Jasmine Sheppard, University of Toronto

Jasmine Sheppard (1), Jorn Stok (1,2), Edyta Marcon (3), Jack Greenblatt (1,3), Lori Frappier (1)1. Department of Molecular Genetics, University of Toronto, Toronto, Canada 2. UMCU Medical Centre, Universiteit Utrecht, The Netherlands 3. Donnelly Centre, University of Toronto, Toronto, Canada

Suppressing & Conquering

Epstein-Barr Virus (EBV) infects >95% of adults and is a causative agent for several diseases including infectious mononucleosis, multiple sclerosis and several cancers. While EBV-induced tumour cells are latently infected, they also express select lytic EBV transcripts, suggesting that the encoded proteins contribute to oncogenesis. These include a transcript encoding the uncharacterized lytic protein, LF1. We have found that LF1 is expressed late in lytic infection, and that knocking out LF1 impairs late protein expression and virion production. To gain insight into cellular interactions of LF1, we performed affinity purification-mass-spectrometry (AP-MS) and discovered that LF1 interacts with Rae1 and Nup98, components of the nuclear pore complex with roles in mRNA nuclear export. Interestingly, LF1 homologues in Kaposi's Sarcoma-Associated Herpesvirus (KSHV; ORF10) and Murine Gammaherpesvirus 68 (MHV-68) have also been found to interact with Rae1/Nup98 and to inhibit nuclear export of a subset of host mRNAs, including transcripts encoding tumor suppressors, suggesting an oncogenic mechanism. To determine if this is also true for LF1, we performed Poly(A) fluorescence in situ hybridization (FISH) and confirmed that LF1 causes mRNA nuclear retention. Based on homology with ORF10, we designed an LF1 mutant that is defective in Rae1/Nup98 binding, and found that this mutant was defective in mRNA nuclear retention and nuclear membrane localization. To ascertain which cellular mRNAs are affected by LF1, we performed mRNA-Seq on nuclear and cytoplasmic fractions. We are currently analyzing this data to determine how LF1 may be contributing to viral infection and oncogenesis by affecting mRNA nuclear export.