Abstract
Marek’s disease (MD) is an immunosuppressive and highly contagious lymphoproliferative disease caused by Marek’s disease virus (MDV) in poultry. Lymphoblastoid cell lines (LCLs) generated ex vivo from MD lymphomas are considered excellent models to study virus-host molecular interactions. LCLs mostly have latently infected MDV genome, but many of them also have varying populations of lytically-infected cells, thus making them very suitable to examine the molecular events associated with the switch from latent to lytic infection. MDV-encoded phosphoprotein 38 (pp38) is readily detectable in lytically-infected LCLs and hence considered as a biomarker for lytic infection. Whilst previous studies have suggested that pp38 is essential for the early cytolytic infection of B-cells, its role in the switch from latent to lytic infection of LCLs is still unclear. pp24, another phosphorylated protein in the same protein complex, shares the same promoter and N-terminal 65 amino acids as pp38. In this study we employed CRISPR activation (CRISPRa) technology for targeted activation of pp38/pp24 in LCLs to investigate their role in inducing lytic infection. Our results show that enforced expression of pp38/pp24 through CRISPRa induces orchestrated upregulation of other MDV genes including ICP4, gB, Meq and pp14 as well as differential expression of host genes thereby facilitating lytic infection. Our results also show that pp38/pp24 expression induces the lytic switch through inhibiting apoptosis.
Highlights
Published: 8 August 2021Marek’s disease (MD) is caused by Marek’s disease virus (MDV), a cell-associated, highly oncogenic alphaherpesvirus belonging to the genus Mardivirus [1]
These results confirm that targeted activation of pp38/pp24 by CRISPR activation (CRISPRa) induces differential host gene expression, the same set of genes affected during the latent to lytic switch of MDV infection reported previously [4]
Having demonstrated that the CRISPRa-induced activation of pp38/pp24, in the clone 4 of 4523T-dCas9-VP64 (C4) clone of the 4523T cell line, upregulated other MDV lytic genes and induced differential expression of several host genes as observed previously in the latent to lytic switch, we examined the role of increased pp38/pp24 expression in virus reactivation
Summary
Marek’s disease (MD) is caused by Marek’s disease virus (MDV), a cell-associated, highly oncogenic alphaherpesvirus belonging to the genus Mardivirus [1]. During the lytic stage of infection, the virus is in an active state overriding the various restrictive factors such as epigenetic regulation (DNA methylation, histone modification) [3], host cell niche [4] and expression of viral latency-associated transcripts (LATs) [5,6]. Differential expression of several genes, including upregulation of 82 viral genes such as pp, ICP4, gE and gI, was identified in populations undergoing a spontaneous switch to lytic infection in LCL derived from lymphomas induced by recombinant pRB1B-UL47eGFP virus [4]. We employed the CRISPRa system to activate pp38/pp expression in LCL 4523T cells to investigate its role in the induction of MDV lytic replication. The upregulation of other MDV lytic genes, differential expression of host genes and increased MDV reactivation of 4523T in chicken embryo fibroblast (CEF) cells indicated that pp38/pp induces lytic replication in MDV transformed LCLs
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