Characterization of receptor use and entry mechanisms in two KSHV infection systems

Viruses initiate infection at the cell surface, where they use viral proteins to contact and manipulate naturally occurring host receptors in the plasma membrane. Through this interaction, viruses negotiate internalization and begin their infection cycle. These virus-receptor interactions can be surprisingly complex, sometimes coordinating many receptors using several viral proteins simultaneously. Cytoskeletal rearrangements, a multitude of intracellular signaling cascades, and even transcriptional changes can be triggered through the host receptors by this initial interaction and influence the outcome of the attempted infection. Thus, viral entry is a nuanced process evolved to ensure that viruses can infect the right cells at the right time, while successfully evading host defenses. Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an important human pathogen. It is the causative agent of several cancers and inflammatory disease which together, in the context of the global HIV epidemic, are a major public health burden. KSHV is the most recent of the human herpesviruses to be discovered, but research on KSHV entry mechanisms has almost a twenty-year history. Eight receptors for KSHV have been described, and it has become apparent that the step-by-step details of KSHV entry mechanisms are likely to be unique in every cell line. By interacting with the same set of receptors on human foreskin fibroblasts or primary microvascular endothelial cells, for example, the virion is internalized by clathrin-mediated endocytosis or clathrin-independent macropinocytosis, respectively. Here we investigated KSHV receptor usage in cell types that are relatively understudied in the field: epithelial cells and lymphocytes. We uncovered novel variability in receptor use across many susceptible cell lines, particularly that infection of epithelial cells and lymphocytes was independent of known KSHV integrin receptors and likely all known integrins. Additionally, we found that infection of Caki-1 and HeLa cells did not require EphA2 signaling, and infection of primary oral keratinocytes did not depend on Eph receptor interactions whatsoever. We hypothesize that there is at least one more KSHV receptor required for infection in the epithelial cells we studied. Furthermore, we showed that coculture-mediated infection of BJAB cells required heparan sulfate and Eph receptor interactions, despite the fact that BJAB cells do not express heparan sulfate and manipulation of Eph receptor expression did not affect infection. These results are evocative of a “transfer infection” mechanism akin to Epstein-Barr Virus, which requires receptor interactions on adjacent cells to promote infection of an otherwise non-susceptible cell type. We identified KSHV orf28 as a potential player in determining lymphocyte tropism.Our work reveals another layer of complexity beyond receptor availability on cells. It is now clear that even when KSHV receptors are expressed by a cell, additional contextual factors determine whether they play a role during infection. Going forward, this will be very important to understand, especially since virus-receptor interactions are often targeted by small molecules or biologics in the hopes of slowing viral dissemination

Characterization of receptor use and entry mechanisms in two KSHV infection systems

Viruses initiate infection at the cell surface, where they use viral proteins to contact and manipulate naturally occurring host receptors in the plasma membrane. Through this interaction, viruses negotiate internalization and begin their infection cycle. These virus-receptor interactions can be surprisingly complex, sometimes coordinating many receptors using several viral proteins simultaneously. Cytoskeletal rearrangements, a multitude of intracellular signaling cascades, and even transcriptional changes can be triggered through the host receptors by this initial interaction and influence the outcome of the attempted infection. Thus, viral entry is a nuanced process evolved to ensure that viruses can infect the right cells at the right time, while successfully evading host defenses. Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an important human pathogen. It is the causative agent of several cancers and inflammatory disease which together, in the context of the global HIV epidemic, are a major public health burden. KSHV is the most recent of the human herpesviruses to be discovered, but research on KSHV entry mechanisms has almost a twenty-year history. Eight receptors for KSHV have been described, and it has become apparent that the step-by-step details of KSHV entry mechanisms are likely to be unique in every cell line. By interacting with the same set of receptors on human foreskin fibroblasts or primary microvascular endothelial cells, for example, the virion is internalized by clathrin-mediated endocytosis or clathrin-independent macropinocytosis, respectively. Here we investigated KSHV receptor usage in cell types that are relatively understudied in the field: epithelial cells and lymphocytes. We uncovered novel variability in receptor use across many susceptible cell lines, particularly that infection of epithelial cells and lymphocytes was independent of known KSHV integrin receptors and likely all known integrins. Additionally, we found that infection of Caki-1 and HeLa cells did not require EphA2 signaling, and infection of primary oral keratinocytes did not depend on Eph receptor interactions whatsoever. We hypothesize that there is at least one more KSHV receptor required for infection in the epithelial cells we studied. Furthermore, we showed that coculture-mediated infection of BJAB cells required heparan sulfate and Eph receptor interactions, despite the fact that BJAB cells do not express heparan sulfate and manipulation of Eph receptor expression did not affect infection. These results are evocative of a “transfer infection” mechanism akin to Epstein-Barr Virus, which requires receptor interactions on adjacent cells to promote infection of an otherwise non-susceptible cell type. We identified KSHV orf28 as a potential player in determining lymphocyte tropism.Our work reveals another layer of complexity beyond receptor availability on cells. It is now clear that even when KSHV receptors are expressed by a cell, additional contextual factors determine whether they play a role during infection. Going forward, this will be very important to understand, especially since virus-receptor interactions are often targeted by small molecules or biologics in the hopes of slowing viral dissemination

A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

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.IMPORTANCE 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

A Kaposi’s Sarcoma-Associated Herpesvirus Infection Mechanism is Independent of Integrins α3β1, αVβ3, and αVβ5

ABSTRACTHost receptor usage by 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, then interacts with a complex of EphA2, xct, and integrins α3β1, αVβ3, 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 while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.IMPORTANCEOur data reveals an integrin-independent route of KSHV infection and suggests 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.</jats:sec