Defining the Role of EBNA-LP in Epstein-Barr Virus Infection and Transformation of B Cells

Abstract

Epstein-Barr Virus (EBV) is double stranded DNA gammaherpesvirus that latently infects almost all adults worldwide. EBV infection is associated with diseases including multiple sclerosis and cancers including lymphomas in immune compromised individuals. EBV encodes viral proteins which are required for the establishment of viral latency. In the context of an immature immune system, the viral proteins can act as oncoproteins to drive uncontrolled cellular proliferation as can be modeled in vitro by EBV transformation of primary B cells into Lymphoblastoid Cell Lines (LCLs). While the viral protein EBNA-LP is essential for naïve B cell transformation, the function of the EBNA-LP has not been well characterized. We therefore developed a trans-complementation assay in which an episomal DNA construct expressing EBNA-LP is capable of complementing EBNA-LP Knockout (LPKO) virus infected cells to screen EBNA-LP mutants at highly conserved regions. This approached identified highly conserved leucine-rich motifs within EBNA-LP of the sequence LXX(X)LL that are essential for naïve B cell transformation. Co-immunoprecipitation and Cut&Run were used to determine that the leucine-rich motifs in EBNA-LP are engaged in association with the transcription factor and DNA looping modulator YY1, and that EBNA-LP stabilizes YY1 at regions of active transcription in LCLs. To further elucidate the role of EBNA-LP during early infection, single cell RNA-sequencing (scRNA-seq) was performed on WT and LPKO infected B cells during the first 8 days of infection. Key points of EBV restriction in the absence of EBNA-LP were identified, including increased cellular stress and arrest; and failure to re-capitulate all of the states required for WT virus transformation. This failure of LPKO infection is due to reduced expression of the essential viral protein LMP1, reduced expression of gene involved in cellular proliferation, and elevated expression of ant-viral genes in the absence of EBNA-LP. Using a Cas9/RNP-based screening approach we identify the Speckled proteins Sp100 and Sp140L as restriction factors which EBV infection requires EBNA-LP to overcome. The function of Sp140L was previously unknown, therefore we examined whether it restricts other herpesviruses and found Sp140L indeed restricts and is targeted by the related primate virus Herpesvirus Saimiri – suggesting SP140L is broadly restrictive across DNA virus infection. We also more fully elucidate the degree to which EBNA-LP is regulated by posttranslational modifications. We identified prolyl hydroxylated residues on EBNA-LP and found phosphorylation and prolyl hydroxylation as mutually exclusive modifications that may alter EBNA-LP stability or function. Overall, these results identify novel roles for EBNA-LP in overcoming the intrinsic and innate restriction of initial EBV infection and in promoting expression of genes required for cellular transformation.