Analysis of host gene expression changes reveals distinct roles for the cytoplasmic domain of the Epstein-Barr virus receptor/CD21 in B-cell maturation, activation, and initiation of virus infection

Abstract

Epstein-Barr virus (EBV) attachment to human CD21 on the B-cell surface initiates infection. Whether CD21 is a simple tether or conveys vital information to the cell interior for production of host factors that promote infection of primary B cells is controversial, as the cytoplasmic fragment of CD21 is short, though highly conserved. The ubiquity of CD21 on normal B cells, the diversity of this population, and the well-known resistance of primary B cells to gene transfer technologies have all impeded resolution of this question. To uncover the role(s) of the CD21 cytoplasmic domain during infection initiation, the full-length receptor (CD21=CR), a mutant lacking the entire cytoplasmic tail (CT), and a control vector (NEO) were stably expressed in two pre-B-cell lines that lack endogenous receptor. Genome-wide transcriptional analysis demonstrated that stable CD21 surface expression alone (either CR or CT) produced multiple independent changes in gene expression, though both dramatically decreased class I melanoma-associated antigen (MAGE) family RNAs and upregulated genes associated with B-cell differentiation (e.g., C2TA, HLA-II, IL21R, MIC2, CD48, and PTPRCAP/CD45-associated protein). Temporal analysis spanning 72 h revealed that not only CR- but also CT-expressing lines initiated latency. In spite of this, the number and spectrum of transcripts altered in CR- compared with CT-bearing lines at 1 h after infection further diverged. Differential modulation of immediate early cellular transcripts (e.g., c-Jun and multiple histones), both novel and previously linked to CD21-initiated signaling, as well as distinct results from pathway analyses support a separate role for the cytoplasmic domain in initiation of intracellular signals.

Importance: Membrane proteins that mediate virus attachment tether virus particles to the cell surface, initiating infection. In addition, upon virus interaction such proteins may transmit signals to the interior of the cell that support subsequent steps in the infection process. Here we show that expression of the Epstein-Barr virus B-cell attachment receptor, CD21, in B cells that lack this receptor results in significant changes in gene expression, both before and rapidly following EBV-CD21 interaction. These changes translate into major signaling pathway alterations that are predicted to support stable infection.

FIG 1

Pre-B-cell lines Nalm6 and Laz221, which lack endogenous CR and variably express CR complex members, can stably express recombinant CR and CT and bind EBV. (A) Flow cytometric analysis (FC) of CD21, CD35, CD19/CD81/Leu13(CD225), and HLA-II (DR, DP, and DQ) expression on Nalm6 and Laz221 cells. x axis, log fluorescence intensity; y axis, cell number. An LCL is displayed as a staining control. (B) Expression of CR on two Nalm6 clones (1 and 2) compared with a lymphoma line (BL41) and a LCL that express endogenous CR at different levels. (C) Verification of CR and CT expression by immunoblot (left) and by PCR (right). (D) Demonstration that expression of associated proteins is unmodified in Nalm6CR although coreceptor, HLA-II expression is increased ∼3-fold (left shift of log mean fluorescence intensity). (E) Top four histograms, demonstration that EBV attaches to cells expressing CR and also CT by FC. CR and CT were directly detected with MAb HB-5 anti-CD21 (black line) compared with an isotype control (block). MAb 2L10 anti-EBVgp350/220 was used for detection of bound EBV (black line). No virus served as the negative control (block). An LCL is displayed as a positive control. Bottom histogram, EBV blockade, confirms that rabbit anti-CR blocks EBV attachment to Laz221CR and CT (dark gray block), whereas normal rabbit serum does not (light gray block). See Materials and Methods for details. (F) Demonstration that CT is not cleaved by phosphatidylinositol-specific phospholipase C (PIPLC) (not PI linked) by FC. Top, the GPI-linked proteins CD48 and CD59 are cleaved by PIPLC in HPB-ALL, a control T-cell line, resulting in decreased surface expression. Neither HLA-I nor CR (endogenous), which lack PI linkages, is altered. Bottom, concurrent analysis of Nalm6CT showed no evidence of CT cleavage. CD48 and CD59 are absent and HLA-I uncleaved on Nalm6CT.

