Progress 02/15/14 to 02/14/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Identifiedviral protein, Meq, binding sites on chicken genome in infected T-cells
Publications
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Progress 02/15/16 to 02/14/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Kazushi Nakano, aresearch associate in my lab attends three days bioinformatics training program prepared by UC Davis Bioinformatics Core. He is now capable of visualing next generation sequence data and have prepared many figures. Dr. Lyu, a postdoctral fellow attends two day workshop to learn R-statics program, which allows him to analyze data with diffierent approches and generate new figures. Collaborations between UC Davis Medical School and Texas A&M Veterinary School allowed us to exchange students between the two schools and train researchers in different environments. A graduate student ontained a scholarship from Texas A&M and visited our laboratory (UC Davis Medical School) for three weeks. He learned biochemical and genetic research techniques in our laboratory. Our lab member are planning to visit Texas A&M veterinary school to perform in vivo studies in coming months. An international collaboration with Kyoto Pharmaceutical University has also been established to facilitate recombinant DNA techniques. Students obtained multiple scholarships for travel and research awards. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Goal1/2: Next reporting period, we will analyze association between MDV reactivation and local histone modifications. We will study effects on MDV reactivation in presence of multiple epigenetic drugs. Goal3: We are planning to perform capture Hi-C experiments before/after of MDV infection. We identified that KSHV genome forms unique genomic structure and the structure has significant effects on viral gene expression. Because MDV genome is integrating host telomere region, we are interesting to examine effects on the chicken chromosome strucrue by viral genome integration. We are expecting that MDV genome forms genomic loops with chicken genome and the genomic interactin deregulate hst gene expression program. We also speculate that generation of new genomic loops at the host telomere repeat sequence may induce genome instability; this may lead to tumorigenesis. As we have done same experiment with KSHV, we have all protocol to be set and ready to perform the experiments in coming year. Goal4: We will start to generate point mutant Meq, which no longer binds to MEQ. We will start to construct recombinant MDV, which encodesmutant Meq protein.
Impacts
What was accomplished under these goals?
Our dual use of animals for dual benefit research program is to use oncogenic chicken herpesvirus as a model to study cancer development. Marek's disease virus (MDV) develops T-cell lymphomas in 99.9% of infected chickens within a month. Accordingly, the virus infection is among the most economically important all of infectious disease affecting poultry production worldwide. However, the infection also allows us to synchronize the initiation of T-cell lymphoma, which provides us a chance to monitor changes made during tumor development. MDV encodes defined oncogene, Meq gene; this is also significant advantage to our study by focusing to characterize action of Meq during tumor development in vivo. In my laboratory, we also study human oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV) in parallel. Both herpesviruses have common features and protein homologues for viral replication. In addition, they have same life style, latent infection and lytic replication. Our approach is to perform experiments in parallele and apply our finding in advanced human cancer research into MDV research field. Important feature identifiedin vitro studies will then be confirmed in MDV infected chicken in vivo. In this project year, we finished genetic studies with MDV infected T-cell lymphoma. Same technique was also applied to KSHV infected B-cell lymphoma in parallele. Goal 1: We performed ChIP-sequence analyses to identify MEQ binding sites in both chicken genome as well as MDV genome. We identified that MEQ binds to viral transactivator (ICP4) promoter in addition to own (Meq) promoter. With detailed Meq binding sites in hands, we have prepared primer sets targeting these regions and verified the ChIP-sequence studies. We have perfored with three biological replicates and ontained similar results. To further analyze detail of MDV transcriptional program, we desigbned MDV PCR array, which contains primer pairs targeting every MDV open reading flame. With the PCR array, we can conveniently studies every MDV ORF expression in one 96 well plates. We also verified utility of the array with RNA extracted from MDV reactivated chicken T-cells. Goal 2: We could not obtain sufficient materials to perform ChIP-sequencing with actual chicken tumors. We infected MDV again in Dr. Reddy's lab and are waiting for tumor development. Goal 3: ChIP-sequence analyses with antiodies for specifichistone modification marks were also performed. The results showed that MDV genomes were heavily marked by both H3K4me3 and H3K27me3 as similar to KSHV. These results indicated common strategy for the herpesvirus family used to establish latent infection. Goal 4: Biochemical studies were conducted to identified PA200 binding sites on Meq protein. We also generated PA200 specific antibodies in rabbit, which recognizes endogenous PA200 of chicken T-cells. Subsequently, we performed ChIP-sequence analyse with anti-PA200 