Targeted Editing of the pp38 Gene in Marek's Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System.

Yaoyao Zhang,Jun Luo,Man Teng,Zhiqiang Shen,Venugopal Nair,Katy Moffat,Yongxiu Yao,Na Tang,Vishwanatha R.A.P Reddy

doi:10.3390/v11050391

Yaoyao Zhang, Jun Luo + Show 7 more

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https://doi.org/10.3390/v11050391

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Marek’s disease virus (MDV), a lymphotropic α-herpesvirus associated with T-cell lymphomas in chickens, is an excellent model for herpesvirus biology and virus-induced oncogenesis. Marek’s disease (MD) is also one of the cancers against which a vaccine was first used. In the lymphomas and lymphoblastoid cell lines (LCLs) derived from them, MDV establishes latent infection with limited gene expression. Although LCLs are valuable for interrogating viral and host gene functions, molecular determinants associated with the maintenance of MDV latency and lytic switch remain largely unknown, mainly due to the lack of tools for in situ manipulation of the genomes in these cell lines. Here we describe the first application of CRISPR/Cas9 editing approach for precise editing of the viral gene phosphoprotein 38 (pp38), a biomarker for latent/lytic switch in MDV-transformed LCLs MDCC-MSB-1 (Marek’s disease cell line MSB-1) and MDCC-HP8. Contradictory to the previous reports suggesting that pp38 is involved in the maintenance of transformation of LCL MSB-1 cells, we show that pp38-deleted cells proliferated at a significant higher rate, suggesting that pp38 is dispensable for the transformed state of these cell lines. Application of CRISPR/Cas9-based gene editing of MDV-transformed cell lines in situ opens up further opportunities towards a better understanding of MDV pathogenesis and virus-host interactions.

Highlights

Marek’s disease (MD) is a lymphoproliferative disease of chickens with a complex pathogenesis, characterized by neoplastic transformation of T cells that infiltrate lymphoid tissues, visceral organs, and even peripheral nerves
We have demonstrated that CRISPR/Cas9 editing can be applied efficiently in targeted editing of the Marek’s disease virus (MDV) genome during lytic stages of infection in infected chick embryo fibroblasts (CEF) cultures [37,39]
We chose to target the pp38 gene in the MDV-transformed cell line MSB-1 using the same gRNAs that were successfully used for pp38 deletion from CVI988 genome in CEF culture system [39]

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Introduction

Marek’s disease (MD) is a lymphoproliferative disease of chickens with a complex pathogenesis, characterized by neoplastic transformation of T cells that infiltrate lymphoid tissues, visceral organs, and even peripheral nerves. MDV undergoes a cytolytic infection of B and T cells in lymphoid organs followed by establishment of latency in CD4+ T cells and integration of its genome into the telomeres of host chromosomes [1,2,3]. The integration process is very efficient as up to 15 chromosomes in tumor cells has been observed harboring the MDV genome detected by either fluorescent in situ hybridization (FISH) or pulsed-field gel electrophoresis (PFGE) [2,4,5,6,7]. Due to factors or events that are still largely unknown, some of the latently infected CD4+ T cells in susceptible and unvaccinated birds subsequently get transformed into neoplastic cells, resulting in the development of lymphomas, leading to high levels of mortality [8,9].

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