FIG 2

EBV infection of CR-bearing pre-B-cell lines analyzed by RNA blot hybridization. (A) Top, expression of LMP1 and EBER in Nalm6CR and Laz221CR cells was detected 24 to 72 h after EBV incubation. Nalm6NEO cells served as a negative control. Expression of BHRF1, a highly expressed lytic RNA, was absent. Ak (Akata) cells served as the positive control. GAPDH or 28S RNA (arrow) provided loading controls. Bottom, preincubation of cells with OKB7, a MAb that blocks binding of EBV gp350/220 to CR, prevented infection of Nalm6CR, whereas UPC10, an isotype-matched control, did not. (B) EBNA-1 (top), EBNA-2 (middle), and LMP-2A (bottom) transcripts were also detected, as shown in Nalm6CR cells. (C) Cellular RNAs encoding RAG-1, TdT, and CD10 declined upon EBV infection, as previously observed (22). (D) Infection of CT-bearing cells induced LMP1 (Nalm6 and Laz221) and EBNA2 (Nalm6) expression with a time course similar to that of CR.

FIG 3

Unsupervised clustering and principal-component analysis of the gene expression data. (A) An unsupervised Euclidean distance-based cluster of arrays after normalizing the data demonstrates that B-cell lines that express CT, CR, or NEO form separate clusters that correlate better with one another than with cells expressing an alternate receptor form. (B) PCA analysis demonstrates that CT, CR, and NEO cells have distinct gene expression patterns. (C) Heat map of the differentially expressed genes by comparing NEO, CT, and CR at 1 min and 60 min demonstrates that NEO, CR, and CT cells have a set of unique differentially expressed genes. Rows represent genes, and columns represent samples from CT, CR, and NEO groups. Genes are clustered using row-normalized signals and mapped to the [−1,1] interval (shown in scales beneath each heat map). Red and green represent high and low expression values, respectively.

FIG 4

Differentially expressed genes in NEO, CR, and CT cells at 1 min (constitutive changes). (A) Venn diagrams of differentially expressed (downregulated and upregulated) genes in NEO, CT, and CR cells. (B) Heat map of selected top genes constitutively altered (1 min) after recombinant receptor expression. Left, NEO versus CT; middle, NEO versus CR; right, CT versus CR. Genes are clustered using row-normalized signals and mapped to the [−1,1] interval (shown in scales beneath each heat map). Red and blue represent high and low expression values, respectively.

FIG 5

Comparison of genes differentially altered in NEO, CT, and CR at 1 versus 60 min after incubation with EBV. (A) Venn diagrams of differentially expressed (downregulated and upregulated) genes in NEO, CT, and CR cells. (B) Heat map of selected top genes changing from 1 to 60 min within the cell lines NEO, CT, and CR. (C) Heat map depicting selected top genes changing at 60 min in NEO versus CT, NEO versus CR, and CT versus CR. Genes are clustered using row-normalized signals and mapped to the [−1,1] interval (shown in scales beneath each heat map). Red and blue represent high and low expression values, respectively.

FIG 6

Comparison of top signaling pathways affected in different receptor-bearing cell types. (A) CT; (B) CR. Sets of differentially expressed genes (LCB of >1.2) between cell lines were uploaded onto Ingenuity Pathway Analysis and corresponding signaling pathways predicted. Statistical significance was set at −log P = 2.

FIG 7

Comparison of predicted upstream regulators of NEO, CT, and CR transcripts at 60 min after EBV infection. Differentially expressed genes were processed through Ingenuity Pathway Analysis to predict the downstream regulators whose activation status was affected following EBV interaction. No regulators reached the significant activation z score of −2 or +2 in NEO (A), whereas several regulators were predicted to be activated (z-score of >2) in CT (B) and a significantly larger number of regulators was either activated or inhibited (z score of <−2) in CR (C).

FIG 8

RT-PCR validation of selected differentially expressed genes, including MAGE family, immediate early proto-oncogenes, histones, and novel candidates.

FIG 9

Schematic of CD21 complex formation on normal resting (primary) B cells compared with transfectant cell line models (CR and CT) used in this work.