antiboy and found that Meq binding site and PA200 recrutment sites were significantly overlapped in chicken T-cell lymphoma cell lines. We are planning to peerform same experiments with chicken T-cell lymphomas isolated from MDV infected chikens. Goal 5: In addition to PA200 binding mutant, we are currently constructing a few more MDV mutant viruses. These are (1) MDV transactivator inducible MDV, in which MDV transactivator can be induced in presence of tetracycline. With themutant virus, we are hoing to regulate MDV reactivation in infected chicken and analyze "oncolytic" strategy for viral induced malignancies. (2) Color-coded recombinant MDV. We are inserting GFP and RFP marker under viral promoter or constitutively active promoter. With this recombinant virus, we can monitor MDV lytic replication as a RFP signals and infected cells with GFP signals. We have similar system with KSHV and we found it is very useful to study gene regulatory mechinism in vitro. With MDV, we may be able to expand such gene regulatory study in vivo, and analyze relationshipwith tumorigenesis. Other progress with new discovery and deveopment of novel technique: Imaging based analyses of viral gene regulation in an infected cells: It is well established that locally concentrated nuclear factors can ensure efficient binding to the DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable RNA-polII pre-initiation complexes. In this project period,we have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around viral genomes in the host cell nucleus. Using immunofluorescent labeling of viral latent protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate-early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At single cell level, we found that not all viral genomes were uniformly transcribed following external stimuli. However, those viral genomes that were being transcribed, would spontaneously aggregate to form transcriptional "factories", which recruit a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular genes transcription, which consequently impairs cellular gene expression globally. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be another aspect of viral gene regulation by re-purposing a limited supply of RNA polymerase II in infected cells. This study visualized transcribing viral genome in a infected cells first time and established new platform of herpesvirus gene expression studies. These results were submitted to a journal for publication.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Marek's disease vaccines: Current status, and strategies for improvement and development of vector vaccines.
Sanjay M. Reddy, Yoshihiro Izumiya, Blanca Lupiani
Vet. Microbiol PMID: 28038868DOI: 10.1016/j.vetmic.2016.11.024
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Kaposis Sarcoma-associated Herpesvirus Hijacks RNA Polymerase II to Create a Viral Transcriptional Factory
Christopher Phillip Chen, Yuanzhi Lyu, Frank Chuang, Kazushi Nakano, Chie Izumiya, Mel Campbell, Yoshihiro Izumiya. Journal of Virology (In revision)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Zic2 Is Essential for Polycomb-Mediated Maintenance of KSHV Latency and a Target of K-Rta
Yuanzhi Lyu, Kazushi Nakano, Ryan R. Davis, Clifford G. Tepper, Mel Campbell, Yoshihiro Izumiya PLoS Pathogens (In revision)
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Acceleration of Highly Oncogenic Viral Parthenogenesis by Infection Phase Regulation via a Virus-Encoded MicroRNA Cluster
Guoqing Zhuang, Aijun Sun, Yoshihiro Izumiya, Sanjay M Reddy, Blanca Lupiani
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Progress 02/15/15 to 02/14/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Postdoctoral fellows attended three days bioinformatics training program held at UC Davis Bioinformatics Core. The Galaxy training workshop allowed postdoctoral fellows to analyze and visualize RNA and ChIP-seq data. One postdoctoral fellow also attend training to perform proteomics analyses and learned how to use Mass Spectrometer. Collaboration was also established with biochemist to visualize dynamics of epigenetics changes with genetically encoded small illuminates. Our group provided experimental platform, which is generated by this grant,for the biological studies andour collaboration allowed usobtained U01 grant from NIH. I am serving as a co-PI for the 4D Nucleome imaging program. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Goal1/2: Large number of samples was submitted to next generation sequencing. Next reporting period, we will analyze those data. If necessary, we willrepeat the experiment to validate the results. Goal3: We would like to perform Hi-C experiments before/after of MDV infection. We noticed that viral genome can interact with host genome by forming genomic loops. We like to know if MDV genome can form genomic loops with host chromosome. Because MDV genome is frequently integrated at telomere repeat region, we speculate that generation of new genomic loops at the host telomere repeat sequence may induce genome instability; this may lead to tumorigenesis. As we have done Hi-C experiment with human herpesvirus, we have all protocol to be set and ready to perform the experiments. Goal4: We will continue to perform interaction assays. In next reporting period, we will confirm the interaction in transient transfection analyses. In addition, we will start to generate point mutant in order to generate recombinant MDV for in vivo studies in next project period.
Impacts
What was accomplished under these goals?
