Cooperation between Epstein-Barr Virus Immune Evasion Proteins Spreads Protection from CD8 ⁺ T Cell Recognition across All Three Phases of the Lytic Cycle

Abstract

CD8 ⁺ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8 ⁺ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8 ⁺ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8 ⁺ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE<E<<L). Moreover, double-knockdown experiments showed that BILF1 and BNLF2a co-operate to further inhibit antigen presentation of L epitopes. Together, these data firstly indicate which potential immune-evasion functions are actually relevant in the context of lytic virus replication, and secondly identify lytic-cycle phase-specific effects that provide mechanistic insight into the immunodominance pattern seen for CD8 ⁺ T cell responses to EBV lytic antigens.

Author Summary

Epstein Barr Virus (EBV), an oncogenic herpesvirus, infects and persists asymptomatically in the majority of humans. In immunocompetent individuals, EBV co-exists with its host as a lifelong infection in the face of strong anti-viral CD8 ⁺ T-cell responses. Evasion of this immune-response is presumed to be due in part to immune-modulating mechanisms of certain EBV-encoded proteins expressed during lytic cycle replication. Three such proteins (BNLF2a, BGLF5 and BILF1) have been identified biochemically as able to interfere with HLA-class I antigen presentation. In this study we investigated these proteins in the context of EBV-infected cells in lytic cycle, and their functional recognition by EBV virus-specific CD8 ⁺ T-cells. A novel feature of EBV biology was revealed; rather than demonstrating simple redundancy, evasion proteins effect optimum temporal protection at different phases of lytic cycle. BNLF2a strongly inhibited CD8 ⁺ T-cell recognition immediately after the EBV-infected cells entered lytic cycle, with its influence waning upon progression to later phases of lytic cycle. Conversely, BILF1 strongly inhibited recognition predominantly at the late phase of lytic cycle. Unexpectedly, despite its well-characterised molecular functions, BGLF5 had relatively little effect on recognition at any stage of lytic cycle. Our results help to explain the previously-identified unusual pattern of immunodominance of anti-EBV CD8 ⁺ T-cell responses.