Impaired Transporter Associated with Antigen Processing-Dependent Peptide Transport during Productive EBV Infection

Abstract

Human herpesviruses, including EBV, persist for life in infected individuals. During the lytic replicative cycle that is required for the production of infectious virus and transmission to another host, many viral Ags are expressed. Especially at this stage, immune evasion strategies are likely to be advantageous to avoid elimination of virus-producing cells. However, little is known about immune escape during productive EBV infection because no fully permissive infection model is available. In this study, we have developed a novel strategy to isolate populations of cells in an EBV lytic cycle based on the expression of a reporter gene under the control of an EBV early lytic cycle promoter. Thus, induction of the viral lytic cycle in transfected EBV+ B lymphoma cells resulted in concomitant reporter expression, allowing us, for the first time, to isolate highly purified cell populations in lytic cycle for biochemical and functional studies. Compared with latently infected B cells, cells supporting EBV lytic cycle displayed down-regulation of surface HLA class I, class II, and CD20, whereas expression levels of other surface markers remained unaffected. Moreover, during lytic cycle peptide transport into the endoplasmic reticulum, was reduced to <30% of levels found in latent infection. Because steady-state levels of TAP proteins were unaffected, these results point toward EBV-induced interference with TAP function as a specific mechanism contributing to the reduced levels of cell surface HLA class I. Our data implicate that EBV lytic cycle genes encode functions to evade T cell recognition, thereby creating a window for the generation of viral progeny.

Silencing the shutoff protein of Epstein–Barr virus in productively infected B cells points to (innate) targets for immune evasion

“…In control cells, EBV reactivation caused a minor reduction in CD71 surface levels, while HLA I and II were strongly downregulated (Figs 2a and S4a, upper panels), which is in line with earlier observations (Ressing et al, 2005). In lytic AKBM-shBGLF5 cells, surface display of HLA I and II was partly rescued (Figs 2a and S4a, lower panels), supporting a contribution of shutoff to evasion from T-cell detection during EBV replication in B cells.…”

“…Based on our current data, two groups of host surface proteins can be discriminated. The first group comprises proteins that are downregulated to a limited extent during EBV replication; the co-stimulatory molecules CD80 and CD86 can be included in this group (Ressing et al, 2005). The second group is more strongly downregulated, likely by multiple EBV lytic proteins, and their surface levels remain substantially reduced when BGLF5 is silenced.…”

“…Here, we have employed the EBV + Akata B cell line AKBM, in which cross-linking of the B-cell receptor with anti-human IgG reactivates EBV in 10-40 % of cells. Productively infected B cells can be identified and sorted on the basis of induced expression of a reporter protein, rat CD2-GFP (rCD2-GFP) (Ressing et al, 2005). Using this system, we have elucidated several immune evasion mechanisms acting during the productive phase of EBV infection (Horst et al, 2009;Ressing et al, 2005;van Gent et al, 2011.…”

“…Productively infected B cells can be identified and sorted on the basis of induced expression of a reporter protein, rat CD2-GFP (rCD2-GFP) (Ressing et al, 2005). Using this system, we have elucidated several immune evasion mechanisms acting during the productive phase of EBV infection (Horst et al, 2009;Ressing et al, 2005;van Gent et al, 2011. As our approach to suppress BGLF5-mediated shutoff during productive infection in B cells, lentivirus-delivered shRNAs were introduced into these EBV + AKBM cells.…”

Cyclical Expression of EBV Latent Membrane Protein 1 in EBV-Transformed B Cells Underpins Heterogeneity of Epitope Presentation and CD8+ T Cell Recognition

“…For these experiments, we selected LCLs transformed with a rEBV isolate lacking the BZLF1 gene, thus blocking the switch from latency into lytic cycle. EBV lytic cycle is associated with progressive down-regulation of HLA class I expression (28,29), and we wished to avoid such cells contributing to the HLA low fraction. The HLA high and HLA low subpopulations sorted from BZLF1-deficient LCLs were then analyzed for their sensitivity to CD8 ϩ T cell-mediated lysis, and for their expression levels of various cellular and viral proteins.…”