Our dual use of animals for dual benefit research program is to use oncogenic chicken herpesvirus as a model to study cancer development. Marek's disease virus (MDV) develops T-cell lymphomas in 99.9% of infected chickens within a month. Accordingly, the virus infection is among the most economically important all of infectious disease affecting poultry production worldwide. However, the infection also allows us to synchronize the initiation of T-cell lymphoma, which provides us a chance to monitor changes made during tumor development. From initiation of cancer cells to development of tumors, which then impacts our health, is estimated to take about 15 years. Thus it is very difficult to examine dynamic changes made by oncogene during development of cancers in other system. Importantly, MDV encodes defined oncogene, Meq gene; this is also significant advantage to our study. In my laboratory, we also study human oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV) in parallel. Both herpesviruses have common features and protein homologues for viral replication. In addition, they have same life style, latent infection and lytic replication. Accordingly, our approach is to apply our findingof advanced human cancer research into MDV research field and new insights in MDV in vivo studies into the KSHV research. In this project year, we met a few difficulties to obtain T-cell lymphoma from infected chicken due to renovation of chicken SPF facilities at Texas A&M University. Accordingly, we needed to focus more on biochemical and genetic studies in vitro. Goal 1/2: Two chicken T-cell lymphoma cell lines were generated from two different chickens, which were infected with identical recombinant MDV generated from MDV BAC DNA. The cell lines from two independent birds allow us to avoid clonal variation of cancer cells. Large amount of each cell linewas cultured and performed chromatin immunoprecipitation with anti-Meq antibody. Precipitated DNA was submitted to direct sequence analyses. We are currently waiting to receive the sequence data. In same time, we are repeating the ChIP analyses in order to validate the recruitment sites. To identify Meq binding site during infection, we infected MDV to obtain tumor from chicken and we will perform same experiments with actual tumors from chickens. The experiment was delayed due to renovation of SPF facility at Texas A&M University. Transcriptome analyses were also coupled to the ChIP-sequencing analyses, and we submitted total RNA, which was extracted from the cell lines for RNA-seq analyses. Large quantity of data is currently analyzed in my laboratory. These sets of results should provide us how Meq binding associates with gene expression. Goal3: In a light of recent advancement of knowledge in epigenetic gene regulation, three-dimensional genomic structure was found to have significant impact on gene expression. We noticed that latently-infected herpesvirus genome provides exciting model to study such regulation, because herpesvirus genome is much smaller than host genome and viral gene regulation is relatively simple when we compared with human gene regulation. In addition, viral genome is epigenetically modified same way with human genome and located in infected cell nucleus. Accordingly, we have analyzed latently-infected herpesvirus genome as a model to study relationship between 3D structure and viral gene expression. In the study we found that herpesviruse coordinate a unique genomic structure for their own transcriptional factors. Genome-wide ChIP-chip analysis showed a limited number of viral transactivator enriched sites in the genome, and the 3D genomic structure allows for the viral transactivator to access to distal viral promoter. We engineered viral genome to harbor point mutations at the direct binding site of viral transactivator with BAC technology and we found that the mutations significantly attenuated not only the direct downstream gene expression, but also distal viral gene expression in a genomic domain-specific manner. Our finding suggest first time that herpesvirus genome forms active chromatin hubs by clusteringviral transactivator binding sitesvia formation of genomic loops. We submitted these results to Cell Host & Microbe, which is currently in revision. Goal4: Our studies identified that MDV oncogene interacts with proteasome activator, PA200. Biochemical analyses showed that the interaction facilitates degradation of tumor suppressor in infected cells. To identify the significance of the interaction, we are generating mutant Meq protein, which no longer interacts with PA200 and prepare recombinant MDV to examine functions in tumorigenesis in vivo. Recombinant proteins of both chicken PA200 and Meq have been prepared. In vitro interaction analyses were performed. The results showed that N-terminal region of Meq interacts with PA200. We are generating point mutilations at N-terminal region of Meq in order to reveal significance of the interaction between Meq and PA200 in vivo. In addition, interaction between Meq and PA200 will be confirmed in transient transfection analyses in DF-1 cells. The experiments will be performed in next funding period.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Acceleration of Highly Oncogenic Viral Pathogenesis by Infectious Phase Regulation via a Virus-Encoded MicroRNA Cluster
Guoqing Zhuang, Aijun Sun, Yoshihiro Izumiya, Sanjay M. Reddy, Blanca Lupiani
KSHV Chromatin Looping Facilitates Effective Gene Expression; Gene Cluster Activation via Direct K-Rta Binding at a Viral Chromatin Hub
Mel Campbell1, Tadashi Watanabe, Kazushi Nakano, Ryan Davis,
Clifford G. Tepper, Masahiro Fujimuro, and Yoshihiro Izumiya
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Progress 02/15/14 to 02/14/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Activities include training of two postdoctral fellows and one grduate student. Postdoctral fellows attended an international meeting to present their research. Collaorations were also established with other scientists working in cancer research field. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Goal1: In order to have larger size of tumor samples, we will infect more chickens to get enough amounts of tumors to perform ChIP-sequencing analyses. This strategy will avoid amplification steps that may cause bias in ChIP-seq data. Nevertheless, first ChIP samples will be used ChIP-sequencing analyses after amplification with WGA kit, which is widely used for ChIP-sequence analyses. Next samples with larger size of tumor samples will be used to confirm first ChIP-sequence analyses. Goal 2: Once we identify Meq binding sites on chicken genome, we will take time course to isolate chicken T-cells and examine Meq binding sites. Goal 3: Once we identify Meq binding sites on chicken genome, we will examine histone modification at Meq binding sites and compare with non-infected T-cells by qt-PCR. This experiment should allow us to understand effects of Meq binding in histone modification. We plan to use many specific histone antibodies that recognize different histone modification status. Total RNA from tumor smples will be isolated and correlate the local histone modification and gene expression. Goal4: In next year, purified PA200 protein will be generated from recombinant baculovirus infected cells and used for biochemical studies to map Meq binding sites. By using purified proteins, we will also confirm direct binding between viral onco-protein and proteasome activator.
