Epstein-Barr virus isolates retain their capacity to evade T cell immunity through BNLF2a despite extensive sequence variation

Abstract

The Epstein-Barr virus (EBV)-encoded immune evasion protein BNLF2a inhibits the transporter associated with antigen processing (TAP), thereby downregulating HLA class I expression at the cell surface. As a consequence, recognition of EBV-infected cells by cytotoxic T cells is impaired. Here, we show that sequence polymorphism of the BNLF2a protein is observed with natural EBV isolates, with evidence for positive selection. Despite these mutations, the BNLF2a variants efficiently reduce cell surface HLA class I levels. This conservation of BNLF2a function during evolution of EBV implies an important role for the viral TAP inhibitor in preventing T cell recognition during viral infection.

Fig 1

EBV isolates retain BNLF2a-mediated HLA I downregulation. (A) MJS cells were transiently transfected to express the control protein GFP or to coexpress wild-type BNLF2a and GFP. After 48 h, cells were stained for intracellular expression of BNLF2a (monoclonal antibody [MAb] 8E2). Subsequently, the cells were analyzed by flow cytometry using CellQuest Pro software (BD Biosciences). (B) MJS cells were transiently transfected to express the control protein GFP, wild-type BNLF2a (wt), or one of the following BNLF2a mutants: A8T/R40K, H3Q/A8T/R40K, A8T/S39R/R40K, A8T/V46A, or V46A. After 48 h, cells were stained for cell surface expression of HLA I (MAb B9.12.1) and HLA II (MAb L243) and analyzed by flow cytometry using CellQuest Pro software (BD Biosciences). (C) Quantification of flow cytometry data. Cell surface expression levels of HLA I were correlated with GFP expression for cells transfected to express the control protein GFP, wild-type (wt) BNLF2a, or the A8T/R40K BNLF2a mutant. To this end, values were corrected for cell surface expression of HLA I in GFP-negative cells. The standard deviations are represented by the error bars. *, P < 0.01 as determined by a t test. (D) Graphical display of the results shown in panel B. The mean fluorescence index of HLA I expression is plotted against the mean fluorescence index of GFP expression. The results of one representative experiment out of at least three independent experiments are shown. For panel A, the experiment was performed in duplicate.

Fig 2

N-terminal amino acids of BNLF2a affect its immune evasion function. (A) MJS cells were transiently transfected to express the control protein GFP, wild-type BNLF2a (wt), BNLF2a-A8T (A8T), BNLF2a-H3Q/A8T (H3Q/A8T), or BNLF2a-H3P (H3P). After 48 h, cells were stained for cell surface expression of HLA I (MAb B9.12.1) and HLA II (MAb L243) and analyzed by flow cytometry using CellQuest Pro software (BD Biosciences). (B and D) Graphical display of flow cytometry data. The mean fluorescence index of HLA I expression is plotted against the mean fluorescence index of GFP expression. (C and E) Quantification of flow cytometry data. Cell surface expression levels of HLA I were correlated with GFP expression. To this end, values were corrected for cell surface expression of HLA I in GFP-negative cells. The standard deviations are represented by the error bars. *, P < 0.01 as determined by a t test. The results of one representative experiment out of three independent experiments are shown.