscript describing these studies was published this year. The domains of ORF2 that inhibit apoptosis and interfere with Notch1 mediated activation of gene expression was also performed. A manuscript describing these studies was recently accepted for publication in the Journal of Virology. We have also identified two viral encoded micro-RNA that inhibit bICP0 protein expression. Dexamethasone consistently initiates reactivation from latency. Wild type BHV-1, but not the LR mutant virus, express all viral genes during reactivation from latency. Gene chip studies have been employed to identify cellular genes that are induced during reactivation from latency. This study demonstrated that during dexamethasone induced reactivation from latency expression of several cellular transcription factors are increased. These cellular transcription factors were subsequently shown to stimulate viral gene expression and productive infection. We suggest that dexamethasone induction of cellular transcription factors in sensory neurons promotes reactivation from latency. A manuscript describing these studies was recently submitted.

Publications

  • Workman, A., D. Sinani, D. Pittayakhajonwut, and C. Jones. 2011. A Protein (ORF2) Encoded by the Latency Related Gene of Bovine Herpesvirus 1 Interacts with Notch1 and Notch3. J Virology, 85: 2536-2546.
  • Gaudreault, N. and C. Jones. 2011. Regulation of promyelocytic leukemia (PML) protein levels and cell morphology by bovine herpesvirus 1 infected cell protein 0 (bICP0) and mutant bICP0 proteins that do not localize to the nucleus. Virus Research, 156:17-24.
  • Frizzo da Silva, L. and C. Jones. 2011. Infection of cultured bovine cells with bovine herpesvirus 1 (BHV-1) or Sendai virus induces different beta interferon subtypes. Virus Research, 157: 54-60.
  • Workman, A. and C. Jones. 2011. Analysis of the cell cycle regulatory protein (E2F1) after infection of cultured cells with bovine herpesvirus 1 (BHV-1) or herpes simplex virus type 1 (HSV-1). Virus Research 160: 66-73.
  • Frizzo da Silva, L.F., N. Gaudreault, and C. Jones. 2011. Cytoplasmic localized infected cell protein 0 (bICP0) encoded by bovine herpesvirus 1 inhibits beta interferon promoter activity and reduces IRF3 (interferon response factor 3) protein levels. Virus Research 169:143-149.
  • Chowdhury, S.I., M.C.S. Brum, C. Coats, A. Doster, H. Wei, C. Jones. 2011. Bovine Herpesvirus Type 1 envelope protein Us9 acidic domain is crucial for anterograde axonal transport. IN PRESS, Vet Micro.
  • Sinani, D. and C. Jones. 2011. Localization of sequences in a protein encoded by the latency related gene of bovine herpesvirus 1 (ORF2) that inhibits apoptosis and interferes with Notch1 mediated trans-activation of the bICP0 promoter. In Press, J Virol.


Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: Yeast two-hybrid analysis has revealed that ORF-2 sequences specifically interact with two cellular proteins that regulate cell survival and differentiation (Notch 1 and Notch 3). We have confirmed that ORF2 interacts with Notch 1 and 3 by confocal microscopy and pull-down assays. Interestingly, ORF2 inhibits the ability of Notch 1 to trans-activate promoters. We recently identified two viral encoded micro-RNA that inhibit bICP0 protein expression. Dexamethasone consistently initiates reactivation from latency. Wild type BHV-1, but not the LR mutant virus, express all viral genes during reactivation from latency. C/EBP-alpha is induced during reactivation from latency. C/EBP-alpha trans-activates viral promoters and is induced by dexamethasone. PARTICIPANTS: Several graduate students in my lab helped to perform these studies. These include Aspen Workman, Tareq Jaber, and my technician (Gail Henderson). TARGET AUDIENCES: Herpes virologists, ranchers, pharmaceutical companies, and veterinarians will be interested in the results of these studies. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our goal is to identify viral and cellular factors that regulate the latency-reactivation cycle. The ability of the LR gene to inhibit apoptosis and interact with cellular regulatory proteins is crucial. Finally, we believe that an understanding of virus host interactions in sensory neurons may lead to better strategies to develop superior modified live vaccines.

Publications

  • Jaber, T., A. Workman, and C. Jones. 2010. Small non-coding RNAs encoded within the bovine herpesvirus 1 latency related gene can reduce steady state levels of infected cell protein 0. J Virology, 84:6297-6307.