Host receptor usage by Kaposi's sarcoma-associated herpesvirus (KSHV) has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding and then interacts with a complex of EphA2, xCT, and integrins α3β1, αVβ3, and αVβ5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of αV- and β1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2. Our data reveal an integrin-independent route of KSHV infection and suggest that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.

Host receptor usage by KSHV has been best studied using endothelial and fibroblast cells in 24 tissue culture. In these models, KSHV adheres to the cell though heparan sulfate (HS) binding, then 25 interacts with a complex of EphA2, xct, and integrins α3β1, αVβ3, αVβ5 to catalyze viral entry. We 26 dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor 27 usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS 28 and EphA2, but is independent of integrin expression. Furthermore, infection appears to be 29 independent of EphA2 signaling, despite the reliance on clathrin-mediated endocytosis. We also 30 demonstrated while two other endogenous Eph receptors were dispensable for infection, transduced 31 EphA4 and EphA5 significantly enhanced KSHV infection of cells lacking EphA2. 32 33 IMPORTANCE Our data reveals an integrin-independent endocytic route of KSHV infection and suggests 34 that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph 35 receptors are large protein families with diverse expression patterns across cells and tissues, we propose 36 that KSHV may engage with several proteins from both families in different combinations to negotiate 37 successful entry into diverse cell types. 38 39 40 41 42 43 44 45 46 47 48 49