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Tracking viral entry into target cells by virological and immunological methods

Epstein-Barr virus is a γ-herpesvirus endemic (>90%) in the human population and implicated in a variety of lymphomas and solid tumours including Hodgkin’s lymphoma, Burkitt’s lymphoma and post-transplant lymphoproliferative disorder. Despite the prevalence of EBV, little is known about the mechanisms of early infection, including the role of the various membrane glycoproteins to entry, fusion and escape. Additionally, although the long term immune response is well characterised, the contribution of CD4+ and CD8+ T cell recognition to surveillance of primary cellular infection is not known. In order to further understand the processes of virus entry and processing we have used a panel of glycoprotein knockout viruses and viral epitope-specific CD4+ CD8+ T cell clones to study EBV infection of primary B cells. We have shown that following binding uptake is rapid yet selective, with suggested roles for gp42 and gp85 in attachment and initiation of fusion. Endocytosis results in rapid up-regulation of primary resting B cell endosomal activity, although no further surface-bound EBV is internalised. Endosomal trafficking of endocytosed virus is rapid, reaching the late endosomal compartment within the first hour, and subsequent HLA class II loading and presentation within 8 hours of binding. Further, both membrane and capsid proteins were seen in the late endosome by confocal, indicating relatively low fusion efficiency. The endocytic/HLA class II pathway may therefore offer a promising target for CD4+ T cell immunotherapy offering a first line of defence during de novo infection.

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