Cell type-specific KSHV entry mediators for infection of epithelia and fibroblasts

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Kaposi’s sarcoma-associated herpesvirus (KSHV) is a major cause of morbidity and mortality in Sub-Saharan Africa and poses a threat to immunocompromised individuals worldwide. KSHV enters host cells through mechanisms involving a variety of interactions of viral glycoproteins with cellular receptors, among those the interaction with integrins through glycoprotein B and the interaction with molecules of the Eph family of receptor tyrosine kinases through the gH/gL glycoprotein complex. Previous studies from our group and others have shown that, while important, these known receptor interactions do not fully explain KSHV entry into host cells, also depending on the target cell. We therefore focused on the less well-studied glycoprotein K8.1, a highly glycosylated and immunogenic protein of the KSHV envelope. We observed that deletion of the gene coding for K8.1 leads to strongly reduced infectivity on epithelial cells. We speculated that this is due to the interaction of K8.1 with a so-far unknown receptor on these cells and therefore have identified a number of K8.1-interacting cellular proteins by affinity purification and mass spectrometry that we will now test for function in the KSHV entry process through gene knockout. We will further pinpoint the exact function of K8.1 and its interaction with cellular receptors with regard to the attachment step, endocytosis, and membrane fusion. In a second approach, we performed preliminary studies to identify cellular proteins that interact with the known KSHV entry receptor EphA2. We identified both known and novel interactors and analyzed these candidates through gene knockout. Knockout of one of these factors, a small GTPase-binding protein, caused a significant reduction in the susceptibility to KSHV infection of a number of different cell lines and primary cells, but not of all cells that were tested. The reduction in infection upon knockout was most pronounced in fibroblasts but not observable e.g. in an epithelial cell line. We now want to study the exact function of this GTPase-binding protein for both endocytosis of KSHV and of the EphA2 receptor.