Impacts
What was accomplished under these goals?
The association between viruses and lymphoma has long been recognized. In addition to lymphoma, approximately 12% of all human cancers are caused by oncoviruses; however, the complex nature of the disease and its heterogeneity of cause, make it difficult to study. In most cases, it takes years to decades after theinitial infection for cancer to develop in the infected individual; this reflects the multistep nature of viral oncogenesis and host genetic variability. In fact, the viruses may contribute to a portion of the oncogenic events. Hanahan and Weinberg defined the hallmarks of cancer to encompass key biological capabilities that are acquired and essential for the development of cancers. These capabilities include sustained proliferation, evasion of growth suppression, death resistance, replicative immortality, induced angiogenesis, initiation of invasion, deregulation of cellular energetics, avoidance of immune destruction and chronic inflammation. How does oncovirus infection lead infected cells to acquire such capability? Are there any good models to systematically study development of cancer by viral infection? Marek's disease virus (MDV) is a very potent oncogenic herpesvirus, which induces a highly contagious T-cell lymphoma (Marek's disease, or MD) in nearly 100% of infected chickens, its natural host, within 3-4 weeks of infection. Accordingly, it is among the most economically important of all infectious diseases affecting poultry production worldwide. A single viral oncoprotein (Meq), whose function is absolutely required for tumorigenesis has been identified and is currently the focus of research in our laboratories. In addition, a genetic system to manipulate viral genes, as well as an infection model to evaluate oncogenicity of the virus have been well-established. These features provide us a unique opportunity to explore oncogenic molecular events rather systematically in vivo. Among the essential features associated with cancer development, we are currently focusing on how a single viral oncoprotein (Meq) shuts-off the host tumor suppressor pathway. We have isolated host cellular proteins that are associated with the viral oncoprotein in naturally infected cells. Among the associated proteins we identified PA200, a proteasome activator, and found that PA200 is important for elimination of tumor suppressor proteins, such as p53, in infected cells. We will continue to use the defined viral oncoprotein as a tool to study molecular events in vitro and evaluate the effects on the tumor development in vivo. Goal 1. Identify viral protein, Meq, binding sites on chicken genome in infected T-cells For mapping of Meq binding sites on chickenand MDV genome, we infected MDV BAC delieved virus to chickens. Tumors from multiple organs were isolated. Meq expression was examined in tumors form different organs and found that tumors in spleen showed highest expression of Meq protein. We next performed chromatin immunoprecipitation with tumor samples from spleen. However, amount of precipitated DNA was too little for direct-sequencing. To circumvent the problem, we will amplify and perform the direct-sequence analyses. In addition, we are re-infected larger number of chickens and isolating tumors. Goal 4. Generation of recombinant Marek's disease virus, which harbors Meq mutant protein lacking the ability to bind proteasome activator, PA200. During last funding period, we have made significant progress by isolating chicken PA200 cDNA and generated prufied protein as well as expression plasmids. In next year, the purified protein will be used to map the Meq binding site in order to generate recombinant MDV to analyze biological function of the interaction between viral oncoprotein and proteasome activator.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Guoqing-Zhuang, Ai-jun Sun, Yoshihiro Izumiya, Sanjay M. Reddy, Blanca Lupiani
The Role of MDV1-miR-M8-M10 in the Very Virulent Plus (vv+), 686 Strain of Mareks Disease Virus
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