Gammaherpesviral tegument proteins - crucial effectors of viral infection

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All Gammaherpesviruses encode tegument proteins with homology to cellular PFAS (FGARAT), a purine nucleotide synthesis pathway enzyme conserved in all life forms. The viruses, however, have evolved these enzyme homologs toward diverse effector functions that interfere with ND10 mediated intrinsic immunity. Since high-resolution structural information is lacking from both viruses and eukaryotes, we will perform a structure-based analysis of viral FGARAT interactions (KSHV ORF75, RRV ORF75, and HVS ORF3) with their cellular counterpart, the human PFAS. Hitherto, protein expression of viral ORF75 (KSHV, RRV) and cellular PFAS (human) for structural analysis is in progress. The KSHV ORF75 protein is essential for replication and thus a target for the development of a new class of antiviral inhibitors. The molecular and biochemical analysis will include MS-based technologies and CRISPR/Cas9 mediated knockout as well as synthetic activation mediator (SAM) technologies established in the laboratory. The broader effects of viral FGARATs on the nuclear and cytoplasmic proteome will be quantitatively analyzed by SILAC in cells conditionally expressing the viral effectors. MS-based identification of interaction partners has already revealed SMCHD1, cellular PFAS, and USP15 as important targets. Notably, the cellular tumor suppressor SMCHD1 is being relocated by KSHV ORF75 to ND10. SMCHD1 is a crucial mediator of X-chromosome compaction and inactivation, and it is involved in DNA-Damage response, suggesting a link between viral interference with SMCHD1 and cancer. USP15 is a known promoter of TGFbeta signaling and oncogenesis. From the current results, we propose that viral FGARATs tackle several lines of cellular defense, and the project plan will address the following hypotheses:- Rhadinoviral vFGARAT tegument proteins target distinct ND10 associated chromatin remodeling factors to achieve a chromatin status favorable for viral gene expression during de-novo infection and during reactivation.- KSHV vFGARAT contributes to the development of viral cancers through interactions with cellular targets that have roles in human cancer and DNA repair.Uncovering the mechanisms of newly identified viral effector interactions with host cell proteins will extend knowledge on viral and cellular replication and central biosynthesis pathways, and on predisposition to virus-associated cancer.