Methods for the Manipulation of Herpesvirus Genome and the Application to Marek's Disease Virus Research.

Herpesviruses are a group of double-strand DNA viruses that infect a wide range of hosts, including humans and animals. In the past decades, numerous methods have been developed to manipulate herpesviruses genomes, from the introduction of random mutations to specific genome editing. The development of genome manipulation methods has largely advanced the study of viral genes function, contributing not only to the understanding of herpesvirus biology and pathogenesis, but also the generation of novel vaccines and therapies to control and treat diseases. In this review, we summarize the major methods of herpesvirus genome manipulation with emphasis in their application to Marek's disease virus research.

The role of Meq-vIL8 in regulating Marek's disease virus pathogenesis.

Marek's disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes T cell lymphoma in chickens. MDV-encoded Meq and vIL8 proteins play important roles in transformation and early cytolytic infection, respectively. Previous studies identified a spliced transcript, meq-vIL8, formed by alternative splicing of meq and vIL8 genes in MDV lymphoblastoid tumour cells. To determine the role of Meq-vIL8 in MDV pathogenesis, we generated a recombinant MDV (MDV-meqΔSD) by mutating the splice donor site in the meq gene to abrogate the expression of Meq-vIL8. As expected, our results show that MDV-meqΔSD virus grows similarly to the parental and revertant viruses in cell culture, suggesting that Meq-vIL8 is dispensable for MDV growth in vitro. We further characterized the pathogenic properties of MDV-meqΔSD virus in chickens. Our results show that lack of Meq-vIL8 did not affect virus replication during the early cytolytic phase, as determined by immunohistochemistry analysis and/or viral genome copy number, but significantly enhanced viral DNA load in the late phase of infection in the spleen and brain of infected chickens. In addition, we observed that abrogation of Meq-vIL8 expression reduced the mean death time and increased the prevalence of persistent neurological disease, common features of highly virulent strains of MDV, in inoculated chickens. In conclusion, our study shows that Meq-vIL8 is an important virulence factor of MDV.

Role of Marek's Disease Virus (MDV)-Encoded US3 Serine/Threonine Protein Kinase in Regulating MDV Meq and Cellular CREB Phosphorylation.

Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Alphaherpesvirinae Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, US3. The functions of US3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV US3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV US3 and identified chicken CREB (chCREB) as a substrate of MDV US3. We show that wild-type MDV US3, but not kinase-dead US3 (US3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using US3 deletion and US3 kinase-dead recombinant MDV, we identified US3-responsive MDV genes during infection and found that the majority of US3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some US3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which is also regulated by US3. Furthermore, biochemical studies demonstrated that MDV US3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, in vitro kinase studies revealed that Meq is a US3 substrate. MDV US3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression.IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. US3 encoded by alphaherpesviruses is a multifunctional kinase involved in the regulation of various cellular pathways. Using an MDV genome quantitative reverse transcriptase PCR (qRT-PCR) array and chromatin immunoprecipitation, we elucidated the role of MDV US3 in viral and cellular gene regulation. Our results provide insights into how viral kinase regulates host cell signaling pathways to activate both viral and host gene expression. This is an important step toward understanding host-pathogen interaction through activation of signaling cascades.